partial purification and characterization of selected enzymes of bovine nitrogen metabolism

PARTIAL PURIFICATION AND CHARACTERIZATION OF SELECTED ENZYMES OF

BOVINE NITROGEN METABOLISM:

COMPARISON OF THE NGUNI AND HEREFORD BREEDS

By

LUTENDO MICHAEL MATHOMU

Submitted in accordance with the requirements for the degree of

MASTER OF SCIENCE

In the subject

LIFE SCIENCES

At the

UNIVERSITY OF SOUTH AFRICA

SUPERVISOR: DR M S MYER

CO-SUPERVISOR: PROF C KENYON

NOVEMBER 2012

ii

DECLARATION

I, Lutendo Michael Mathomu hereby declare that the dissertation, “Partial purification and

characterization of selected enzymes of bovine nitrogen metabolism: Comparison of the Nguni and

Hereford breeds”, is my own unaided work. It is being submitted for the Master of Science degree

at the University of South Africa, Pretoria. It has not been submitted before for any degree or

examination in any other university.

________________________

(Signature of candidate)

______day of _____________ 2012

________________________ ________________________

(Signature of supervisor) (Signature of co-supervisor)

______ day of _____________ 2012 ______ day of _____________ 2012

iii

ABSTRACT

Ruminant animals consuming low N-diet have been reported to have increased urea reabsorption

with the Nguni being categorized as N-recycling ruminant. The enzymes associated with N-cycling

are hypothesized to contribute to survival of the Nguni in harsh conditions. Enzymes responsible

for such a function needed to be characterized in order to determine their effect in the functioning

of the Nguni as opposed to Hereford breed. Crude enzymes from both breeds were separated from

most or some contaminants by sephadex G-25, DEAE sephacel, and different affinity column

chromatography. CPS and GDH were successfully purified and characterized by LC-MS/MS and

further analysed by ProteinPilot™, blasted and matched >95% with those of Bos Taurus.

Comparison of characterized enzymes and those which failed to ionise such as ARG, GS and GA

was done using kinetics and graphs annotating specific activities. Partial purification and

characterization was in part achieved.

KEY TERMS

Cattle breeds, Purification, Characterization, Nitrogen metabolism, Arginase, Carbamoyl phosphate

synthetase, Glutamine synthetase, Glutamate dehydrogenase, Glutaminase, Chromatography, Rate

of maximal velocity, Enzyme activity and Kinetics.

iv

ACKNOWLEDGEMENTS

I would like to appreciate my supervisors: Dr. Martin Myer for his valuable support without

compromise and Professor Colin Kenyon for his enthusiastic mentorship and profound knowledge

of science.

My sincere gratitude goes to Mrs Caroline Kunene and Mr Albert Ngwako Mabetha for their

unfading availability and assistance during my laborious laboratory work.

I also wish to thank Dr. Robyn Roth for being there when I needed her advices and for always

listening to my error beeps and offering to help, I whole heartedly appreciate.

Thank you to Dr. Stoyan Stoychev and Mr Sipho Mamputha for always landing a helping hand

during most of my analysis.

The generous financial support of the following organizations is gratefully acknowledged:

University of South Africa and Technology Innovation Agency.

I am thankful to Devon farming, Hereford breeders of South Africa together with Dr. Hans van Zyl

for the provision of samples.

I am grateful to have had you to listen to my troubles Sharon. I always knew you will give a

valuable advice, Dr. Tsepo Tsekoa.

Whenever I thought of giving up you said it would work out. Thank you for listening to my

complaints whenever I got frustrated Rirhandzu.

For your unconditional support and allowing me to pursue my interests, I say you remain the best

mother (…tombo li nyadzwa nga vhafhati, Avhafunani Gladys Mathomu).

I told you I believe in your promises. God, you are good.

v

ABBREVIATIONS & ACRONYMS

AD Anno Domino

AL Argininosuccinic acid Lyase

ARC Agricultural Research Council

ARC-AII Agricultural Research Council Animal Improvement Institute

ARG Arginase

AS Argininosuccinic acid Synthase

ADP Adenosine diphosphate

AMP Adenosine monophosphate

ACN Acetonitrile

AESBF 4-(-2-Aminoethyl)-Benzenesulfonyl Fluoride

ATP Adenosine triphosphate

BC Before Christ

CPS Carbamyl phosphate synthetase

CITR Citrulline

Ca Calcium

Cd Cadmium

Co Cobalt

Cα Alpha-carbon

cAMP Cyclic Adenosine monophosphate

CNBr Cyanogen Bromide

C Carbon

DTT Dithioerythritol

DNA Deoxyribonucleic acid

DEAE Diethylaminoethyl

DMSO Dimethyl sulfoxide

EUN Endogenous urinary nitrogen

EDTA Ethylenediaminetetraacetic acid

EGTA Ethyleneglycoltetraacetic acid

FAO Food and Agricultural Organisation

vi

GS Glutamine synthetase

GOGAT Glutamine oxoglutarate aminotransferase

GA Glutaminase

GABA Gamma-aminobutyric acid

GI Gastrointestinal

GDH Glutamate dehydrogenase

GLUD Glutamate dehydrogenase

GTP Guanosine triphospahate

H Hereford

HCO3-

Bicarbonate

HPLC High performance liquid chromatography

HCl Hydrochloric

HLARG Hereford liver arginase

HLGA Hereford liver glutaminase

HLGS Hereford liver glutamine synthetase

HLCPS Hereford liver carbamoyl phosphate synthetase

HLGDH Hereford liver glutamate dehydrogenase

ID Identification

K2HPO4 Dipotassium phosphate

KH2PO4 Potassium dihydrogen phosphate

Km Michaelis-Menten constant

KDa Kilodalton

KCN Potassium cyanide

KCl Potassium chloride

LC-MS/MS Liquid chromatography - Mass Spectrophotometer/ Mass Spectrophotometer

Li2+

Lithium

M Marker (Protein page ruler)

MFN Metabolic faecal nitrogen

MnCl2 Manganese chloride

MgCl2 Magnesium chloride

MgSO4 Magnesium sulphate

vii

MgCl2.6H2O Magnesium chloride anhydrous

MeOH Methanol

Min Minute

mM

Millimolar

Mg Milligrams

Ml Millilitres

Mn Manganese

Mg Magnesium

N Nitrogen

NAGS N-Acetyl-Glutamate Synthase

NaHCO3 Sodium Hydrogen Carbonate

NLARG Nguni liver arginase

NLGA Nguni liver glutaminase

NLGDH Nguni liver glutamate dehydrogenase

NLGS Nguni liver glutamine synthetase

NPN Non-protein nitrogen

NO Nitrogen oxide

ODC Ornithine decarboxylase

ORNT Ornithine

OTC Ornithine Transcarbamylase

PDG Phosphate-dependent glutaminase

PMSF Phenylmethylsulfonyl-fluoride

SCD Spinocerebellar degeneration

SDS-PAGE Sodium dodecylsulphate polyacrylamide gel electrophoresis

TCA Tricarboxylic acid

UK United Kingdom

UniprotKB Universal protein Knowledgebase

Vmax Maximum velocity

α-KG Alpha Ketoglutarate

viii

LIST OF FIGURES

Figure 1.1. Dispersal of domesticated cattle in Africa

1

Figure 1.2. Frequency of taurine and indicine Y chromosome allele in sub-Saharan

Africa.

2

Figure 1.3. The primary enzymatic steps of the urea cycle

11

Figure 1.4. The mechanism of ammonia detoxification forming urea

14

Figure 1.5. Transport of ammonia to the liver for urea synthesis, using either glutamine

(most tissues) or alanine (muscle) as the carrier.

15

Figure 1.6. End products of the utilization of amino acids are ammonia and urea

excreted by the animal in urine.

16

Figure 2.1: A general overview of L-arginine-connected pathways

21

Figure 2.2. Summarised reaction mechanism in the biosynthesis of Carbamoyl

phosphate and three other unstable intermediates

23

Figure 2.3. An indication of the place of GS in the complex matrix of nitrogen and

carbon metabolism.

27

Figure 2.4. Crystal structure of Arginase complexed with 2Mn2+

ions and the arginine

analogue nor-N-omega-hydroxy-L-arginine

33

Figure 2.5. Structure of Carbamoyl phosphate synthetase.

34

Figure 2.6. Stereo view representation of the large and small subunits of CPS.

35

ix

Figure 2.7. Crystal structures of mammalian glutamine synthetase with 10 identical sub

units

37

Figure 2.8. Structure of bovine GDH.

39

Figure 2.9. The crystal structure of kidney glutaminase showing glutamate in the active

site.

41

Figure 4.1. Indication of the effect of the incubation times on the specific activity of

Nguni and Hereford liver arginase (0.2 – 0.3 mg) at 37oC.

65

Figure 4.2. Effect of the manganese concentration on the specific activity of Nguni

arginase.

66

Figure 4.3. Effect of the manganese concentration on the specific activity of Hereford

arginase.

67

Figure 4.4. Effect of the arginine concentration on the specific activity of Nguni

arginase.

68

Figure 4.5. Effect of the arginine concentration on the specific activity of Nguni

arginase.

69

Figure 4.6. Comparison of the effect of the manganese concentration on the specific

activity of Nguni and Hereford liver arginase presented as average of each increasing

concentration of manganese.

70

Figure 4.7. Comparison of the effect of the arginine concentration on the specific

activity of Nguni and Hereford liver arginase presented as average of each increasing

arginine concentration.

71

x

Figure 4.8. Effect of the selected divalent cations on the specific activity of Hereford

and Nguni arginase.

74

Figure 4.9: SDS-PAGE (10%) analysis of partially purified liver arginase 75

Figure 4.10. The effect of the concentration of Mg-ATP on the specific activity of

Nguni liver carbamoyl phosphate synthetase.

77

Figure 4.11. The effect of the concentration of Mg-ATP on the specific activity of

Hereford liver carbamoyl phosphate synthetase.

78

Figure 4.12. The effect of the concentration of NH4Cl on the specific activity of Nguni

liver carbamoyl phosphate synthetase.

79

Figure 4.13. The effect of the concentration of NH4Cl on the specific activity of

Hereford liver carbamoyl phosphate synthetase.

80

Figure 4.14. Comparison of the effect of the concentration of Mg-ATP on the specific

activity of Nguni (NLCPS) and Hereford (HLCPS) liver carbamoyl phosphate

synthetase presented as average of each increasing Mg-ATP concentration.

81

Figure 4.15. Comparison of the effect of the concentration of NH4Cl on the specific

activity of Nguni (NLCPS) and Hereford (HLCPS) liver carbamoyl phosphate

synthetase presented as average of each increasing NH4Cl concentration.

82

Figure 4.16. SDS-PAGE (10%) analysis of partially purified liver carbamyl phosphate

synthetase

85

Figure 4.17. QSTAR Elite ESI Generated peptide profile for CPS. Peptides of interest:

Precursor MS regions analysed by ProteinPilot™ Software 3.0

86

xi

Figure 4.18. A QSTAR Elite ESI Generated: Carbamoyl-phosphate synthetase I. -

Homo sapiens (Human) is displayed. Peptides of interest: Precursor MS regions

analysed by ProteinPilot™ Software 3.0

85

Figure 4.19. Specific activity of Nguni liver GS versus Mg-ATP concentration. Specific

activity as a function of Mg-ATP concentration.

88

Figure 4.20. Specific activity of Hereford liver GS versus Mg-ATP concentration.

Specific activity as a function of Mg-ATP

89

Figure 4.21. Comparison of the specific activity of Nguni (NLGS) and Hereford

(HLGS) liver glutamine synthetase versus Mg-ATP concentration presented as average

of each increasing Mg-ATP concentration.

90

Figure 4.22. SDS-PAGE (10%) analysis of partially purified liver glutamine synthetase

91

Figure 4.23. Effect of the variation of α-ketoglutarate concentration on the specific

activity of Nguni liver glutamate dehydrogenase at 37oC.

93

Figure 4.24. Effect of the variation of α-ketoglutarate concentration on the specific

activity of Hereford liver glutamate dehydrogenase at 37oC.

94

Figure 4.25. Effect of the variation of ammonium sulphate concentration on the specific


95



96

Figure 4.27. Comparison of the effect of the variation of α-ketoglutarate concentration

on the specific activity of Nguni and Hereford liver GDH presented as an average of

each increasing concentration of α-ketoglutarate

97

Figure 4.28. Comparison of the effect of the variation of ammonium sulphate 98

xii

concentration on the specific activity of Nguni and Hereford liver GDH represented as

evarage of each increasing concentration of ammonium sulphate.

Figure 4.29 SDS-PAGE (10%) analysis of partially purified Nguni liver glutamate

dehydrogenase

100

Figure 4.30 SDS-PAGE (10%) analysis of partially purified Hereford liver glutamate

dehydrogenase

101

Figure 4.31. QSTAR Elite ESI Generated peptide profile for GDH. Peptides of interest:


102



103

Figure 4.33. Effect of glutamine concentration on the specific activity of Nguni liver

glutaminase at 37oC.

106

Figure 4.34. Effect of glutamine concentration on specific activity of Hereford liver


107

Figure 4.35. Comparison of the effect of glutamine concentration on the specific

activity of Nguni (NLGA) and Hereford (HLGA) liver glutaminase at 37oC.

108

Figure 4.36: SDS-PAGE (10%) analysis of partially purified liver glutaminase

111

Figure 4.37. SDS-PAGE (10%) analysis of partially purified liver glutaminase

112

Figure 5.1. Detoxification of ammonia released via transdeamination by its conversion to urea 125

Figure 5.2. The urea cycle showing the two mitochondrial and 3 cytosolic reactions 126

xiii

LIST OF TABLES

Table 4.1. Purification of Nguni liver and Hereford liver Arginase

62

Table 4.2. Comparison of Vmax and Km of Nguni and Hereford liver arginase with

respect to manganese is calculated by graph-pad prism.

63


respect to arginine is calculated by graph-pad prism.

64

Table 4.4. Summary of purification of carbamoyl phosphate synthetase from Nguni

(N) and Hereford livers (H).

74

Table 4.5. Comparison of Vmax and Km of Nguni liver CPS with respect to Mg-ATP

and ammonium is calculated by graph-pad prism.

81

Table 4.6. Comparison Vmax and Km of Hereford liver CPS with respect to Mg-ATP

and ammonium is calculated by graph-pad prism.

82

Table 4.7. Blast information for Nguni liver CPS generated from NCBI database of

proteins.

85

Table 4.8. Blast information for Hereford liver CPS generated from NCBI database of

proteins.

86

Table 4.9. Purification of Nguni liver (N) and Hereford liver (H) GS, indication from

both liver samples. (Units = μmoles ferric hydroximate complex/minute/mg protein).

87

Table 4.10. Comparison of Vmax and Km of Nguni and Hereford liver GS with respect

to Mg-ATP is calculated by graph-pad prism.

87

Table 4.11. Summary of purification of Nguni liver and Hereford liver glutamate

dehydrogenase.

92

Table 4.12. Comparison of Vmax and Km of Nguni and Hereford liver GDH with

respect to substrate α-ketoglutarate is calculated by graph-pad prism.

99

xiv


respect to cofactor manganese is calculated by graph-pad prism.

99

Table 4.14. Blast information for Nguni liver GDH generated from NCBI database of

proteins. Blast Information

103

Table 4.15. Blast information for Hereford liver GDH generated from the NCBI blast database

of proteins. Blast Information.

104

Table 4.16. Purification of Nguni liver and Hereford liver Glutaminase synthetase.

Summarised results.

104

Table 4.17. Comparison of Vmax and Km of Nguni liver glutaminase with respect to

substrate is calculated with the use of Graph-Pad™ prism 5.02.

109

Table 4.18. Comparison of Vmax and Km of Hereford liver glutaminase with respect to

glutamine is calculated with the use of Graph-Pad™ prism 5.02.

109

xv

TABLE OF CONTENTS

Declaration …………………………………………………………………... ii

Abstract ………….………………………………………………………....... iii

Key terms ……………………………………………………………………. iii

Acknowledgement …………………………………………………………… iv

Abbreviations and Acronyms ………………….……………………………. v

List of figures ………………………………………………………………... viii

List of tables …………………………………………………………………. xiii

Table of Contents ……………………………………………………………. xv

CHAPTER 1: BACKGROUND ......................................................................................................... 1

1. Introduction .............................................................................................................................. 1

1.1. Renewed Interest in the Nguni Breed ............................................................................... 6

1.2. Overview of the Nguni cattle project in South Africa ...................................................... 7

1.3. Mammalian nitrogen metabolism ..................................................................................... 8

1.4. Urea cycle ......................................................................................................................... 9

1.5. Problem statement .......................................................................................................... 13

1.6. Expansion of the Problem Statement.............................................................................. 13

1.7. Justification of the Study ................................................................................................ 16

1.8. Aims and Objectives ....................................................................................................... 17

1.8.1. Aims ........................................................................................................................ 17

1.8.2. Objectives ................................................................................................................ 18

CHAPTER 2: UREA CYCLE ENZYMES ...................................................................................... 19

2.1. Enzymology of urea cycle enzymes ............................................................................... 31

CHAPTER 3: MATERIALS AND METHODS .............................................................................. 42

3.1. Purification of bovine liver arginase .............................................................................. 42

xvi

3.1.1. Materials .................................................................................................................. 42

3.1.2. L-Arginine-AH-Sepharose 4B affinity preparation ................................................ 43

3.1.3. Purification of bovine liver arginase procedure ...................................................... 43

3.1.4. Effect of divalent cations......................................................................................... 45

3.1.5. Effect of incubation time ......................................................................................... 45

3.1.6. Arginase Assay: Estimation of enzyme activity ..................................................... 45

3.1.7. Data analysis ........................................................................................................... 46

3.2. Purification of carbamoyl phosphate synthetase ............................................................ 46

3.2.1. Materials .................................................................................................................. 46

3.2.2. Purification of bovine CPS procedure ..................................................................... 47

3.2.3. Specific activity of Carbamoyl phosphate synthetase: Analysis by High

Performance Liquid chromatography (HPLC). ..................................................................... 48

3.2.4. Data analysis ........................................................................................................... 49

3.3. Purification of glutamine synthetase .............................................................................. 49

3.3.1. Materials .................................................................................................................. 49

3.3.2. Purification of bovine GS procedure ....................................................................... 50

3.3.3. Glutamyl Transferase Assay: Estimation of enzyme activity of GS ....................... 51

3.3.4. Specific activity of GS: Analysis by High Performance Liquid chromatography

(HPLC) 51

3.3.5. Data analysis ........................................................................................................... 52

3.4. Purification of glutamate dehydrogenase ....................................................................... 52

3.4.1. Materials .................................................................................................................. 52

3.4.2. Purification of bovine GDH procedure ................................................................... 53

3.4.3. GTP Sepharose Column chromatography: Preparation of GTP Sepharose ............ 54

3.4.4. Glutamate dehydrogenase Assay: Estimation of enzyme activity. ......................... 55

3.5. Purification of glutaminase ............................................................................................. 55

xvii

3.5.1. Materials .................................................................................................................. 55

3.5.2. Purification of bovine glutaminase procedure ........................................................ 56

3.5.3. Glutaminase Assays: Estimation of enzyme activity. ............................................. 57

3.5.4. Data analysis ........................................................................................................... 58

3.6. Protein concentration assay ............................................................................................ 59

3.7. SDS-PAGE ..................................................................................................................... 59

3.8. Bio-analysis of extracted proteins with Liquid Chromatography Mass Spectrometry

(LC-MS/MS) ............................................................................................................................. 59

3.8.1. Introduction to QSTAR®

Elite Liquid Chromatography Mass Spectrometry (LC-

MS/MS) 59

3.9. Database search .............................................................................................................. 60

3.10. Kinetic constants ......................................................................................................... 61

CHAPTER 4: INTEGRATED RESULTS........................................................................................ 62

4.1. Purification of bovine arginase ....................................................................................... 62

4.1.1. Kinetics.................................................................................................................... 63

4.1.2. Enzyme Incubation time.......................................................................................... 64

4.1.3. Effect of substrate concentrations ........................................................................... 64

4.1.4. Effect of divalent cations......................................................................................... 72

4.1.5. Estimation of arginase molecular weight on SDS-PAGE ....................................... 73

4.2. Purification of carbamoyl phosphate synthetase ............................................................ 74

4.2.1. Kinetics.................................................................................................................... 75

4.2.2. Estimation of CPS molecular weight on SDS-PAGE ............................................. 82

4.2.3. Trypsin digest and LC/MS/MS for CPS band Identification .................................. 83

4.3. Purification of glutamine synthetase .............................................................................. 86

4.3.1. Kinetics ....................................................................................................................... 87

4.3.2. Estimation of GS molecular weight on SDS-PAGE ............................................... 90

xviii

4.4. Purification of glutamate dehydrogenase ....................................................................... 92

4.4.1. Kinetics.................................................................................................................... 92

4.4.3. Trypsin digest and LC-MS/MS for GDH band ID ................................................ 102

4.5. Purification of glutaminase ........................................................................................... 104

4.5.1. Kinetics.................................................................................................................. 105

4.5.2. Estimation of glutaminase molecular weight on SDS-PAGE ............................... 110

CHAPTER 5: INTERGRATED DISCUSSION AND CONCLUSION ........................................ 113

5.1. Arginase ........................................................................................................................ 114

5.2. Carbamoyl phosphate synthetase (CPS) ....................................................................... 116

5.3. Glutamine synthetase (GS) ........................................................................................... 118

5.4. Glutamate dehydrogenase (GDH) ................................................................................ 120

5.5. Glutaminase .................................................................................................................. 122

5.6. General Conclusion ....................................................................................................... 123

CHAPTER 6: SUMMARY AND RECOMMENDATIONS ......................................................... 127

6.1. Summary ....................................................................................................................... 127

6.2. Recommendations ........................................................................................................ 128

REFERENCES ............................................................................................................................... 130

APPENDICES ................................................................................................................................ 163

Chapter 1: Background

1

CHAPTER 1: BACKGROUND

1. Introduction

African cattle have been surveyed and the continent is home to 145 cattle breeds with 22% of the

original breeds having become extinct in the last 100 years while 27% of the remainder are at

varying degrees of risk (Rege, 1999). Out of the livestock breeds existing in 1999, 70% were in

developing countries where the risk of loss is at an alarming level (Rege, 1999).

The records of domestication of cattle in Africa show that the Nguni breed was present in the Nile

Valley by 400 BC (Epstein, 1971). Presumably cattle migrated southwards from the northern

regions of Africa in the course of barter and avoiding environmental pressures and war as shown in

Figure 1.1 below.

Figure 1.1. Dispersal of domesticated cattle in Africa (Hanotte et al., 1998)

Cattle were found in the Luangwa Valley in Zambia around 300 BC. By 300 AD communities

which had settled with cattle were living in southern Africa in areas of eastern Botswana, in


2

Gauteng as far as the Hartebeespoort dam area, the eastern lowveld as well as the coastal region of

Natal (Plug, 1980).

It was during the movement of domesticated cattle from the north of the continent that the animals

were exposed to the harsh extreme climatic conditions and the tropical diseases of Africa.

Presumably, natural selection favoured animals genetically suited to the hostile environment at the

time. Different ecotypes developed as the result of adaptations to the extreme climate of the

continent.

Figure 1.2. Frequency of taurine and indicine Y chromosome allele in sub-Saharan Africa. For each

breed, the proportion of taurine (grey) or indicine allele (black) is indicated. Representation of

South African breeds concentrated in the north of the country (red oval) with numbers 1)

Drakensberger, 2) Afrikaner, 3) Nguni and 4) Pedi (Hanotte et al., 2000).


3

The Nguni ecotype developed during the time of dispersal (Figure 1.2). It can be separated on the

basis of genetic distancing from other ecotypes which had also developed during the migration

(Hanotte et al., 2000; Bester et al., 2003). Indicine is seen to be more dominant in the central to

Northern parts of the African continent than Taurine which is only dominant in the Southern Africa

amongst other ruminant livestock (Hanotte et al., 2000).

Ruminant livestock have the ability to convert crop residues and fibrous materials of no value to

other herbivores, into protein of high quality. They can also facilitate the use of marginal lands of

little or no value for crop agriculture (Rege & Gibson, 2003). This ability can in part be attributed

to their symbiotic relationship with ruminal microorganisms (Lobley, 1992).

The ability of ruminant livestock to utilise forage materials is not accomplished without cost,

however, as it includes the relatively inefficient use of both ingested energy and protein (Lapierre

et al., 1997). This has been singled out as a focal point for those arguing against the poor usage of

natural resources that occurs with current agricultural practices (Bester et. al., 2003). There is also a

general interest in decreasing nitrogen excretion by livestock to reduce pollution in the air as well

as in the soil and in water (Lapierre et al., 1997). It is thus necessary to improve the metabolic

efficiency of productive animals, with consequent advantages for agricultural economics and better

conservation of ecosystem (Lobley, 1992).

Studies need to increase the efficiency with which ruminants convert feed nitrogen into animal

protein. Part of this improvement must involve strategies to reduce “obligate” catabolism

associated with maintenance of nitrogen equilibrium. Further gains will accrue from a better

understanding of how the hormonal and substrate regulation of protein metabolism and retention

occurs, which will subsequently allow for novel nutritional, endocrinological and genetic

approaches to be adopted in future (Lobley, 1992).

A selective grazer and browser, the Nguni is able to obtain optimal nutritional value from the

available natural vegetation, thus enabling it to survive under conditions that bulk grazers, such as

the European cattle breeds, would find extremely testing (Bester et. al., 2003). Temperamentally,


4

the Nguni is very docile – another characteristic of an animal in harmony with its total environment

(Ramsay, 1985).

Most studies done to date, mainly under station or improved production conditions in tropical and

sub-tropical countries, have shown that indigenous breeds can be as productive, if not more

productive than European breeds, especially if account is taken of viability and maintenance

requirements, as measured in terms of live-weight (Mpofu, 2002a). In addition, evidence exists in

the literature which indicates that specialised imported breeds cannot compete under conditions in

which indigenous African livestock are predominantly kept; specialised imported breeds cannot

compete (Mpofu, 2002b). The profile of the Nguni, in particular, shows that this breed developed

under a process of natural selection in highly challenging environmental circumstances. Overall,

these animals have a genetic potential to perform better under optimal production environments,

being a medium frame animal with a high tick tolerance and disease resistance (Bester et. al., 2003;

Collins-Lusweti, 2000).

Almost 70% of the world‟s rural poor depend on livestock as an important component of their

livelihoods (Hoffmann, 2010). The African continent where many of the rural poor live, is home to

over 230 million cattle (FAO, 2008) being made up of 145 breeds, most of them indigenous to the

African continent (Rege and Bester, 1998). These breeds have unique genetic attributes, such as

adaptation and tolerance to drought, heat, diseases, as well as having the ability to utilize low-

quality indigenous forages (Lamwaka, 2006; Wurzinger et al., 2007). The Nguni breed falls into

the latter category, being typically kept under pastoralism and the traditional crop-livestock system.

After having entered the growing commercial sector early on the 20th

century, this breed is now

become an excellent source of genetic material well suited to the management style and needs of

the livestock industry. This is especially true for the emergent farmer who requires low

maintenance and relatively high output animals. In addition, the adapted genes of this breed are

used by commercial farmers as a source of “hardy” genes (Bester et. al., 2003).

Under adverse conditions, which are common during the dry season in most rural areas of Southern

Africa, Nguni meat quality is as good as that from other cattle breeds, such as Angus and

Bonsmara. Therefore, besides being a smaller and multipurpose breed, the Nguni can compete


5

favourably with established breeds in terms of productive performance and meat quality. There is a

need, however, to perform a sensory evaluation of the meat and assess other meat aspects, such as

fatty acid profiles, against Angus and Bonsmara cattle breeds (Muchenje et al., 2008).

Furthermore, the global drive for the development of biotechnology into patentable and marketable

products has sparked interest from international investigators into the genetics of the Nguni breed,

which, even under harsh and arid environmental constraints has high fecundity and also produces

high quality meat (Collins-Lusweti, 2000).

One of the phenomenon that enable the Nguni to survive under adverse conditions is nitrogen

recycling pathway, resulting in animals being substantially less dependent on dietary protein than

other breeds. This attribute is also of importance to the developed world, where nitrogen

contamination of the environment limits intensive animal production. Elevated blood urea levels in

these cattle reflect this phenomenon, particularly when the animals are maintained on poor quality,

low protein grazing food sources (Linington & Osler, 1992; Bester et. al., 2003).

Policies and development efforts to improve livestock production in the communal areas have been

based on the use of fast growing imported breeds (Collins-Lusweti, 2000; Muchenje et al., 2008).

These are perceived to be superior to native breeds, because of their large body size (Bester et al.,

2003). Contrary to this presumption, exotic breeds are failing to cope with the harsh environmental

and socio-economic conditions prevalent in the communal areas in Africa where, among other

constraints, disease is rampant, animal feed is scarce and livestock management is poor (Collins-

Lusweti, 2000; Scholtz, 1988; Schoeman, 1989). As such, farmers raising imported breeds under

these kinds of adverse conditions are more likely to incure high meat production costs.

When compared with other breeds, especially in relation to cow mass and reproductive

performance, the Nguni cattle have proved themselves to be the most fertile beef breed in South

Africa, being ideally suited as a dam line in terminal cross breeding. In addition, its traits of heat,

tick and disease tolerance make it ideal breed for large scale systems (Bester et. al., 2003). This

breed has also shown high reproductive performance and good walking and foraging ability

(Muchenje et al., 2008).


6

Consumers are increasingly becoming concerned about the quality of meat they eat. There is also

an increasing requirement for information on the origin of the meat and the type of production

system used (Revilla & Vivar-Quintana, 2006). As a way to encourage production systems which

are acceptable and which conform to consumer demands, the use of the adapted indigenous Nguni

cattle breed for beef production in rural South Africa is promoted. In general, there are limited and

inadequate forage management practices in rural areas in Africa, which result in overgrazing and

overstocking with the concomitant sudden loss of weight especially in the dry season, which may

also drastically affect meat quality (Bester et. al., 2003; Muchenje et. al., 2008).

The Nguni breed is known to maintain a high blood urea level when the nitrogen content of the

pasture drops (Chimonyo et al., 2002) and especially during adverse conditions (Linington &

Osler, 1992). It is postulated that this characteristic has been pivotal to the Nguni becoming the

“superior” breed on the African continent. Therefore an understanding of variations which can

occur in the urea cycle, especially focussing on the key enzymes, is crucial for gaining an adequate

understanding of the physiological responses related to Nguni adaptation to arid environments. In

addition, a better understanding of these mechanisms is also essential for the development of

improved strategies to better realise the potential of these breeds. As such, an overall understanding

as to how these enzymes are regulated is therefore crucial in coming to any conclusion as to what

separates the Nguni breed along the lines of the urea cycle, from less “hardy” breeds. It is

generally believed that elevated levels of urea in the Nguni blood sera, probably arises from

increased conversion of glutamine to glutamate in the liver by glutaminase. It is possible that the

Nguni are capable of creating glutamine stores in the muscle in the way sheep do (Marais & Van

der Walt, 1983).

1.1. Renewed Interest in the Nguni Breed

Most studies conducted within the Nguni have concentrated on a limited number of factors, such as

meat quality (Muchenje et al., 2008). Cattle provide diverse functions to farmers and the general

community in Africa and abroad, despite low productivity (Scoones & Graham, 1994). In rural

farming areas, most domesticated animals also provide draught animal power and also serve as


7

security and risk reduction in rural households, thereby enhancing the sustainability of small land

holder farming systems (Scholtz, 1988). Of the various factors which reduce cattle productivity, the

major ones are low quality of grazing fields during the dry seasons, poor health management and

under-nutrition (Bester et. al., 2003; Mapiye et al., 2007).

European breeds, including the Hereford, Aberdeen Angus as well as Sentimental breeds, have

been distributed throughout Southern Africa. Because of the importation of the less adapted

European breeds, together with their perceived higher income; indigenous breeds were looked upon

as inferior. The perception of indigenous breeds, such as the Nguni, was turned around by scientific

motivations, as well as the committee appointed in 1985 on the desirability of keeping a germ

plasm bank to preserve the hardiness (Hanotte et al., 2000). The Nguni‟s popularity under

commercial breeders has increased exponentially over the last decade. There are also projects in

South Africa which currently aim to increase the number of the indigenous Nguni cattle in the

communal areas. One such project in Eastern Cape aims to up-grade the existing cattle population

in the communal areas by eliminating all non-Nguni bulls within a given community. The Nguni

breed has thus been attracting increasing international interest, mainly due to their resistance to

ticks and tick-borne diseases, high reproductive performance and good walking and foraging ability

(Strydom et al., 2001; Muchenje et al., 2008).

1.2. Overview of the Nguni cattle project in South Africa

The Nguni Cattle Development Project is currently underway in four provinces in South Africa,

having been initiated by Agricultural Research Council (ARC) and the University of Fort Hare,

together with the collaboration of rural development agencies in the country. In a separate project,

farmers in selected communities were given two bulls and 10 in-calf heifers, to allow them to build

up a nucleus herd (Fuller, 2006). In effect, the existing bulls in each community were replaced by

registered Nguni bulls. In a programme consisting of five year cycles, the selected communities

give back to the project two bulls and 10 heifers, which are then passed on to another community

(Raats et al., 2004). One of the conditions of the project is that communities should have fenced

grazing areas, together with a Range Land Management Committee to supervise rotational resting

at specified stocking rates (Mapiye et al., 2007). The model adopted has a long-term goal which is


8

to develop a market for Nguni beef, Nguni skins and to position the communal farmers for the

global beef market through organic production and product processing (Raats et al., 2004). This

project discourages the use of unknown cows and enforces the removal of the existing exotic bulls

order to replace them with pure registered Nguni bulls. A project development committee made up

of interested stakeholders is in charge of the development of infrastructure, training of farmers and

the redistribution of animals. The implementation of the model in communal areas is conducted in

collaboration with the Department of Agriculture (Raats et al., 2004).

1.3. Mammalian nitrogen metabolism

The ability of mammals to transfer urea from the blood to the gastrointestinal (GI) tract is common

to most species. In ruminants, this phenomenon can also supplement the nitrogen supply to the

ruminal microorganisms, who in turn will also supply the host animal with essential amino acids.

This “protein regeneration cycle” (Houpt, 1959) is of great significance to the survival of ruminants

under unfavourable nutritional conditions. Animals consuming low-nitrogen diets by preventing the

excretion of urea have been reported to have physiological changes such as reduced plasma

filtration by the kidney and an increase in urea re-absorption, thus preventing urea excretion (Leng

et al., 1985; Cirio & Boivin, 1990). There is also an increase in the GI tract clearance rate of urea in

animals which are fed low-nitrogen diets (Marini & Amburgh, 2003), but the specific mechanism

of action in the clearance of urea from GI is yet to be described. Urea transporters in the GI tract of

ruminants might be the mechanism responsible for this increase in the transfer of urea into the GI

tract (Marini & Amburgh, 2003; Ritzhaupt et al., 1998).

The second requirement to avoid high output and loss of nitrogen is that the energy intake by the

animal should be great enough to protect the nitrogenous constituents of the tissues from

catabolism. Under these conditions, the nitrogen output in the urine is reduced to a minimum, and

may be regarded as a measure of the requirement of nitrogen, by the animal, for the maintenance of

life (Mitchell, 1926; McClellan & Hannon, 1932).

Combination of both Endogenous Urinary Nitrogen (EUN) as well as Metabolic Faecal Nitrogen

(MFN) helps to maintain the requirements of an animal for absorbed nitrogen. EUN, being the

nitrogen lost in the urine, resulting from normal turnover of body nitrogen is defined as the


9

minimum loss of nitrogen through the urine when consuming a nitrogen-free diet (Smuts 1935).

MFN, being the portion of the faeces that is composed of microbial cells, mucus, and other eroded

cells from the gastrointestinal tract, is defined as the nitrogen cost which can be attributed to the

digestive processes (Smuts 1935). Digestibility coefficients for plant nitrogen are high for

ruminants, although variability can occur if soluble digestion inhibitors, such as phenolics, are

present within the plant cells (McLeod, 1974).

In ruminant amino acid nutrition the utilization of protein is influenced by a number of factors.

These include the following, being (a) energy and amino acids, which are absorbed from different

sites in the alimentary tract, such as from the rumen and intestine respectively, (b) the rate of

digestion of specific amino acids from microbial protein (c) amino acid composition effect upon

absorption and (d) selective absorption or digestion of essential amino acids, as compared with

non-essential amino acids (Purser, 1970).

There have been concerns in the past about environmental pollution by nitrogen from domesticated

and commercialised animal enterprises; as such, there has been raised interest in the use of low-

protein diets for pigs, cattle, sheep and other animals (Kornegay & Verstegen, 2001). It has been

estimated that each one percentage unit reduction in dietary protein, accompanied by appropriate

amino acid supplementation, results in around 8% less nitrogen excreted in manure (Kerr & Easter,

1995). However this has not been found to be the case in some experimental settings, which show

no reduction in excreted nitrogen, although the dietary protein requirements were substantially

reduced (Cromwell, 1996).

1.4. Urea cycle

The ruminant stomach is divided into four compartments, namely, the reticulum, rumen, omasum

and abomasum. Feed is passed into the rumen, which may be regarded as a large fermentation

chamber providing a suitable environment for the continuous culture of the microbial population.

The microbial population acts symbiotically by producing the enzymes required for the degradation

of the cellulose material that is ingested by the animal. The ruminant species are therefore of great

value to man because they provide a means of capturing the solar energy stored in the cellulosic

bonds of plants. The function of the omasum is poorly understood. The function of the abomasum


10

is similar to the stomach of monogastrics, in that it is a glandular compartment where hydrochloric

acid and enzymes partially hydrolyse the protein arising from the feed, as well as the protein arising

from the microbial activity in the rumen. The abomasums secretes lysozyme, which is an enzyme

that efficiently breaks down bacterial cell walls, thereby making the bacterial protein produced in

the rumen available for subsequent proteolysis absorption by the animal.

The majority of soluble protein entering the rumen is degraded to ammonia. Less soluble proteins

which escape rumen proteolysis are passed into the small intestine where they undergo continued

proteolysis, thereby making them available for absorption. The major goal of the ruminant feeding

system, is to maximize the energy conversion and the utilization of the cellulosic biomass, so as to

maximize the conversion of ammonia to microbial biomass (microbial protein) as well as to

minimize the ammonia loss from the rumen by absorption. The ammonia produced and reabsorbed

is detoxified by the liver by synthesizing urea, thereby preventing ammonia toxicity. The rumen

ingesta of the ruminant comprise the feed as well as saliva, with the ruminant animal producing 100

to 150 litres of saliva per day. Depending on the production factors, between 19 and 96 % of the

endogenous urea produced by the liver is recycled to the gut, with 15 to 95% of the transfer

occurring via the saliva (Huntington et al., 2007). Some urea is also transferred across the gut wall.

Urea genesis by the liver is closely linked to the degradability of the dietary nitrogen (dietary N)

and subsequent absorption of ammonia. The ruminant may secret from 50 to 130g of urea per day

in the saliva. The urea, on entering the rumen, is then converted to CO2 and ammonia by the urease

enzymes produced by the rumen microorganisms.

The urea cycle plays an essential role in human and other ureotelic organisms for the efficient

removal of toxic ammonia from body tissues. As such, as already stated, an understanding of the

overall regulation of urea biosynthesis would be greatly improved by elucidation of the individual

regulatory components of the pathway.

The urea cycle (also known as the ornithine cycle) is a cycle of biochemical reactions occurring in

many animals that produces urea ((NH2)2CO) from ammonia (NH3). In mammals, the urea cycle

takes place primarily in the liver, and to a lesser extent in the kidney (Figure 1.3). The urea cycle

consists of five reactions: two mitochondrial and three cytosolic. During these reactions two amino


11

groups, one from NH4+ and one from aspartate, and a carbon atom from HCO3

- are converted to the

relatively nontoxic excretion product urea at the cost of four "high-energy" phosphate bonds (3

ATP hydrolyzed to 2 ADP and one AMP). Ornithine is the carrier of these carbon and nitrogen

atoms. The presence of arginine in animal protein was shown in 1895 by Hedin (Rogers & Visek,

1985). Arginine has been considered the only intermediate metabolite in the process of nitrogen

detoxification for the years, but other workers have suggested that the urea cycle has other

functions, such as the control of pH homeostasis (Haussinger et al., 1988) which is performed by

other bi-products in the cycle.

Figure 1.3. The primary enzymatic steps of the urea cycle are shown in capital letters, i.e. N-

Acetyl-Glutamate Synthase (NAGS), Carbamyl Phosphate Synthase (CPS-I), Ornithine

Transcarbamylase (OTC), Argininosuccinic acid Synthase (AS), Argininosuccinic acid Lyase

(AL), and Arginase (ARG). Ornithine (ORNT) and Citrulline (CITR) respectively (Tuchman and

Batshaw, 2002).


12

These bi-products are also involved in the nitric oxide synthase pathway (Moncada et al., 1989).

All this simply suggests that there are many other amino acids involved in ureagenesis and nitrogen

detoxification (Figure 1.3). Scientists continue to explore and evaluate the wide implications of

urea cycle.

Evidence of the modulation of ureagenesis, by arginine, is best understood when considered within

the context of all the urea cycle intermediates: arginine is mostly taken up by the liver, while

ornithine is taken up a lot less (De Bandt et al., 1995), and citrulline uptake is almost insignificant

(Drotman & Freedland, 1972). In the liver of ureotelic animals, arginine activates N-acetyl-

glutamate synthase, thereby catalysing the synthesis of N-acetyl-L-glutamate, which is an allosteric

obligatory activator of carbamyl phosphate synthetase. Another line of evidence suggests that the

modulating effect of arginine is dependent upon the nutritional status of the animal, rather than

depending on an increase in local arginine synthesis through arginno-succinate synthase activations

(Saheki et al., 1977). Arginine has no action on mitochondria from fasted rats, whereas it increases

6.5 to 7.5 fold in mitochondria from rats fed 0% to 60% casein. Feeding rats with a high protein

diet increases CPS and OCT (Wakabayashi et al., 1995), as well as the activity of N-acetyl-

glutamate synthase, together with the modulatory effect of arginine (Morimoto et al., 1990). It is

therefore likely that arginine acts as a key signal for the activation of ureagenesis during high-

protein feeding.

When considering the net balance of ureagenesis:

2NH4+ + 2HCO3

- Urea + CO2 + 3H2O, it is usual to focus on the fact that urea formation allows

for the removal of ammonia. Accordingly, it can be considered that hepatic ureagenesis plays a

considerable role in the maintenance of bicarbonate homeostasis, because bicarbonate is removed

in an equimolar fashion (Haussinger & Gerok, 1988). Under acidotic conditions, the decrease in

glutamine consumption leads to a decreased ureagenesis rate and therefore to a sparing of HCO3-.

Although the role of arginine in the process of adaptation to acidosis is not known, it is interesting

to note that administration of ornithine α-ketoglutarate counteracts acidosis in starved rats (Ziegler

et al., 1992).

Absorbed nitrogen in animals can be used directly for productive processes, or can be converted to

urea in the liver by urea cycle processes; therefore knowledge of urea kinetics is important in

understanding the total nitrogen economy of the animal. Urea recycling is usually considered to be


13

the process whereby urea in the blood escapes urinary excretion and re-enters the gastrointestinal

tract. Recycled urea in the body of the animal can serve both as a source of protein, as well as a

buffer to the rumen microbial environment against dietary nitrogen deficiency (Houpt 1959).

Once in the rumen, urea is readily hydrolysed by bacterial urease, to produce carbon dioxide and

ammonia. The ammonia may then be reabsorbed and reconverted to urea in the liver, or

synthesized into bacterial protein, or excreted in faeces (Mould & Robbins, 1981).

1.5. Problem statement

Protein in cattle diets is the most expensive major component of cattle feed and insufficient

quantity of protein is the major limiting factor in animal production. In developing world, protein

additives could be better utilized directly by humans, whilst in the developing world excreted

nitrogen leads to massive land pollution problems.

Nguni breed of cattle are less dependent on protein than other breeds in a challenging environment

and maintain elevated levels when the nitrogen content of pasture drops (Linington & Osler, 1992;

Chimonyo et al., 2002). Despite the high blood urea, which is normally considered detrimental to

fertility in cattle, the Nguni is the most fertile breed in South Africa (Scholtz & Theunissen, 2010).

The following question arises: “Is there a difference in the enzyme associated with nitrogen

recycling in the livers of the Nguni breed when compared with a European breed such as the

Hereford?”

1.6. Expansion of the Problem Statement

The rumen and the associated compartment, the reticulum, comprise an anaerobic fermentation of

1011

bacteria and 1010

protozoa. These microorganisms are continually passed into the abomasum

which is acidic, and where digestion of the bacteria is initiated. These microorganisms are therefore

the free food of the ruminant. The ruminant secretes copious quantities of saliva that conveys

sodium hydrogen carbonate (NaHCO3) and urea to the rumen. In this way, the pH of the bacterial

fermentation in the rumen is controlled by neutralising the excess fermentation acids. The ammonia

released by urease activity is either absorbed through the rumen wall, transported to the liver where

it is converted to urea, or the ammonia is utilised by the rumen bacteria to synthesise protein. The


14

urea may either diffuse back into the rumen via the blood, or is returned to the rumen via saliva.

Approximately 70% of the nitrogen in the saliva is urea. It is believed that elevated blood urea

levels in Nguni cattle play a significant role in allowing this breed to survive in adverse conditions.

Understanding the variation in the urea cycle in various breeds of cattle is therefore crucial to select

for probes that may be used in genetic selection of enzyme polymorphisms that confer resistance to

adverse conditions in the Nguni breed. The mechanism of ammonia detoxification forming urea in

mammals is outlined in Figures 1.4 - 1.5.

Figure 1.4. The mechanism of ammonia detoxification leading to formation of urea in animals

http://themedicalbiochemistrypage.org/nitrogen-metabolism.html#urea


15

The ammonia released by the reverse reaction of glutamate dehydrogenase is converted to

carbamyl phosphate synthetase I, in the mitochondria. Arginine is converted to urea by arginase,

regenerating ornithine and completing the urea cycle.

Glutamine synthetase in the liver also plays a role in ammonia detoxification. Glutamine is a major

blood amino acid. Glutamine synthesized may also be transported to the liver where the amide-N is

converted to urea. The amide-N of glutamine is released as intra-mitochondrial ammonia by the

enzyme glutaminase. The ammonia may also be converted to citrulline.

The overall regulation of these enzymes is therefore crucial in the understanding of what separates

the Nguni breed urea cycle from other less “hardy” breeds, such as Herefords. The elevated urea

level in the Nguni blood sera possibly arises from increased conversion of glutamine to glutamate

in the liver by glutaminase.

Figure 1.5. Transport of ammonia to the liver for urea synthesis, using either glutamine (most

tissues) or alanine (muscle) as the carrier.

(http://www.campbell.edu/faculty/nemecz/323_lect/Nitrogen_metabolism/images/ammonia-transport.jpg)

The genetic mechanism underlying this unique phenomenon in the Nguni has not been

characterized and can therefore not be fully exploited in the Nguni or other cattle breeds. In order

http://www.campbell.edu/faculty/nemecz/323_lect/Nitrogen_metabolism/images/ammonia-transport.jpg


16

to be able to include this trait in a selection programme, polymorphisms in the genes involved in

the efficiency of nitrogen metabolism of cattle must still be elucidated.

1.7. Justification of the Study

In livestock production, nutritional shortages relate directly to a decrease in food production

through increased susceptibility of production animals to disease, decreased fertility and milk

production, retarded growth and a subsequent decrease in meat quality.

One of the most important limiting factors in this cycle is the dependency of most production

animals on dietary protein. Ruminants are less dependent on dietary protein than monogastrics, as

bacteria in the rumen can utilize non-protein nitrogen (NPN) sources. The ruminant then in turn

utilizes proteins in the bacteria formed from NPN, as shown in Figure 1.6.

Figure 1.6. End products of the utilization of endogenous NH3 and amino acids in urea cycle are

ammonia and urea excreted in urine.

(http://ahdc.vet.cornell.edu/clinpath/modules/chem/images/urea.gif)

http://ahdc.vet.cornell.edu/clinpath/modules/chem/images/urea.gif


17

Proteins from fodder and proteins in gut bacteria are degraded to amino acids, which after

absorption from the gut, are metabolised by a variety of tissues. At this point, the fate of nitrogen is

in the hands of intermediary metabolism of particular animal, where subsequent metabolic

efficiency of the animal dictates whether it is wasted or utilized. The end-products of the utilization

of amino acids are ammonia and urea, which are excreted by the animal in the urine are depicted in

Figure 1.6, where in some animals ammonia and urea excreted in dung.

In the developed world, nitrogen in urine and dung (as ammonia) is a major cause for concern, as

this leads to pollution of the environment. In contrast, livestock production in the developing world

is severely limited by insufficient protein to sustain production year round.

As previously stated, the Nguni cattle breed developed as a result of a process of natural selection

in a highly challenging environment. As such, these animals have an excellent ability to maintain

their condition in winter as a result of a lower dependency on protein supplementation during

adverse conditions. The Nguni breed has also shown refusal of NPN licks when feeding on low

quality roughage, despite adverse environmental conditions (Le Roux et al., 1999). This may be

due in part to the maintenance of a high blood urea levels under similar conditions. Overall, Nguni

cattle appear to have an ability to recirculate nitrogen when other breeds merely lose nitrogen

through excretion in urine or dung.

Breeders world-wide are keen to improve the animals they keep. Competition amongst breeders

and breed societies is fierce and the introduction of a test to determine the efficiency of protein

utilization would be of major consequence within the livestock industry world-wide.

1.8. Aims and Objectives

1.8.1. Aims

The aim of this research was to characterize and compare the enzymes associated with nitrogen

recycling in the livers of the Nguni and Hereford breeds. Genetic polymorphisms in the key

enzymes were characterized, based on the variation in the functionality of these enzymes.


18

1.8.2. Objectives

a. To characterize key enzymes in nitrogen metabolism of Nguni and Hereford breed.

b. To investigate the biochemical and basis for nitrogen recirculation.

In order to do this the following enzymes were studied: arginase, carbamoyl phosphate

synthetase, glutamine synthetase, glutamate dehydrogenase and glutaminase.

Chapter 2: Urea Cycle Enzymes

19

CHAPTER 2: UREA CYCLE ENZYMES

Enzymes of urea cycle include amongst others arginase which is an enzyme of nitrogen metabolism

also called L-arginine amidino hydrolase (EC 3.5.3.1), a binuclear manganese metallo-enzyme that

catalyses the hydrolysis of L-arginine to form L-ornithine and urea (Figure 1.3, Page 11 and

Figure 2.1, Page 21). It is the terminal enzyme of the urea cycle among the six enzymes involved

in the urea cycle (Cederbaum et al., 2004).

Another enzyme of urea cycle is carbamoyl phosphate synthetase (CPS) which catalyses the

reaction of ammonia or glutamine with bicarbonate and two molecules of magnesium ATP to form

carbamoyl phosphate, which is indicated in the reaction 1 below:

2ATP + HCO3-

+ NH4+ 2ADP + H2NCOOPO3

2- + H3PO4………………………………...........

(Marshall et al., 1958; Pierard & Wiame, 1964).

Glutamine synthetase (L-glutamate ammonia ligase: EC 6.3.1.2., GS) forms part of urea cycle and

it catalyses the ATP-dependent incorporation of ammonia into glutamate to form glutamine, which

inturn is an important component of urea synthesis, as indicated in reaction 2 below:

L-Glutamate + NH3 + ATP L-Glutamine + ADP + Pi………………….

Hydroxylamine can replace ammonium in an ATP-dependent synthetase reaction, such as the one

catalysed by glutamine synthetase above (Maurizi et al., 1987; Ronzio et al., 1969).

Glutamate dehydrogenase as an enzyme of urea cycle, also known as ([L-glutamate-NAD (P) +

oxidoreductase (deaminating)], EC 1.4.1.3, GDH) is found in all organisms, where it catalyses the

reversible oxidative deamination of L-glutamate to 2-oxoglutarate and free ammonia (Fang et al.,

2002; Hudson & Daniel, 1993). In the oxidative deamination reaction, GDH feeds the tricarboxylic

acid (TCA) cycle by converting L-glutamate to 2-oxoglutarate, whereas the reductive amination

reaction supplies nitrogen for several biosynthetic pathways (Smith et al., 2001) in the mammalian

system, as indicated in reaction 3 below:

2-oxoglutarate + NH+

4 + NAD(P)H L-glutamate + H2O + NAD(P).................

2

1

3


20

The activity of GDH may result in oxidative deamination of glutamate, or in reductive amination of

2-oxoglutarate as the reaction is reversible (Garnier et al., 1997).

Glutaminase catalyzes the first reaction of the primary pathway for ammonia synthesis (Goldstein,

1967) forming glutamate and ammonia. This enzyme also forms part of urea cycle, where it

catalyses the hydrolytic cleavage of glutamine to form glutamate and ammonium ions in

mammalian systems (Watford et al., 2002), as well as bacteria, moulds and yeasts (Imada et al.,

1973). The role of the catalytic activities of glutaminase (Garnier et al., 1997) and GDH in the

synthesis of ammonia are shown in a coupled reaction 4 and 5 below.

L-glutamine + H2O L- glutamate + NH3....................

Glutamic acid + ß-NAD 2-ketoglutarate + ß-NADH + NH3……………….

The Urea cycle has six reactions following each other and works together to rid the body of waste

nitrogen (Figure 2.1). Arginase is the sixth and final enzyme of this cycle, being considered to be

the most recently evolved of all the enzymes in the cycle (Brusilow & Horwich, 2001). When

arginase catalyses the conversion of arginine to urea and ornithine, ornithine is recycled to continue

the cycle, while urea is excreted out of the cell as excess nitrogen (Brusilow & Horwich, 2001).

There have also been reports of urea re-absorption being a useful supply of nitrogen (Cirio &

Boivin, 1990). The first three enzymes in the cycle, namely, N-acetyl-glutamate synthase (NAGS),

carbamoyl phosphate synthase I (CPSI), and ornithine transcarbamylase (OTC), function inside the

mitochondria, whereas the other three, namely argininosuccinic acid synthase (AS),

argininosuccinic acid lyase (AL), and arginase (ARG), act in the cytosol of the cell (Brusilow &

Horwich, 2001). The best characterized arginase of animal origin is from rat and rabbit livers,

where highly purified forms of this enzyme with similar physicochemical properties were

suggested to have an oligomeric native structure (Reyero & Dorner, 1975).

In mammals, arginase has also been purified and characterized from different organs, with each

having different characteristic properties with regard to molecular weight, subunit structure and

their regulation in urea cycle (Carvajal, et al., 1982). In general, this enzyme has been found to

exist in two forms, having a broad tissue distribution, including liver and muscles. Although the

two forms of arginase are encoded by distinct genes with subcellular localization, they nevertheless

GDH

4

?

? 5


21

possess similar enzymatic properties. A cytosolic form of arginase (Arginase I) is highly expressed

in the liver or hepatic cells, playing an important role in ureagenesis. Extrahepatic arginase

(Arginase II) is thought to be involved in the biosynthesis of polyamines, ornithine, proline,

glutamate and also in the inflammatory process. Arginase II is a mitochondrial protein that is

mainly concentrated in the kidney (Cederbaum et al., 2004). Some studies have documented the

presence of arginase I and II in blood vessels from numerous animal species (Berkowitz et al.,

2003).

In addition, the type II isoenzymes have also been reported from rat kidneys (Kaysen & Strecker,

1973) and mammary glands (Jenkinson & Grigor, 1994).

Figure 2.1. A general overview of L-arginine-connected pathways. (1) Arginase, (2) ornithine

carbamoyl transferase, (3) argininosuccinate synthetase, (4) argininosuccinase (5) NO synthase, (6)

ornithine decarboxylase, (7) ornithine transaminase, (8) glutamine synthase and (9) glutaminase

(Cynober et al., 1995).

4

Arginosuccinate

3

Arginine

NO

5

Citrulline

2

Ornithine

1

6

α-amines

7

9

Glutamate

Glutamate semi-

aldehyde

8

Glutamine


22

Type II arginase isoenzyme has a wide tissue distribution and subsequently plays significant roles

in arginine metabolism, unlike the type I arginase isoenzyme, whose primary function is only the

net production of urea for excretion. The main function of Type II arginase isoenzyme is believed

to be net production of ornithine, a precursor for the biosynthesis of glutamate or polyamines (Li, et

al., 2001), as also found by Cederbaum in 2004.

Previous studies have shown that type I and type II arginases are located extrahepatically, with the

possibility that both enzymes might be involved in the regulation of nitric oxide production in

mammals. Elevated levels of type II arginase activity have been reported to be involved in the

production of ornithine and subsequently of proline and polyamines, all of which are necessary for

wound healing, while expression of the type I isozyme helps in the prevention of excessive

amounts of toxic nitric oxide (Salimuddin et al., 1999; Louis et al., 1998).

Arginine has been reported to be synthesized in only one way for mammalian cells; this mechanism

involves enzymes argininosuccinate synthase (EC 6.3.4.5) and argininosuccinate lyase (EC 4.3.2.1)

which transforms citrulline into arginine via arginosuccinate (Figure 2.1, Page 21). These enzymes

are found predominantly in liver tissues, occurring intracellularly across mitochondrial and

cytosolic barriers. The very high hepatic content of arginase which splits arginine into ornithine and

urea (Figure 1.3, Page 11) prevents the release of any arginine from the liver into the circulation.

The major source of endogenously synthesized arginine is derived from citrulline, which is taken

up by the kidney, with citrulline itself being mainly derived from glutamine and glutamate

metabolism (Tuchman & Batshaw, 2002; Saheki et al., 1977).

Three isoenzymes exist for carbamoyl phosphate synthetase, namely CPS1, CPS2 and CPS3

(Lawson et al., 1996). CPS1 utilizes ammonia rather than glutamine and is activated by

acetylglutamate (Marshall 1976; Anderson 1980). CPS2 is glutamine-dependant enzyme which

catalyzes the first committed step in pyrimidine biosynthesis, and subsequent arginine biosynthesis

in bacteria and fungi (Lawson et al., 1996). CPS3 has also been discovered in fish and in some

invertebrates. It is an acetyl-glutamate-activated enzyme which is related to CPS1 (Hong et al.

1994).


23

The interest of many researchers was drawn to a glutamine-dependent form of CPS, after it was

discovered in the mushroom Aguricus bisporus (Levernberg, 1962). Another glutamine dependent

form of CPS was also found in extracts of Escherichia coli (Pierard & Wiame, 1964), mammalian

liver mitochondria (Marshall et al., 1958) and frog liver mitochondria (Marshall et al., 1961).

Carbamoyl phosphate plays an integral part as intermediate in the biosynthesis of pyrimidine

nucleotides, including arginine from glutamine, which also involves bicarbonate, and two

molecules of Mg-ATP, as shown in Figure 2.2.

Figure 2.2. Summarised reaction mechanism in the biosynthesis of carbamoyl phosphate via three

unstable intermediates (Adopted from Lund et al., 2010).

The ability of CPS from E. coli to utilise either ammonia or glutamine, as the amino group donor,

was demonstrated by Kalman et al, (1965). Genetic evidence from the well studied Escherichia

coli has also indicated that the most important mechanism for the synthesis of carbamoyl phosphate

utilizes L-glutamine as a nitrogen donor instead of NH4+

(Pierard et. al., 1965).

In the reaction mechanism for CPS requiring glutamine for enzymatic activity, ATP phosphorylates

bicarbonate to form carboxy phosphate, while glutamine is hydrolysed to glutamate and ammonia.

The ammonia then reacts with the carboxy phosphate intermediate to form carbamate. In the final

HCO3-

Bi-carbonate

ATP

ADP

CO.O.PO32-

Carboxy phosphate

H2O

NH3

L-Glu

L-Glutamine

CO2-.NH2

Carbamate ADP

ATP NH2.CO.O.PO3

2-

Carbamoyl phosphate


24

step, a second molecule of ATP phosphorylates carbamate to produce carbamoyl phosphate. There

are three unstable intermediates formed in this process of four separate reactions (ammonia,

carboxy phosphate, and carbamate), before formation of carbamoyl phosphate, as indicated in

Figure 2.2 (Page 23).

Carbamoyl phosphate is of particular importance, because it is a precursor of arginine which, in

turn, is the last in the formation and excretion of urea (Tuchman & Batshaw, 2002). CPS has been

well studied from mammals, plants, yeast and bacteria. These studies revealed that in all cases at

least two distinct enzymes catalyse the synthesis of carbamoyl phosphate (Elliott & Tipton, 1974;

Kalman et al., 1965; Hiller, 1964; Levenberg, 1962).

CPS catalyses a reaction which results from the previous catalysis of three steps in sequence:

bicarbonate phosphorylation, carbamate formation from carboxy-phosphate and ammonia, with

subsequent carbamate phosphorylation (Lusty, 1983; Marshall et al., 1958). A partial bicarbonate-

dependent ATPase reaction reflects a step of bicarbonate phosphorylation, while a partial reaction

of ATP synthesis from ADP and carbamoyl phosphate, reflects the reversal of the step of

carbamate phosphorylation (Figure 2.2, Page 23).

Carbamoyl phosphate synthetase I (CPSI) deficiency, is an autosomal recessive inborn error of the

urea cycle that causes generally lethal hyperammonaemia, protein intolerance, as well as impaired

physical development (Summar et al., 1995; Brusilow & Horwick, 2001). Two clinical patterns of

CPS deficiency are described as neonatal form and delayed onset form (Gelehrter & Snodgrass,

1974; Arashima & Matsuda, 1972; Russell et al., 1962). Enzyme defects are more severe in the

neonatal form and a partial deficiency can occur in the late onset form (Hoshide et al., 1993).

A variety of diseases have been attributed to a deficiency in CPS, including ornithine

transcarbamoylase deficiency (Palmer et al., 1974), as well as congenital hyperammonemia

(Snodgrass & Delong, 1976).

The nature and the properties of the glutamine synthetase (GS) have been purified from different

sources, including bacteria (Adler et al., 1975), blue green alga (Orr et al., 1981), yeast (Mitchell &

Magasanik, 1983), sheep brain (Ronzio et al., 1969), pig brain (Stahl & Jaenicke, 1972), rat liver


25

(Deuel et al., 1978), and chick retina (Sarkar et al., 1972), amongst others. In bacterial GS, the

cellular activity of GS is finely regulated by repression/derepression mechanisms, mainly by

adenylylation and deadenylylation of a specific tyrosine residue (Stadtman et al., 1968), although

this process does not happen in mammals (Brown et al, 1971). GS plays a key role in nitrogen

assimilation, because it regulates the synthesis of several other enzymes involved in nitrogen

metabolism, including glutaminase, which requires glutamine synthesised by GS from glutamate

and ammonium (Magasanik et al., 1974; Tyler, 1978). The glutamate-to-glutamine conversion

(reaction 5) by glutamine synthetase (GS) provides the nitrogen donor for the first step in the

biosynthesis of many amino acids, purines, pyrimidines, and amino sugars (Robinson et al., 2001).

(http://themedicalbiochemistrypage.org/nitrogen-metabolism.php)

GS is found at high levels in mammalian brain and liver (Robinson et al., 2001). In mammalian

liver, GS is reported to be expressed exclusively in 1-3 cell layers surrounding the central vein of

the liver lobules (Gebhardt & Mecke, 1983).

Some of the earlier research on the activity of GS in fish focussed mainly on the control of

ammonia disposal and urea formation, such as the study by Anderson (1989), which demonstrated

that high GS activity could be found within the brain, presumably to detoxify ammonia which

might cross the blood-brain barrier (Wilson & Fowlkes 1976).

…………6

http://themedicalbiochemistrypage.org/nitrogen-metabolism.php


26

Bacterial, mammalian and chicken GS have remarkable similarities, but none of these are

physically or chemically identical. Moreover, there are many discrepancies concerning the

regulatory effect of different metal ions on GS activity (Ip et al., 1983; Pishak & Phillips, 1980).

The ability of different divalent cations to activate and regulate of GS has been extensively studied.

Among eight cations tested for ovine brain GS (Ca2+

, Co2+

, Fe2+

, Li2+

, Mg2+

, Mn2+

, Ni2+

, Zn2+

) only

Co2+

, Mg2+

and Mn2+

supported GS activity; the order of activity being Mg2+

> Co2+

> Mn2+

in

ovine brain. The maximum activating effect of Mn2+

occurs only within a very narrow range of

concentration, with an excess of cation causing strong inhibition of GS activity (Tholey et al.,

1987). Mn2+

may modulate the Mg2+

dependent GS activity as proposed in a regulatory system

under scrutiny, via the interactions of the two cations, which may be of significance in the

intracellular control of glutamine synthesis (Tholey et al., 1987).

High acidity in the liver diminishes the rate of glutamate and glutamine metabolism via the

phosphate dependent glutaminase (PDG) and GDH pathway, but stimulates glutamine synthesis via

GS by recycling glutamine. This process happens in humans (Nissim, 1999), but it is not known

what exactly occurs in bovine physiology with regard to acidosis in the liver, except that levels of

glutamine are lowered (Phromphetcharat et al., 1981). It has been demonstrated that where

activities of the hepatic urea cycle enzymes CPS, OTC, and AS are increased in developing

Holstein calves, they appeared to contribute to increased urea synthesis as well as recycling

thereof (Tagaki et al., 2008). Increased activities of urea cycle enzymes may also be secondarily

caused by the demand on urea synthesis after weaning, such as by an increased production of

ammonia by ruminal microbes, or gluconeogenesis from amino acids.

Irreversible inactivation of GS in vivo with L-Met-S-sulfoximine leads to convulsions and death of

the animal, thus indicating that the activity of the GS enzyme is essential. Two reasons for this, are

the removal of toxic NH4+ plus the modulation of levels of L-glutamate and γ-aminobutyric acid,

which are neurotransmitters (Norenberg, 1979).

The glutamine synthetase reaction is important in several respects. First it produces glutamine, one

of the 20 major amino acids. Second, in animals, glutamine is the major amino acid found in the

circulatory system (Maurizi et al., 1986). Its role is to carry ammonia to and from various tissues,


27

but principally from peripheral tissues to the kidney, where the amide nitrogen is hydrolysed by the

enzyme glutaminase; this process regenerates glutamate and free ammonium ion, which is excreted

in the urine (Juurlink, 1982). The position of GS in the complex matrix of nitrogen and carbon

metabolism is shown in Figure 2.3.

Figure 2.3. An indication of the position of GS in the complex matrix of nitrogen and carbon

metabolism. The role of GS (6) is encompassed in this figure amongst other enzymes such as

glutaminase (7) and glutamate dehydrogenase (5) (Robinson et al., 2001; Rennie, 2011).

GS also aids in the synthesis of glutamate the precursor of the glutamine pool, by combined action

with glutamate synthase (GOGAT; L-glutamine: 2-oxoglutarate aminotransferase, EC 2.6.1.53)

(Janssen et al., 1980). Within the urea cycle, the high affinity of GS for ammonia allows removal of

low concentrations of ammonia (Laemmli, 1970; Juurlink, 1982). Thus GS may presumably play a

modifying role in urea-cycle disorders that can result in hyperammonaemia. A second very

important site for ammonia detoxification by GS is in muscle tissue (Maurizi et al., 1986).


28

Glutamine is an important and most abundant amino acid in mammalian blood, making up as much

as 20% of the total amino acid content (Meister, 1984). It is also a significant fuel source utilized

by lymphocytes in rats (Ardawi & Newsholme, 1983) and an important precursor for nucleotide

synthesis (Krebs, 1980). In catabolically stressful disease states, where the immune system is also

placed under high stress, levels of glutamine in the body are lowered, particularly during starvation

scenarios (Cahill 1970), trauma (Souba et al., 1988), acidosis (Phromphetcharat et al., 1981) and

wound healing (Caldwell, 1989). Highly stressful states and suppression of the immune system

during these periods have been linked with lowered availability of glutamine, which has led to the

theory that during severe stress and coinciding with decreased glutamine levels in the circulation,

the immune system is compromised and the mammal becomes more susceptible to infection (Parry-

Billings et al., 1990; Keast & Morton, 1992). In the mammal, glutamine synthetase activity has

been found to be absent from cells and organs of the immune system (Ardawi & Newsholme, 1984)

or nearly so (Szondy & Newsholme 1990). This finding suggests that the immune system is

normally dependent on circulating glutamine. As glutamine can be synthesized de novo, it is

considered a nonessential amino acid that is believed to play a significant role in protein balance

(Lacey & Wilmore, 1990).

There are three types of glutamate dehydrogenase (GDH) that vary according to the coenzyme they

use: NAD(H)-specific, NADP(H)-specific, and GDH, with mixed specificity. NAD(H)-specific

GDH is believed to participate mainly in the catabolism of glutamate, whereas NADP(H)-specific

GDH has mainly an anabolic role (Smith et al., 2001).

GDH is present in the brain at about one-tenth of the activity of GDH in the liver and

immunological studies have already suggested that GDH from the liver as well as from the brain

may have similar properties (Talal & Tomkins, 1964; Smith et al., 2001). The situation concerning

the role of glutamate dehydrogenase, in the brain, is complicated because glutamate is believed to

act as an excitatory neurotransmitter, as well as playing a major role, as a precursor of the

neurotransmitter, γ-aminobutyrate, in the brain (McCarthy, 1980). The role of GDH in the liver is

not as complicated, since the involvement of GDH in serving to balance both nitrogen assimilation

with amino acid catabolism, has been clearly demonstrated (Hudson & Daniel, 1993). Mammals


29

have only one form of this inner mitochondrial enzyme that uses either NAD(H) or NADP(H) with

comparable efficacy (Peterson & Smith, 1999).

Mammals express three isoforms of glutaminase, which are encoded by two distinct but structurally

related genes (Labow et al., 2001). Phosphate-dependent glutaminase is mitochondrially situated

(Guha, 1961) and accounts for higher percentage of the glutamine-hydrolysing activity of the liver

than any other enzyme (Horowitz & Knox, 1968) in the mammalian system. This enzyme appears

to be associated with the inner membrane of the mammalian mitochondria according to McGivan et

al., 1980.

Glutaminase has been isolated from most organs of the mammalian body including the rat kidney

(Curthoys & Weiss, 1974), rat liver (Smith & Watford, 1988), brain mitochondria (Kvamme et al.,

2001), as well as hog small intestinal mucosa (Gelfand et al., 1968).

Glutaminase is the major enzyme of glutamine breakdown in most mammalian tissues including

brain and liver (Kvamme et al., 1988; Orloff & Burg, 1971). Glutamine is the most abundant free

α-amino acid found in the body (Medina, 2001). In essence, glutaminase plays an integral part in

the deamidation of glutamine, which has an important role from the point of view relating to meat

production, specifically in the Beef Industry, through hydrolysis of the glutamine amide group

(Dura et al., 2004). Glutamine deamidation produces ammonia and glutamate (Watford et al.,

2002), which respectively becomes an acidity neutraliser and flavour enhancer in meat production

(Dura et al., 2004). Glutamine is also the main respiratory fuel of the small intestine (Windmueller

& Spaeth, 1978). Carbon derived from glutamine is either channelled into the biosynthesis of other

amino acids, or degraded to yield chemical energy, while ammonia derived from glutamine is

either exported to the liver or used for the biosynthesis of pyrimidine nucleotides (McCauley et al.,

1999).

Glutamine deficiency in animal models, which were induced by glutaminase infusion, was found to

be associated with diarrhoea, dysentery and bowel necrosis, particularly in the studies conducted on

rabbits and rhesus monkeys (Hambleton et al., 1980) and also in the study of glutaminase toxicity

in rabbits (Baskerville et al., 1980)


30

Mammalian liver possesses a unique isozyme of phosphate-activated glutaminase, which plays an

important role in the balance between nitrogen assimilation and amino acid formation through the

regulation of glutamine catabolism (Smith & Watford, 1990). The existence of two isozymes of

glutaminase was first noted by Krebs in 1935. Through glutamine metabolism, hepatic glutaminase

may be responsible, in part, for the regulation of circulating pH, as Haussinger et al. (1988)

suggested.

Various mammals express two isoforms of glutaminase, that are derived from distinct but

structurally related genes, namely a `hepatic type' which is found only within the liver (Watford,

1993) and a `kidney type', which is widely distributed throughout extra-hepatic sites, but most

active within the kidney and villus enterocytes of small intestines (Sarantos et al., 1993). The two

isoforms of glutaminase function in different areas of mammalian system, at different levels of

activity, including that involved in hepatic ureagenesis and gluconeogenesis (Watford et al., 2002),

renal ammoniagenesis (Curthoys & Gstraunthaler, 2001), the synthesis of excitatory and inhibitory

neurotransmitters (Cooper, 2001), and in the utilization of glutamine as a metabolic fuel (Mithieux,

2001). These isoforms of glutaminase are abundant in rat kidney, brain, intestine, and hepatoma

tissues, but are absent from mammalian liver (Kovacevic & McGivan, 1983). The glutaminase

isoforms contained within intact brain and kidney mitochondria consists of 65- and 68-kDa

peptides, which have been shown to be structurally and immunologically related (Shapiro et al.,

1987; Haser et al., 1985).

Liver glutaminase activity increases in response to diabetes, starvation, and high protein feeding,

but is not affected by changes in acid-base status, as is the case with kidney glutaminase, which is

regulated by changes in acid-base status. The changes in glutaminase activity in liver, during

diabetes, are due to an increased amount of enzyme protein with no change in specific activity

(Smith & Watford, 1988).

Glutamate, being the by-product of the reaction catalysed by glutaminase, is at the centre of the

disposal of the daily protein load. The “glutamate family” of amino acids (glutamate, glutamine,

proline, histidine, arginine and ornithine) comprise ~25% of the dietary amino acid intake, which is

disposed of via conversion to glutamate. As such, the key role of glutaminase, together with the


31

glutamate-linked aminotransferases, is in effecting the removal of α-amino nitrogen from almost all

of the amino acids via transdeamination. Glutamate also plays a key role in N-acetyl-glutamate

synthesis, which ensures that the rate of urea synthesis is in accord with rates of amino acid

deamination (Brosnan, 2000).

2.1. Enzymology of urea cycle enzymes

Arginases, whether of eukaryotic or prokaryotic origin, have a common feature in having a

requirement of divalent cations for their activity (Ash, 2004). Mn2+

has since been reported as the

physiologic activator, although other arginases also use different divalent cations, like Co2+

and

Ni2+

(Mora et. al., 1965; Brown, 1966) and in some instances for Fe2+

and Cd2+

are also used

(Anderson, 1945; Edlbacher & Baur, 1958).

Arginases have an alkaline pH optimum with maximal velocities observed in the range of pH 9.0–

9.5 (Roholt & Greenberg, 1956). Rat liver Arginase in a plot of log Vmax versus pH has provided a

pK value of ~7.8–8.0 (Reczkowski, 1991) while pK values of 7.8 and 7.9 have also been reported

in other studies of rat liver (Kuhn et al., 1991) enzymes.

Substrate specificity of the arginases depends on the presence of an intact guanidinium group, the

proper length and hydrophobicity of the side chain, as well as the stereochemistry and nature of

substituents at the Cα position of the molecule (Reczkowski & Ash, 1994). This phenomenon is

demonstrated by D-arginine and guanidinobutyrate failing to work as substrates for the enzyme.

Such exquisite substrate specificity suggests that a well-defined constellation of hydrogen bond

donors must be present at the active site (Ash, 2004).

Arginase has a trimetric structure (i.e. having three identical subunits), with depending on the

organism each subunit having a molecular mass of 20 to 40kD that can bind two Mn2+

ions (Figure

2.4) (Kanyo et al., 1996). Ornithine, lysine, and branched-chain amino acids are all competitive

inhibitors of arginase. However, this enzyme has such a high maximum specific activity, that even

when inhibited, it will never limit flux through the ornithine cycle. Arginase is found primarily in

the liver and, to a lesser extent, in various other organs and tissues. The ornithine thus generated in

the system, is then metabolized to polyamines through ornithine decarboxylase (ODC), then to


32

glutamate (mediated by ornithine aminotransferase), and finally to citrulline (mediated by ornithine

carbamoyl transferase) (Cohen, 1962).

CPS is a heterodimeric enzyme composed of two subunits of unequal size (Abdelal & Ingraham,

1975) with the heavy and light subunits being ~40kDa and 118kDa, respectively (Thoden et al.,

1997; Mathews & Anderson, 1972). The heavy subunit alone, catalyses the synthesis of carbamoyl

phosphate from ammonia, ATP and bicarbonate, a reaction which is activated by the presence of

ornithine, while the light subunit confers the ability to utilize glutamine as the amide donor (Trotta

et al., 1974; Abdelal & Ingraham, 1975).

CPS has also been shown to be twice as active when Mg2+

was used as the co-factor, compared to

when Mn2+

and Fe2+

were used as co-factors, each on their own (O‟Neal & Naylor, 1969).


33

Figure 2.4. Crystal structure of Arginase complexed with 2Mn2+

ions and the arginine analogue

nor-N-omega-hydroxy-L-arginine (Cox et al., 2001).

Carbamoyl phosphate synthetase (Figures 2.5 - 2.6) as purified from the liver of several

mammalian species, has demonstrated the requirement for N-Acetyl-glutamate, Mg2+

and K+ for

enzymatic activity (Jones, 1965; Marshall et. al., 1961).


34

Figure 2.5. Structure of Carbamoyl phosphate synthetase. The small subunit that contains the

active site for the hydrolysis of glutamine, is shown in green whereas the large subunit, that

contains the active site for the synthesis of carboxy phosphate and carbamate, is shown in red. The

other terminal domain of the large subunit that contains the active site for the synthesis of

carbamoyl phosphate is shown in blue (Lund et al., 2010).


35

Figure 2.6. Stereo view of a ribbon representation of the large and small subunits of CPS. Shown

here are the locations of the glutamine binding site, the carboxyphosphate and carbamoyl

phosphate active sites together with the allosteric sites (Thoden et al., 1997).

Glutamine was found to be inactive as a substrate to rat CPS, while ammonium was found to be

optimal in the pH range 6.8 - 7.6. Mg2+

and Mn2+

were equally effective as cofactor metal ions in

the forward reaction of rat liver CPS (Lusty, 1978), but excess Mn2+

was found to be inhibitory to

rat liver CPS, as was the case of GS (Tholey et al., 1987).

Extensive biochemical and structural studies have resulted in a picture of the mechanism and

regulation of GS (Eisenberg et al., 2000). Structural analysis of GS has demonstrated that all GS

assemblies are composed of two closed ring structures with active sites formed between protomers,

which associate across an interface, being arranged with dihedral symmetry. GS-I enzymes have

always been dodecameric oligomers with 622 symmetry, whereas the assignment of a consensus


36

quaternary structure for the GS-II enzymes, has been controversial. Most recently, crystal structures

of GS from plants (Unno et al., 2006; Seabra et al., 2009), yeast (He et al., 2009), and mammals

(Krajewski et al., 2008) have overturned the octameric assignments from early electron microscopy

studies and revealed a decameric arrangement of subunits with 522 symmetry. To date, however,

only low-resolution structural data exists to describe GS-III enzymes, which are also double-ringed

dodecamers (van Rooyen et al., 2006).

In another study where ß-glutamate and α-glutamate were tested as substrates for GS, activity was

moderately high toward both ß-glutamate and α-glutamate. Specific activities of GS in the test with

ß-glutamate and α-glutamate could be increased when 500mM potassium acetate was added to the

purification buffer (Robinson et al., 2001). As confirmed by high performance liquid

chromatography (HPLC), the product of the reaction with ß-glutamate, ATP and crude enzyme was

ß-glutamine which therefore confirmed the substrate ß-glutamate effectiveness (Lai et al., 1991;

Robinson et al., 2001) in both reactions where either hydroxylamine or ammonium was used. There

is evidence that the activity of GS is dependent on glutamate concentration, this was demonstrated

by a hyperbolic trend followed by GS when glutamate concentrations were varied (Robinsn et al.,

2001).

Mammalian GS is a multimeric enzyme composed of 10 identical subunits (Figure 2.7) with each

subunit varying being between 41 and 58kDa molecular weight, depending on the organism. This

pattern is also apparent in most bacteria, archaea and mammals (Meister, 1980; Tholey et al., 1987;

Lai et al., 1991).


37

Figure 2.7. Crystal structures of mammalian glutamine synthetase with 10 identical sub units

showing the 522 symmetry. (Krajewski et al., 2008).


38

The necessary binding of metal ions affects the state of aggregation of the enzyme when tested in

vitro (Denman & Wedler, 1984). The many studies with GS have shown that there are two essential

metal ion sites per subunit (Figure 2.7, Page 37) that must be saturated for activation to take place

(Hunt et al., 1975) , particularly where metal ions and active-site ligands greatly stabilize inter-

subunit bonding domains (Maurizi & Ginsburg, 1982). Although GS of bacterial origin greatly

differs from the GS obtained from mammalian tissues, as it relates to oligomeric structure, but

comparisons of catalytic activities have nevertheless indicated marked similarities in active sites

(Gass & Meister, 1970).

The structures of bovine GDH have been studied and determined using X-ray crystallography

(Smith et al., 2001). The enzyme is a homo-hexamer with each subunit having a molecular mass of

~56 kDa. The core structure of the mammalian GDH is a stacked dimer of trimers which form the

N-terminal, “glutamate-binding domain” that is composed mostly of α-strands. On top of these

domains is the NAD binding domains that rotate down upon the substrate and coenzyme to initiate

catalysis. This rotation occurs about a helix at the back of the NAD binding domain called the

“pivot helix”. The determined structure of bovine GDH complexed with NADH, glutamate and

GTP are depicted in Figure 2.8 (Smith et al., 2001). NADH has been shown to bind to a second

site on each subunit, but the effect of coenzyme binding to this site is unclear. This second

coenzyme site binds NAD(H) 10-fold better than NADP(H) (Smith et al., 2001).

The structure of GDH (Figure 2.8) is essentially made up of two trimers of subunits stacked

directly on top of each other, with each subunit being composed of at least three domains (Li et al.,

2012; Smith et al., 2001). The bottom domain makes extensive contacts with a subunit from the

other trimer. Resting on top of the bottom domain is the „NAD binding domain‟ that has the

conserved nucleotide-binding motif. Mammalian GDH has a long protrusion or „antenna‟, rising

above the NAD binding domain that is not found in bacteria, plants, fungi, and the vast majority of

protists. The antenna from each subunit lies immediately behind the adjacent, counter-clockwise

neighbour, within the trimer (Li et al., 2012).


39

Figure 2.8. Structure of bovine GDH. On the left is the structure of bovine GDH with each subunit

represented by a ribbon diagram in different colours. On the right side are magnified views of the

regions that exhibit large conformational changes. The arrows highlight the key movement points

(Li et al., 2012).


40

Purified mammalian glutaminase isozymes are of different sizes; with kidney-type glutaminase

being composed of four subunits of 65 and 68 kDa in size, whereas the monomer size of the liver

glutaminase is 58 kDa (Watford, 1993). According to Haser et al, (1985), after comparing

glutaminase protein from rat kidney and brain, it was confirmed that the protein is a hetero-tetramer

composed of three 66-kDa and one 68-kDa peptides. Hepatic glutaminase activity is subject to

acute regulation by hormones, such as glucagon, catecholamines and effectors such as ammonia,

bicarbonate and pH (Szweda & Atkinson, 1989).

When studying the many effects pertaining to activity of these enzymes, such as product inhibition,

many factors have been proposed to influence the activity of mammalian glutaminase, including

ammonia (Haussinger & Sies, 1979), HCO-3; glucagon (Joseph & McGivan, 1978), cAMP and

branched-chain amino acids (Baverel & Lund, 1979).

Glutaminase activity is seen to be negligible in some studies of rat liver when ammonia is not

added as the activator (Haussinger & Sies, 1979). In contrast to its activation by ammonia, the

activity of glutaminase is competitively inhibited by glutamate. This effect can most likely be

attributed to glutamine and glutamate interacting with the same active site on glutaminase enzyme,

although specificity of the site is dependant on the concentration of phosphate in the reaction buffer

(Krebs, 1935; Goldstein, 1966; Shapiro et al., 1982).

The monomeric structure of kidney glutaminase is as shown in Figure 2.9 (Karlberg et al, 2012).

Included in the structure is the location of glutamate in the active site.


41

Figure 2.9. The crystal structure of kidney glutaminase showing glutamate in the active site

(Karlberg et al., 2012).

Chapter 3: Materials and Methods

42

CHAPTER 3: MATERIALS AND METHODS

To determine the extent by which the specific enzyme activities of the urea cycle enzymes of

Nguni and Hereford breeds differ, livers were obtained from both breeds of animals. The extraction

and purification protocols for arginase, GS, GDH, CPS and glutaminase are outlined below. Four

purifications were carried out for each enzyme arising from four different livers from each breed.

The specific activities were determined using steady-state enzyme assays as outlined below. All

enzymes were assessed for purity using SDS-PAGE. The liver samples from the Nguni breed

considered in this study were kindly provided by Hannes van der Merwe, from Devon farming and

Hans van Zyl from Agricultural Research Council Animal Improvement Institute (ARC-AII), Irene

South Africa. The other liver samples from the Hereford breed utilised in the study were kindly

supplied by the Hereford Breeders Association of South Africa with the assistance of Kobus

Grobler.

3.1. Purification of bovine liver arginase

3.1.1. Materials

All solutions were prepared with nano-pure deionised water. Analytical reagent grade chemicals

were used without further purification, being obtained from the following commercial sources:

Sucrose, potassium chloride, Sodium chloride, L-arginine-HCl, MnCl2.4H2O, β-mercaptoethanol,

Sodium hydrogen carbonate, Tri-chloro-acetic acid, urea and ammonium sulphate from Merck

chemicals (RSA); acrylamide/Bis-acrylamide from Sigma aldrich; Trizma (Sigma, Steinheim

Germany); SDS (Boehringer, Germany); Ninhydrin (USB cooperation, UK); Sephadex G-25,

DEAE Sephacel, CNBr-Sepharose 4B from Pharmacia Biotech (Uppsala, Sweden); Zhicheng

Shaking incubator (Shanghai, China); Molecular weight marker (Fermentas); Glacial acetic acid

and methanol (Saarchem, RSA); Perkin Elmer Cetus DNA thermal cycler heating block;

Coomassie brilliant blue (R-250).


43

3.1.2. L-Arginine-AH-Sepharose 4B affinity preparation

Arginase was purified using an L-arginine based affinity resin. The L-Arginine-AH-Sepharose 4B

affinity resin was prepared according to Patil et al., 1990. L-Arginine-HC1 (0.25g) was dissolved

in 20 ml coupling buffer made of 0.1 M NaHCO3, pH 8 containing 0.5M NaCl. Activated resin was

washed with cold 1mM HCl (1 µl/ 10 ml) for 30 minutes at 4°C (reactive groups hydrolyses at high

pH). Ligand solution was mixed with gel for 2 hours at room temperature, while uncoupled

arginine was removed by washing the gel with coupling buffer. Remaining active groups were

blocked by transferring the medium to 0.1M Tris-HCl buffer pH 8.0 and left to stand for 1 hour at

room temperature. Three cycles were used to thoroughly wash the product at alternating pH. Each

cycle consisted of a wash with 0.1M acetic acid/sodium acetate, pH 4.0 containing 0.5M NaCl,

followed by a wash with 0.1M Tris-HCl buffer pH 8 containing 0.5 M NaCl.

The column was equilibrated with Tris-HC1, 10mM, pH7.8, 1mM MnC12 and stored at 4 °C in a

20% ethanol. The medium was always prevented from freezing.

3.1.3. Purification of bovine liver arginase procedure

Purification of arginase was done according to Dabir et al., 2005 with some modifications by

incorporating an extraction method by Reyero & Dorner, 1975. All procedures were performed at

4oC unless otherwise mentioned. The liver samples, which were initially stored in a 3M Sucrose

solution at -70oC, were subsequently thawed at room temperature and lifted out of the sucrose

solution before being washed with purification buffer. Each 15 g mass of liver sample was weighed

and homogenized with 100 ml of chilled Tris-HCl buffer (100mM, pH 7.5) containing 5mM MnCl2

and 100mM KCl. The homogenate was centrifuged at 17,100 x g for 30 minutes and the

supernatant was collected, while the pellet was discarded.

This supernatant was then subject to heat treatment at 60oC for 30 minutes, with swirling in a

shaking incubator. The supernatant was then immediately cooled in an ice bath, with denatured

proteins being removed by centrifugation at 15,800 x g for 20 minutes.


44

The resulting supernatant was adjusted to 30% (197.5 g/L) saturation with solid (NH4)2SO4 and

stirred at 4 ºC for 30 minutes. The solution was then centrifuged and the pellet discarded. The

supernatant was then brought to 60% (385 g/ L) saturation with solid ammonium sulphate and

treated as before, except that the pellet was retained. The pellet was resuspended in 45 ml of 15mM

Tris-HCl buffer pH 8.0 containing 5mM MnCl2 and brought to solution by gently stirring. The

unresolved proteins were removed by centrifugation at 15,800 x g for 20 minutes and the

supernatant dialyzed overnight against 1L of the same Tris-HCl buffer used for homogenization.

Sephadex G-25 Chromatography

The dialyzed material was applied to a 2.5 x 10 cm Sephadex G-25 column, which was equilibrated

with a 5 bed volume of 15mM Tris-HCl, pH8.0. This material was then eluted with 12 ml of the

same buffer. All samples thus prepared were assayed; with fractions containing activity being

further purified using a DEAE Sephacel column.

DEAE-Sephacel chromatography

Combined samples from the Sephadex G-25 column were applied to a 2.5 x 10 cm column of

DEAE-Sephacel (Sigma) at 4oC equilibrated with the Tris-HCl buffer (15mM, pH 8.0) containing

2mM MnCl2. Elution was carried out with a linear gradient of NaCl from 0 to 0.5M in the same

buffer containing 2mM MnCl2 at a flow rate of 2 ml / min. The active fractions containing arginase

activity were pooled together and stored.

L-Arginine-AH-Sepharose 4B affinity column chromatography

The pooled fractions from above step were dialyzed at 4oC in 1 X 2 L of Tris-HCl buffer (15mM,

pH 8.0) containing 2mM MnCl2 and passed through a cyanogen bromide activated Sepharose 4B

column equilibrated with a 5 bed volume of 100mM Tris-HCl, pH 8. Each protein sample was

loaded onto the column, which was then incubated in ice for 30 minutes. The column was then

washed with a single bed volume of equilibration 100mM Tris-HCl, pH 8 and eluted with fresh

elution buffer containing 3.75 ml buffer, 0.4g KCl, and 0.26g L-Arginine-HCl and 6.25 ml H2O in

2 ml fractions.


45

Thereafter, 2 ml column fractions were collected at a flow rate of 2 ml/ min, before assaying for

arginase activity and protein content. Active fractions were pooled and buffer exchange was

undertaken using a Sephadex G-25 column. A 5 ml enzyme solution was further desalted, to

remove the arginine by dialysis in homogenisation buffer, which was done overnight and stored in

4oC.

3.1.4. Effect of divalent cations

To determine the effect of divalent cations on the activity of the Nguni and Hereford liver arginase,

the enzyme was diluted (1:2) with deionised water and 10 ml was extensively dialyzed with

deionised water (2 x 2 L) over a period of eight hours. The enzyme was further dialyzed with a 1L

of 10mM Tris-HCl buffer (pH 7.5), in the absence of manganese, before being assayed for the

effect of various divalent cation metals listed Mn2+

, Mg2+

, Ca2+

, Co2+

, Ni2+

and Cd2+

.Arginase assay

(Section 3.1.6) was followed with the only change being 2.5mM of cation listed above.

3.1.5. Effect of incubation time

To determine the effect of incubation time on the activity of the Nguni and Hereford liver arginase,

the enzyme was assayed for a maximum of 35 minutes at 5 minute intervals. Samples were assayed

as soon as each incubation time ended. Each assay was conducted as in section 3.1.6 below.

3.1.6. Arginase Assay: Estimation of enzyme activity

Arginase activity was measured according to Roman & Ruy‟s (1969) method as based on

estimation of L-Ornithine, with minor modifications. Briefly, the reaction mixture, which consisted

of 10mM sodium bicarbonate buffer (pH 9.6), 2.5mM MnCl2, 25mM L-arginine and enzyme

solution, in a total volume of 1 ml, was incubated for 30 minutes at 37oC. The reaction was

terminated by adding 10% TCA. Protein was removed from this solution by centrifugation; 1 ml of

the supernatant was mixed with 1 ml of anhydrous glacial acetic acid and ninhydrin reagent. The

mixture was kept in 90oC heating block for 30 minutes. The intense red colour developed was

diluted with 1:1 with glacial acetic acid. The reagent blank was run in a parallel manner and the


46

results were compared with control assay. The optical density was read at 492 nm on

spectrophotometer (Labsystems Multiskan MS, Microtitre plate reader, Japan) against reagent

blank. The activity of arginase was expressed as follows: one unit of an enzyme activity was

defined as that amount of an enzyme which produces one μmole of L-ornithine, per milligram of

protein, per minute at 37oC.

3.1.7. Data analysis

Data (absorbance values) obtained from Labsystems Multiskan MS, Microtitre plate reader was

transposed into an MS-Excel workbook for the calculation of specific activity with a formula

adopted from (Davis & Mora, 1968 and Ndolo et al., 2010), as given below:

Where U/mg Enzyme is µmoles per milligrams of enzyme, ODsample is optical density of sample

at 340nm, ODblank is the optical density of blank, ODstd is the optical density of the urea

standard, RV is the reaction volume, SV is the sample volume, t is time of assay and mg is the

sample protein concentration.

3.2. Purification of carbamoyl phosphate synthetase

3.2.1. Materials


were used without further purification. Reagents were obtained from the following commercial

sources: potassium phosphate, ammonium chloride, Sodium EDTA, MgCl2.6H2O, dithioerythritol

(DTT), dimethyl sulfoxide (DMSO), Sodium hydrogen carbonate, Tri-chloro-acetic acid and

ammonium sulphate from Merck chemicals (RSA); acrylamide/Bis-acrylamide from Sigma aldrich;

AMP, ADP, ATP, KCl, glycerol and HEPES (Sigma, Steinheim Germany); SDS (Boehringer,

Germany); Sephadex G-25, DEAE Sephacel, AMP agarose from Pharmacia Biotech (Uppsala,

(ODstd - ODwater)

(ODsample - Odblank) U/mg Enzyme =

UreaSTD (RV x 1000) x

(SV)(t)(mg)


47

Sweden); Zhicheng Shaking incubator (Shanghai, China); Molecular weight marker (Fermentas);

Glacial acetic acid and methanol (Saarchem, RSA).

3.2.2. Purification of bovine CPS procedure

Purification was done according to Price et al., (1978) with a few modifications. All procedures

were done at 4oC unless otherwise mentioned. Approximately 15 g of defrosted bovine-liver was

suspended in 120ml of 50mM KH2PO4 at pH 7, which contained 1mM sodium EDTA, 20%

glycerol and 10mM MgCl2, together with 1mM dithioerythritol (DTT) and homogenised for 2

minutes at full speed using a commercial blender. Homogenates were centrifuged at 14,500g for 10

minutes with a Sorvall RC-50 centrifuge, after which the supernatant was retained.

The supernatant was first treated with solid (NH4)2SO4 to 80% (525g/L) saturation, then stirred for

20 minutes at 4oC , followed by centrifugation for 20 minutes at 34800g. The resulting pellet was

resuspended in 22.5ml of 2mM KH2PO4, pH 7, containing 0.5mM Sodium EDTA, 4% Glycerol

and 15% DMSO together with 1mM DTT. The solution was sonically disrupted at 50% duty cycle

for one minute and then centrifuged at 39100g for 60mins, after which and the supernatant was

carefully removed.

Sephadex G-25 Desalting

A Sephadex G-25 desalting column (2.5 x 10cm) was equilibrated with 5x column bed volumes of

buffer comprising 2mM KH2PO4, 0.5mM Sodium EDTA, 4% glycerol and 15% DMSO together

with 1mM DTT at pH 7. An amount of supernatant equal to 1 column bed volume was applied to

the column and eluted with 12ml same buffer before being collected into 2ml fractions.

DEAE Sephacel (Anion by Gravity), PD-10

The sample from Sephadex G-25 column were combined and subsequently applied to a 2.5 x 10

cm DEAE-Sephacel (Sigma) column at 4oC, which was equilibrated with 2mM KH2PO4 at pH 7

containing 0.5 mM sodium EDTA, together with 1mM dithiothreitol, 15% dimethyl-sulfoxide and

4% glycerol. This column was left stand for 30 minutes at 4oC, before being washed with 5 bed

volumes of the same buffer and eluted with a gradient from 0 M – 0.5 M KH2PO4, pH 7 in 2 mM


48

KH2PO4, pH 7 containing 0.5 mM sodium EDTA, plus 1 mM dithiothreitol, 15% dimethyl-

sulfoxide and 4% glycerol buffer. A series of 2 ml fractions were subsequently collected. Fractions

containing activity were then pooled and combined for use in the next column. A separate aliquot

was also stored for further analysis.

Adenosine Monophosphate Agarose (AMP) chromatography

The combined sample from Sephadex G-25 chromatography was applied to the AMP agarose

column (2.5 x 5 cm column). The AMP agarose column was equilibrated with 2mM KH2PO4 pH 7,

containing 4% Glycerol, 15% DMSO and 1mM DTT. The column was then washed with five

column volumes of the equilibration buffer. Thereafter, 12ml of 2 mM KH2PO4, pH 7, containing

10 mM MgCl2 and 2.5 mM adenosine diphosphate (ADP) was eluted through the column into 2 ml

fractions. Fractions were then assayed for CPS activity by HPLC; those with the highest CPS

activity were pooled, desalted with Sephadex G-25 and subsequently stored at 4oC until required.

3.2.3. Specific activity of Carbamoyl phosphate synthetase: Analysis by High

Performance Liquid chromatography (HPLC).

Enzyme activity assay was adapted from the method of Price et al., 1978. The reaction mixture

contained in a final volume of 0.6 ml was made up of 20 mM of NaHCO3, 2 mM of ATP, 20 mM

of magnesium chloride, 0.3 M of NH4Cl, 100mM of KCl and 50mM of HEPES at pH 7.6. The

reaction was allowed to proceed for 10 min at 37°C, before being terminated with 3.5 μl of 1 M

trichloroacetic acid. Concentration of ADP was measured using HPLC analysis. As such, all assay

solutions were centrifuged prior to analysis for ADP. The assays for adenosine, AMP, ADP ATP

were carried out using Phenomenex 5μ LUNA C18 column with the mobile phase containing PIC-

A

(Waters Coorporation), 250 ml acetonitrile, 7g KH2PO4 per L of water. The flow rate of the

mobile phase was 1 ml/minute with UV detection.


49


Data (absorbance values) obtained from Labsystems Multiskan MS; Microtitre plate reader was

transposed into an MS-Excel workbook and calculated according to the formula 1 below according

to the assay method by Price et al., 1978.

Where U/mg Enzyme is µmoles per milligrams of enzyme, ADP mg/ml is part of the product of the

reaction, t is the time of incubation, and assay mg/ml is:

Where enzyme mg/ml is the sample protein concentration, enzyme volume is the sample volume

added to the reaction, assay volume is the total volume of the reaction, and enzyme dilution is the

dilution factor used to dilute the sample.

3.3. Purification of glutamine synthetase

3.3.1. Materials


were used without further purification. Reagents were obtained from the following commercial

sources: potassium phosphate, ammonium chloride, Sodium EDTA, MgCl2.6H2O, DTT, DMSO,

Sodium hydrogen carbonate, Tri-chloro-acetic acid and ammonium sulphate from Merck chemicals

(RSA); acrylamide/Bis-acrylamide from Sigma aldrich; AMP, ADP, ATP, KCl, glycerol and

HEPES (Sigma, Steinheim Germany); SDS (Boehringer, Germany); Sephadex G-25, DEAE

Sephacel, AMP agarose from Pharmacia Biotech (Uppsala, Sweden); Zhicheng Shaking incubator

(Shanghai, China); Molecular weight marker (Fermentas); Glacial acetic acid and methanol

(Saarchem, RSA).

U/mg Enzyme = (ADP mg/ml)

(t)/(Assay mg/ml)

Assay mg/ml = (Enzyme mg/ml) (Enzyme volume)

(Assay volume/ Enzyme dilution)


50

3.3.2. Purification of bovine GS procedure

Purification of GS was done according to Tholey et al., (1987), with some modifications which

incorporated an extraction method by Smith & Campbell, (1983). All procedures were performed at

4oC, unless otherwise mentioned. Approximately 30 g (wet weight) of fresh bovine liver was

homogenized in 120 ml of 10mM Hepes (pH 6.9) containing 900 mM NaCl, 2 mM MnCl2 plus 5

mM ß-Mercapto-ethanol together with 0.1 mM phenylmethylsulfonyl-fluoride (PMSF) and placed

in a blender processor for 3 minutes at maximum speed. The crude homogenate was then

centrifuged at 15,800 x g for 30 minutes in a Sorvall RC-50C refrigerated super-speed centrifuge

(Du-Point Instruments, USA), after which the debris was discarded, while supernatant was

retained. The resulting supernatant was then sonicated for 5 minutes with a 50% on/off duty cycle

for cooling. The lysate was first centrifuged at 19,800 x g for 20 minutes, after which the pellet was

discarded.

Supernatant was treated with (NH4)2SO4 to 40% saturation. The mixture was stirred for 30 minutes

and then centrifuged at 17300g for 20 minutes. The pellet was discarded and the supernatant was

dialyzed overnight against a 1L of homogenisation buffer containing 10 mM Hepes (pH6.9) plus

900 mM NaCl, 2 mM MnCl2 and 5 mM ß-Mercapto-ethanol together with 0.1 mM PMSF. The

dialysed sample was centrifuged at a high speed of 27000g for 20 minutes after which the

supernatant was decanted and kept for further experiments.

Sephadex G-25 chromatography

The dialysate was loaded on a column (2.5 x 10 cm column) of Sephadex G-25 (Sigma)

equilibrated with the homogenisation buffer. The proteins were eluted with 12 ml of

homogenisation buffer at a constant rate of 0.5ml /min. Thereafter, 2 ml fractions with high enzyme

activity were combined and continued to the next column chromatography and further experiments.

Adenosine Monophosphate Agarose (AMP) chromatography

The combined eluent from Sephadex G-25 column was applied to the AMP Agarose column (2.5 x

5 cm column) equilibrated with 10mM imidazole buffer (pH 7.15) containing 10 mM MnCl2 and


51

150 mM NaCl. The sample was allowed to stand for half an hour at 4oC. The column was then

washed with five column volumes of the equilibration buffer, before elution with 12ml of 10 mM

imidazole (pH 7.15) containing 10 mM MnCl2 450 mM NaCl and 2.5mM adenosine diphosphate

(ADP). Thereafter, 2 ml fractions were collected and subsequently assayed for GS activity using a

γ-glutamyl transferase assay (GT Assay). Fractions with highest GS activity were pooled and

dialysed in homogenisation buffer overnight at 4oC.

3.3.3. Glutamyl Transferase Assay: Estimation of enzyme activity of GS

GS activity was measured according to Tholey et al., (1987) with some adoptions according to the

method of Pamiljans et al., (1962). The reaction was carried out in 50 mM imidazole buffer (pH

7.15) containing 15 mM glutamine, 0.4 mM di-sodium ADP, 30 mM arsenate, 60 mM

hydroxylamine, 0.3 mM MnCl2.4H2O, 60 mM MgCl2.6H2O. This enzyme solution was made up a

total volume of 0.6 ml for a 96 well Microtitre plate. The assay was incubated for 30 minutes at

37oC. The reaction was terminated by addition of 0.09 ml of a solution of 3.32% FeC13.6H20 and

2% trichloroacetic acid. The optical density was read at 540 nm on spectrophotometer (Labsystems

Multiskan MS, Microtitre plate reader, Japan.) against reagent blank. The enzyme activities were

expressed as µmoles of substrate transformed per minute with one unit of enzyme activity defined

as 1.0 µmol of gamma-glutamyl hydroxamate formed (absorbance at 540nm) per min at 37oC.

3.3.4. Specific activity of GS: Analysis by High Performance Liquid

chromatography (HPLC)

The forward reaction of GS was assayed at 37oC by following the quantity of ATP converted into

ADP. The reaction mixture for the HPLC assay was essentially the same as the colorimetric assay

with a few modifications (Wellner & Meister, 1966). The assay mixture contained the following

reagents, in a total volume of 0.6 ml, being 4mM sodium glutamate, 4mM NH4Cl, 1mM MgCl2,

1mM ATP, 50mM Hepes buffer, pH 7.15 and 30µl of 2 - 3 mg/ml enzyme. The assay was

incubated for 30 minutes and terminated with 0.05% 1M TCA. Analysis was performed in

triplicate. One unit of enzyme activity is defined as the amount of enzyme catalysing the hydrolysis

of 1µmol of ADP/min under these conditions. The assays for adenosine, AMP, ADP ATP were


52

carried out using Phenomenex 5μ LUNA C18 column, with the mobile phase containing PIC A

(Waters Coorporation), 250 ml acetonitrile, 7 g KH2PO4 per L water. The flow rate of the mobile

phase was 1 ml/minute with UV detection. Enzyme activities were expressed as µmoles of ADP

formed per minute per mg protein at 37oC.



transposed into an MS-Excel workbook and calculated according to the formulas below according

to the assay method by Tholey et al., 1987.

Where U/mg Enzyme is µmoles per milligrams of enzyme, ADP mg/ml is part of the product of the

reaction, t is the time of incubation, and assay mg/ml is:

Where enzyme mg/ml is the sample protein concentration, enzyme volume is the sample volume

added to the reaction, assay volume is the total volume of the reaction, and enzyme dilution is the

dilution factor used to dilute the sample.

3.4. Purification of glutamate dehydrogenase

3.4.1. Materials

All solutions were prepared with deionised water. Analytical reagent grade chemicals were used

without further purification and obtained from the following commercial sources: potassium

phosphate (KH2PO4), potassium chloride (KCl), potassium cyanide (KCN),

ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), sodium bicarbonate (NaHCO3),

U/mg Enzyme = (ADP mg/ml)

(t)/(Assay mg/ml)

Assay mg/ml = (Enzyme mg/ml) (Enzyme volume)

(Assay volume/ Enzyme dilution)


53

methanol (MeOH), ammonium bicarbonate (NH4HCO3), iodoacetamide and ammonium sulphate

((NH4)2SO4) from Merck chemicals; Trizma and AESBF [4-(2-aminoethyl) benzenesulfonyl

fluoride hydrochloride] (Sigma, Steinheim Germany); sodium dodecylsulphate (SDS) (Boehringer,

Germany); Sephadex G-25, DEAE Sephacel, GTP sepharose and hydrazide gel from Pharmacia

Biotech (Uppsala, Sweden); Zhicheng Shaking incubator (Shanghai, China); Molecular weight

marker and dithiothreitol (DTT) (Fermentas); ammonium bicarbonate (NH4HCO3), ketoglutaric

acid (Calbiochem, USA); glutamate dehydrogenase (GDH), β-nicotinamide adenine dinucleotide

phosphate reduced tetrasodium salt, formaldehyde, acrylamide/Bis-acrylamide, formaldehyde and

trypsin (β-NADPH) (Sigma-Aldrich, USA); acetonitrile (ACN) (Romil pure chemistry); Perkin

Elmer Cetus DNA thermal cycler heating block; Coomassie brilliant blue (R-250).

3.4.2. Purification of bovine GDH procedure

Purification was done according to McCarthy et al., (1980) with some modifications by

incorporating a variation of the method by Tipton, (2002). All procedures were done at 4oC unless

otherwise mentioned. Approximately 15 g (wet weight) of fresh bovine liver was rinsed in 4 mM

KH2PO4, pH 7.4, homogenization buffer containing 0.1 mM AESBF protease inhibitor cocktail and

0.5 mM EDTA. This mixture was then homogenized in 100 ml of homogenization buffer in a

blender for 2 minutes at full speed. Solid (NH4)2SO4 was slowly added to the homogenate in the

blender, to yield a final concentration of 30% saturated solution (113 g/L). Homogenization was

continued for 30 seconds at full speed, after the addition of solid (NH4)2SO4. The crude

homogenate was then centrifuged at 6780g for 30 minutes with a Sorvall RC-50C refrigerated

super-speed centrifuge (Du-Point Instruments, USA), after which the debris were discarded and

supernatant was carefully decanted.

Supernatant was treated with 50% (188g/L) solid (NH4)2SO4 and subjected to mechanical stirring

for 30 minutes at 4oC. After centrifugation at 6780g for 20 minutes, the resultant pellet was re-

suspended in 40 ml of 2 0mM KH2PO4, pH7.4, buffer. The resulting solution was then carefully

stirred for 10 minutes at 4oC and dialyzed in 1L of 20 mM KH2PO4 (pH 7.4) buffer overnight at

4oC. To remove unresolved proteins, the solution was centrifuged at 12100g for 30minutes.


54

Sephadex G-25 column chromatography

The 8 ml dialysate was loaded on a column (2.5 x 10 cm column) of Sephadex G-25 (Sigma)

equilibrated with 20 mM KH2PO4 (pH 7.4) buffer. The proteins were eluted with 15 ml of

equilibrating buffer at a constant rate of 0.5 ml/min. Thereafter, 2 ml fractions with high GDH

enzyme activity were combined and continued to the next column chromatography.

DEAE Sephacel, PD-10 column chromatography

Combined samples making up 5 ml of eluted material from Sephadex G-25 column, was applied to

a 2.5 x 10 cm column of DEAE-Sephacel (Sigma) at 4oC, which was equilibrated with 20 mM

KH2PO4 (pH 7.4) buffer. The column was then washed with equilibrating buffer and eluted with a

linear gradient of KH2PO4 (pH 7.4) from 0.02 to 0.15 M at a flow rate of 2 ml/min. The active

fractions containing GDH specific enzyme activity were pooled and dialysed in a litre of 20 mM

KH2PO4 (pH7.4) overnight at 4oC.

3.4.3. GTP Sepharose Column chromatography: Preparation of GTP Sepharose

GTP sepharose was prepared essentially according to Godinot et al., (1974). A 5g amount of

CNBr-activated sepharose was suspended in 100 ml of cold 1mM HCl for 10 minutes, before being

filtered with a glass filter and washed with 900 ml 1mM HCl. The gel solution was then washed

with 0.75 L of deionised water and equilibrated with 0.5 L of 0.1M NaHCO3 buffer (pH 9.0).

Thereafter, 5 ml of gel was suspended together with 1.5g of L-glutamate γ-methylester, in 20ml of

0.1M NaHCO3 buffer. The mixture was then shaken and allowed to stand at room temperature

(25oC), for 2 hours. The gel was then washed with 0.75 L of deionised water. L-glutamate γ-

methylester sepharose was added, drop wise, to hydrazin hydrate and the mixture then incubated at

70oC for about 15 minutes. The resulting gel was then cooled to room temperature and washed with

0.75 L of deionised water. Thereafter, 0.6 ml of formaldehyde was added to the gel, which was

placed on a shaker for an additional 1 hour. The gel was then washed with 0.5 L of cold 0.1M

phosphate buffer containing 5mM EDTA at pH 6.8, followed by 0.5 L of cold 2M KCl and thirdly

washed with 0.75 L of cold water, which was buffered with 0.75 L of cold 0.05M Tris-HCl

containing 1mM EDTA (pH 7.5).


55

Separation of GDH with GTP-sepharose column

Dialysed sample (8ml) was applied to a GTP-sepharose column (2.5 x 10 cm) that was equilibrated

with 100mM Tris-Acetate pH 7.15 buffers containing 1mM KH2PO4 and 0.1mM EDTA. After the

column was washed with a bed volume of equilibration buffer, proteins were eluted with a KCl

gradient from 0mM – 400mM in equilibration buffer. Active fractions were pooled, combined and

dialysed in 20mM KH2PO4 (pH 7.4) overnight and then stored at 4oC for further use.

3.4.4. Glutamate dehydrogenase Assay: Estimation of enzyme activity.

Glutamate dehydrogenase was routinely spectrophotometrically assayed according to the method of

Garnier et al, (1997) using a Labsystems Multiskan MS, Microtitre plate reader, Japan, at 30oC, by

following the decrease in A340 as ß-NADH was oxidized. The assay mixture contained the

following reagents in a total volume of 0.25 ml, being 0.16 mM ß-NADH, 5 mM α-ketoglutarate,

20 mM (NH4)2SO4, 50 mM potassium phosphate buffer, at pH 8.3, plus enzyme. For the assay of

crude enzyme solutions, the mixture also contained 0.4 mM KCN to decrease NADH oxidase

activity. One unit of enzyme activity is defined as the amount of enzyme catalysing the oxidation of

1µmol of NADH/min/mg of protein under the assay conditions.

3.5. Purification of glutaminase

3.5.1. Materials

All solutions were prepared with deionised water. Analytical reagent grade chemicals were used

without further purification and obtained from the following commercial sources: Mannitol,

sucrose, HEPES, potassium phosphate (KH2PO4), sodium chloride (NaCl), glutamic acid,

Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), Ethylene glycolaminetetraacetic

acid (EGTA), iodoacetamide and ammonium sulphate ((NH4)2SO4) from Merck chemicals; Trizma

and PMSF [Phenylmethylsulfidefluoride] (Sigma, Steinheim Germany); sodium dodecylesulphate

(SDS) (Boehringer, Germany); Sephadex G-25, DEAE Sephacel, GTP sepharose and nickel

nitrilotriacetic acid gel from Pharmacia Biotech (Uppsala, Sweden); Zhicheng Shaking incubator

(Shanghai, China); Molecular weight marker and dithiothreitol (DTT) (Fermentas); Nicotinamide


56

adenine dinucleotide (NAD), Adenosine diphosphate (ADP) (Roche diagnostics, USA); glutamate

dehydrogenase (GDH), acrylamide/Bis-acrylamide, Nessler‟s reagent, trichloroacetic acid (TCA),

formaldehyde and trypsin (Sigma-Aldrich, USA); acetonitrile, hydrochloric acid (Romil pure

chemistry); Perkin Elmer Cetus DNA thermal cycler heating block; Coomassie brilliant blue (R-

250).

3.5.2. Purification of bovine glutaminase procedure

Purification was done according to the method of Patel & McGivan, (1984) with some

modifications incorporating a variation of the method by Smith & Watford, (1988). All procedures

were done at 4oC unless otherwise mentioned. Approximately 30 g (wet weight) of defrosted

bovine liver was rinsed in homogenization buffer containing 210 mM mannitol, 70 mM sucrose,

10mM HEPES, at pH 8, plus 1.5 mM EGTA and 0.1 mM PMSF, being subsequently homogenised

with the addition of a AESBF cocktail for 3 minutes at full speed, using a commercial blender.

Supernatant was slowly treated with the addition of chilled (NH4)2SO4 to yield a final concentration

of 70% saturated solution (254 g/L). The solution was subjected to mechanical stirring for half an

hour at 4oC, followed by centrifugation at 39100g with a Sorvall RC-50C refrigerated super-speed

centrifuge (Du-Point Instruments, USA). Thereafter, the pellet was resuspended in 25 ml of

homogenization buffer, before being stirred at 4oC and dialysed in 1 L of homogenization buffer

overnight at 4oC.

Sephadex G-25 chromatography

The dialysate (8ml) was loaded onto a column (2.5 x 10 cm column) of a Sephadex G-25 column

(Sigma) equilibrated with 210 mM mannitol pH 8.0, buffer containing 50 mM Tris-HCl, 70 mM

sucrose, together with 1mM-dithiothreitol. The proteins were eluted with 15ml of equilibrating

buffer at a constant rate of 0.5 ml/min. Thereafter, 2 ml fractions with high enzyme activity were

pooled and chromatographically analysed using a DEAE sephacel column, after the enzyme

activity assay was completed.


57

DEAE Sephacel, PD-10

A pooled sample which was collected from a Sephadex G-25 column, was then applied to a 2.5 x

10 cm column of DEAE-Sephacel (Sigma) at 4oC equilibrated with 210 mM mannitol, pH 8.0,

buffer containing 50 mM-Tris-HCl, 70 mM sucrose, together with 1 mM DTT. The column was

then washed with equilibrating buffer and eluted with a linear gradient of 0 - 500mM NaCl in

equilibrating buffer at a flow rate of 2 ml / min. The most active fractions were pooled and dialysed

in 210 mM mannitol pH 8.0, buffer containing 50 mM Tris-HCl, 70mM-sucrose, together with

1mM dithiothreitol overnight at 4oC.

Ultrafiltration

Vivaspin 30000MW 50ml tube was used. 5ml of combined samples from DEAE Sephacel column

chromatography were added to the 50ml Vivaspin 30000MW tube and spun at 5000 x g for 30

minutes in a Epperndorf centrifuge 5810R (Merck) refrigerated bench top centrifuge.

3.5.3. Glutaminase Assays: Estimation of enzyme activity.

3.5.3.1. Confirmation calorimetric glutaminase enzyme activity assay

All reagents were added at room temperature. Glutaminase was routinely assayed by the method

described by Heini et al., (1987) calorimetrically (Labsystems Multiskan MS, Microtitre plate

reader, Japan), except that the volume and the concentrations of some reagents were modified for

quantification of ammonia with Nessler‟s reagent. 500µl of 260mM K2HPO4, 63Mm Tris-HCl -

pH8.6 with 2mM EDTA was added to 500µl distilled water and equilibrated to 37oC. Thereafter,

250µl of enzyme extract was added to other reactions, excluding blank, to which distilled water

was added. The solution was mixed by inversion and incubated at 37oC for 30minutes, after which

the reaction was terminated with 250µl 1.5M TCA. Finally, 10% of assay mixture was aliquot into

250µl distilled water, plus 200µl Nessler‟s reagent, to quantify ammonia produced by the reaction.

The solution was transferred in triplicates into 96-well plate and reading was performed at 540nm

using a micro-titre plate reader.


58

3.5.3.2. Kinetic spectrophotometric glutaminase enzyme activity assay

Glutaminase activity was measured by assaying the production of glutamate from glutamine in a

kinetic assay system (Dura et al., 2002). The method included some modifications according to

McGivan & Bradford (1983). Glutaminase was routinely assayed spectrophotometrically with the

aid of a Labsystems Multiskan MS, Microtitre plate reader, Japan. In order to extend the range of

linearity of the oxidation assay, but excluding changes in volume of the reaction mixture,

concentration of some of the substrates were increased up to ten fold, for some of the reagents. The

initial incubation mixture for the phosphate-dependent glutaminase contained 2.6 mM potassium

phosphate, varied glutamine concentrations, plus 2.6 mM Tris-Cl at pH 8. The corresponding

incubation mixture for the phosphate-independent glutaminase contained 3.2 mM boric acid with

enzyme, while the main reaction mixture contained 462 mM Tris-Cl, 266 mM NAD, 2.6 mM ADP,

1.9mM GDH and 660 mM Glutamic acid, for the control reaction. The first mixture was mixed by

inversion and equilibrated to 37oC, before being transferred into a second mixture and mixed by

inversion prior to incubation for 30 minutes at 37oC. The reaction was then stopped with 20µl HCl

and reaction placed on ice for 5 minutes. Out of the combination of first and second mixtures, a

10µl aliquot became third mixture, upon the addition of 12µl of glutamic acid, as a positive

control. A negative control (blank) was made to include 12µl of H2O, instead of glutamine or

glutamic acid.

After stability of the ensuing reaction was achieved, absorbance versus a blank were prepared by

adding water immediately after the enzyme solution, was analysed at 340 nm by recording the first

absorbance value. Increase in the absorbance values were monitored over a 30minute period, when

the last value was recorded.



transposed into an MS-Excel workbook and calculated according to Curthoys & Weiss (1974) as in

the formula below.

U/mg Enzyme = (ΔA340T - ΔA340B) (0.22) (0.6)

(6.22) (mg Enzyme/RM) (0.012) (30)


59

Where U/mg Enzyme is µmoles per milligrams of enzyme, ΔA340 is change of absorbance at

340nm, 0.22 is volume of assay in millilitres of step 1, 0.6 is total volume of assay in millilitres of

step 2, 6.22 is millimolar coefficient, RM is reaction mixture volume, 0.012 is volume of step 1

assay used in step 2, 30 is time of assay in minutes as per units definition.

3.6. Protein concentration assay

The protein content of all samples was determined by the Quint-iT™ protein assay in the Quanti™

fluorometer (Invitrogen detection technologies, USA) method (Appendix 2, Page 170).

3.7. SDS-PAGE

The purity of the various fractions obtained during protein purification was assessed by Sodium

Dodecyl-Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The SDS-PAGE method

used was adapted from Laemmli (1970). A 10% separating gel and 4% stacking gel (Appendix 1,

Page 168) were also prepared.

3.8. Bio-analysis of extracted proteins with Liquid Chromatography Mass

Spectrometry (LC-MS/MS)

3.8.1. Introduction to QSTAR® Elite Liquid Chromatography Mass Spectrometry (LC-

MS/MS)

QSTAR®

Elite LC/MS/MS system is a quadropole time-of-flight (QqTOF) designed for proteomics

and metabolomics research to discover small molecule biomarkers from complex biological

samples. Liquid chromatography mass spectrometry-based approaches for targeted protein

identification and quantification are based on the concept of isotope dilution mass spectrometry

techniques commonly used for the detection of small molecules (Bowers et al., 1993). High-

resolution mass spectrometry provides an approach which can precisely detect wide variety

peptides via high mass accuracy and peptide sequencing. This approach provides high selectivity

and specificity, while technically avoiding most of the problems associated with optimization of


60

multiple assays in a single measurement. This mass spectrometry based technique has a unique

capability to perform candidate-based proteome browsing (Aebersold, 2003; Kuster et al., 2005)

and measure absolute levels of post-translational modifications (Kuster et al., 2005). The in-gel

digestion protocol presented in Appendix 3 (Page 173), was originally introduced in 1996

(Shevchenko et al., 1996), and has become a norm with most workers over the past years. The

protocol can be optimized to balance the time of digestion, plus the speed of the entire protein

identification routine, with the yield of tryptic peptides. The recipe is flexible and can easily be

adapted to meet the specific requirements of any particular proteomics experiment (Havlis et al.,

2003). The method applies with negligible or minor adjustments to gels stained with Coomassie

brilliant blue R250 (Shevchenko et al., 1996). The in-gel digestion procedure is compatible with

downstream MALDI-MS characterization of digests of isolated protein bands or spots.

A variety of mass spectrometric platforms have been used for targeted quantitative proteomics

analysis, including the matrix assisted laser desorption/ionization time-of-flight tandem mass

spectrometer (MALDI TOF) (Pan et al., 2005), and the electrospray ionization (ESI) based QTOF

mass spectrometer (Rivers et al., 2007). The discriminating power as well as maturity of the

technology provided great convenience in adapting the methodology for protein detection.

Protein bands cut out of SDS-PAGE were subjected to tryptic digestion and analysed by LC-

MS/MS according to the methodology indicated in Appendix 3 (Page 173).

3.9. Database search

Protein identification was performed using the ParaghonTM

algorithm thorough search in Protein

Pilot™ software. An identification confidence exceeding 95 % was selected during the search.

Protein Pilot™ software for protein identification supports QSTAR®

Elite reagent workflows. It

incorporates the Paragon™ search algorithm in a single package that increases the speed of

database searches along with advanced data analysis and visualization.

Data generated by QSTAR®

Elite ESI mass spectrophotometer was analysed by Protein pilot™

software and results of peptides obtained were blasted in the http://blast.ncbi.nlm.nih.gov/ website

of proteins database.

http://blast.ncbi.nlm.nih.gov/


61

3.10. Kinetic constants

All enzymes were assayed at a range of substrate concentrations to determine the kinetic constants

using using non-linear regression protocols as defined in the Graph-Pad™ prism 5.02 software

protocols. The calculated enzyme specific activities with their standard deviations were transferred

as values calculated elsewhere into Graph-Pad™ prism 5.02 software for generation of graphs, Vmax

and Km values. When reciprocal plots of velocity against substrate concentration were either linear

or non-linear, the data was fitted to a formula below.

Where v = initial velocity, Vmax = maximal velocity, Km = apparent Michaelis constant,

[S] = substrate (Tveit et al., 1970).

Vmax [S]

Km + [S] v =

Chapter 4: Integrated Results

62

CHAPTER 4: INTEGRATED RESULTS

The data obtained for the purification of arginase, GS, GDH, CPS and glutaminase are outlined

below. Also included is the level of purification obtained for each enzyme at each technique used.

Steady-state enzyme kinetics was used for each enzyme to define the Vmax and Km for each enzyme

substrate used. The GraphPad prism suite of software was used to calculate these kinetic constants

using nonlinear regression analysis.

4.1. Purification of bovine arginase

The purification of arginase from Nguni liver samples and Hereford liver samples was carried out

as described earlier. The results of the enzyme activities obtained at each stage of the purification

are summarized in Table 4.1. The fractional precipitation of arginase with chilled ammonium

sulphate at 4 °C, followed with heat treatment, resulted in good yields of both Nguni and Hereford

arginase. For Nguni liver, the fractional precipitation of arginase with chilled ammonium sulphate

gave a specific activity of 318.29 μmol.min-1

.mg protein, while for Hereford liver; a specific

activity of 510.46 μmol.min-1

.mg of protein was achieved.

Table 4.1. Typical purification of Nguni liver and Hereford liver Arginase (N = Nguni, H =

Hereford) These assays were all run in substrate excess.

Purification

step

Volume

(ml)

Total

protein(mg)

Total activity (U

x103)

Specific activity

(µmol/min/mg)

N H N H N H

Homogenate 110 173 196 218.94 296.34 16.3 22.05

Ammonium

sulphate

50 180 200 318.29 510.46 23.7 37.98

Sephadex G-25 12 205 233 481.05 637.06 35.8 47.4

DEAE

Cellulose

14 196 149 221.8 297.07 17 22.1

Arginine

affinity column

6 185 107 207.49 84.39 15.4 6.28


63

4.1.1. Kinetics

The effect of substrate concentration on the arginase activity was determined using arginine and

manganese. Three different GraphPad Prism calculations were applied to the kinetics of Nguni

liver arginase versus manganese and also versus arginine (Table 4.2), and Hereford liver arginase

versus manganese as well as versus arginine (Table 4.3).

Table 4.2. Comparison of Vmax and Km of Nguni liver arginase with respect to manganese (A) and

also with respect to arginine (B) is calculated by GraphPad Prism.

Non-Linear fit NLARG1 NLARG2 NLARG3 NLARG4 Average STDEV

(A) Manganese

Michaelis-

Menten

Best-fit values

Vmax 9.049 5.201 2.999 3.743 5.248 2.333069

Km 19.26 8.698 4.146 6.125 9.55725 5.829743

(B) Arginine

Michaelis-

Menten

Best-fit values

Vmax 19.22 12.82 19.18 13 16.055 3.145676

Km 51.3 21.1 45.45 18.91 34.19 14.35589


64

Table 4.3. Comparison of Vmax and Km of Hereford liver arginase with respect to manganese (A)

and arginine (B) is calculated by GraphPad Prism.

Non-Linear fit HLARG1 HLARG2 HLARG3 HLARG4 Average STDEV

(A) Manganese

Michaelis-

Menten

Best-fit

values

Vmax 7.766 6.259 4.903 7.869 6.69925 1.2172

Km 5.659 4.118 2.057 4.27 4.026 1.2856

(B) Arginine

Michaelis-

Menten

Best-fit

values

Vmax 20.12 8.996 9.886 22.67 15.418 6.0527

Km 1.057 1.657 1.832 0.7143 1.315075 0.4504

4.1.2. Enzyme Incubation time

The effect of incubation time on the activities of Nguni liver arginase and Hereford liver arginase

was studied over a 30 minute time period to determine the linearity of the enzyme activity (Figure

4.1). On the basis of this all enzyme assays were run as single point assays for 20 minutes.

4.1.3. Effect of substrate concentrations

The effect of the substrate concentrations on the specific activities of arginase obtained from the

Nguni and Hereford breeds was determined. All enzymes were purified from four different livers,

from each breed, to allow a comparison to be made on the statistical significance of the purification

protocol. This was essential if the enzyme kinetics for each breed was to be compared.

Within each breed, the distribution of the specific enzyme activity data showed very little variation.

However, the overall enzyme activity for the Hereford breed was almost double that of the Nguni

breed (Table 4.1) across the purification process other than the final affinity stage.


65

The effect of the concentration of manganese on the activity of arginase is shown in Figure 4.2 -

4.3 for both Nguni and Hereford livers. The steady kinetics for manganese is hyperbolic and yields

a Michaelis-Menten Km of between 6.1 – 19.3mM (Average = 9.557mM) for the Nguni livers and 2

– 5.7mM for the Hereford livers as analysed by GraphPad Prism.

0 10 20 30 400

20

40

60

NLARGHLARG

Incubation time (minutes)

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

in/m

g)

Figure 4.1. Indication of the comparison of the effect of the incubation times on the specific

activity of Nguni (NLARG) and Hereford (HLARG) liver arginase (0.2 – 0.3 mg) at 37oC. These

assays were performed in excess substrate.


66

0 2 4 60.0

0.5

1.0

1.5

2.0

2.5

NLARG1NLARG2NLARG3NLARG4

Nguni

Mn (mM)

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

in/m

g)

Figure 4.2. Effect of the manganese concentration on the specific activity of Nguni arginase in

10mM Sodium bicarbonate, pH 9.6 and 25 mM L-Arginine at 37oC.


67

0 2 4 60

1

2

3

4

5

HLARG2HLARG1

HLARG3HLARG4

Hereford

Mn (mM)

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

in/m

g)

Figure 4.3. Effect of the manganese concentration on the specific activity of Hereford arginase in

10mM Sodium bicarbonate, pH 9.6 and 25 mM L-Arginine at 37oC.


68

0 10 20 30 400

2

4

6

8

10

NLARG1NLARG2NLARG3NLARG4

Nguni

L-Arginine (mM)

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

in/m

g)

Figure 4.4. Effect of the arginine concentration on the specific activity of Nguni arginase in10mM

Sodium bicarbonate, pH 9.6 and 2.5 mM MnC12 at 37oC.

The effect of the concentration of arginine on the activity of arginase is shown in Figure 4.5, that

of manganese concentration on the specific activity of Nguni and Hereford liver arginase in Figure

4.6 and the effect of the arginine concentration on the specific activity of Nguni and Hereford liver

arginase in Figure 4.7.


69

0 10 20 30 400

2

4

6

8

HLARG1HLARG2HLARG3HLARG4

Hereford

L-Arginine (mM)

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

in/m

g)

Figure 4.5. Effect of the arginine concentration on the specific activity of Nguni arginase in 10mM

Sodium bicarbonate, pH 9.6 and 2.5 mM MnCl2 at 37oC.

The steady kinetics for arginine is hyperbolic and yields a Michaelis-Menten Km of between 18.9 –

51.3mM (Average = 34.19mM) for the Nguni livers and 11.8 – 70.2mM (Average = 1.315mM) for

the Hereford livers analysed by GraphPad Prism.


70

0 2 4 60

1

2

3

4

5

NLARGHLARG

Mn (mM)

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

in/m

g)

Figure 4.6. Comparison of the effect of the manganese concentration on the specific activity of

Nguni (NLARG) and Hereford (HLARG) liver arginase presented as average of each increasing

concentration of manganese. This data represents the average for the assays outlined in Figures 4.2

and 4.3.


71

0 10 20 30 400

2

4

6

8

10

NLARGHLARG

L-Arginine (mM)

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

in/m

g)

Figure 4.7. Comparison of the effect of the arginine concentration on the specific activity of Nguni

(NLARG) and Hereford (HLARG) liver arginase, presented as evarage of each increasing arginine

concentration. This data represents the average for the assays outlined in Figures 4.4 and 4.5.


72

4.1.4. Effect of divalent cations

The results as shown in Figure 4.8 were completely inconclusive after several repetitions.

Figure 4.8. Effect of the selected divalent cations at 2.5mM on the specific activity of Hereford and

Nguni arginase in 10mM Sodium bicarbonate, pH 9.6 at 37oC. These assays were run in excess

substrate.


73

4.1.5. Estimation of arginase molecular weight on SDS-PAGE

The molecular weight on SDS-PAGE was marked at 38 kDa for both Nguni and Hereford liver

arginase. There was a 52 kDa visible protein band which was previously reported for buffalo liver

arginase by Dabir et al., 2005 (Figure 4.9). The molecular weight obtained for arginase with both

bovine breeds, is higher than the 35.009 reported for cytoplasmic type 1 bovine arginase, but

similar to 38.616 reported for mitochondrial type 2 bovine arginase. Reports obtained for the

bovine hepatic arginases agree with the reported molecular weights of arginases from buffalo liver

(Dabir et al., 2005). Lane 1 of the Nguni SDS-PAGE indicated approximately 80% purity. All

assays gave very similar responses when compared.

KDa M 1 2 3 4 1 2 3 4

118

66.2 52 52

45 38 38

35

25

18.4

14.4

A B

Figure 4.9. SDS-PAGE (10%) analysis of partially purified liver arginase M: Molecular weight

standard, size in kilo-dalton is shown on the left. A: Partially purified Nguni liver arginase

indicated as 1: NLARG1 2: NLARG2 3: NLARG3 4: NLARG4 and B: Partially purified Hereford

liver arginase indicated as 1: HLARG1 2: HLARG2 3: HLARG3 4: HLARG4.


74

4.2. Purification of carbamoyl phosphate synthetase

The results from the purification of carbamoyl phosphate synthetase from both Nguni and Hereford

liver samples are summarized in Table 4.4. Both ammonium sulphate and low molecular weight

proteins were removed from the enzyme fraction solutions via passage through a Sephadex-G25 gel

column, yielding 1485 µmoles/mg/ml and 1646 µmoles/mg/ml of activity for the Nguni and

Hereford livers respectively. Carbamoyl phosphate synthetase was adsorbed on a DEAE-Sephacel

column and eluted with a KH2PO4, (pH7) gradient, ranging in concentration from 2mM – 500mM

KH2PO4 (pH7), yielding 767 µmoles/mg/ml and 957 µmoles/mg/ml of activity for the Nguni and

Hereford livers respectively. The enzyme fractions were then specifically bound on an AMP

Agarose gel column, yielding 490µmoles/mg/ml and 887 µmoles/mg/ml of activity for the Nguni

and Hereford livers respectively.

Table 4.4. Typical purification of carbamoyl phosphate synthetase from Nguni (N) and Hereford

livers (H). The assays were run in excess substrate.

Purification

step

Volume

(ml)

Total

protein(mg)

Total activity

(µmoles/min/mgx103)

Specific activity

(µmoles/min/mg)

N H N H N H

Homogenate 110 2060 895 1490 1904 3.3 2.05

Ammonium

sulphate

50 1920 772 1313 1512 3.7 2.3

Sephadex G-

25

12 1930 705 1485 1646 5.8 4.59

DEAE

Sephacel

14 2490 695 767 957 2.2 2.1

AMP Agarose 6 1860 665 490 887 0.49 0.887


75

4.2.1. Kinetics

The effect of the concentration of Mg-ATP on the activity of carbamoyl phosphate synthetase

(CPS) is shown in Figure 4.10 - 4.11 for the Nguni and Hereford livers respectively. The steady

state kinetics for Mg-ATP is hyperbolic function and giving a Michaelis-Menten Km of between

0.43 – 0.9mM for the Nguni livers and 0.91 – 8.5mM for the Hereford livers. Although Hereford

liver CPS is more scattered than Nguni CPS, both their goodness of fit is 6/8 plots with the R-

square of 0.94 – 0.98.

0 2 4 6 80.0

0.2

0.4

0.6

NLCPS1NLCPS2NLCPS3NLCPS4

Nguni

[Mg-ATP]

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

g/m

in)

Figure 4.10. The effect of the concentration of Mg-ATP on the specific activity of Nguni liver

carbamoyl phosphate synthetase. The specific activity as a function of Mg-ATP concentration was

measured in 20mM of NaHCO3, 300mM of NH4Cl, 100mM of KCl and 50mM of HEPES pH 7.6.


76

0 2 4 6 80.0

0.5

1.0

1.5

HLCPS1HLCPS2HLCPS3HLCPS4

Hereford

[Mg-ATP]

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

g/m

in)

Figure 4.11. The effect of the concentration of Mg-ATP on the specific activity of Hereford liver

carbamoyl phosphate synthetase. The specific activity as a function of Mg-ATP concentration was

measured in 20mM of NaHCO3, 300mM of NH4Cl, 100mM of KCl and 50mM of HEPES pH 7.6.

The effect of the concentration of ammonium chloride on the activity of CPS is shown in Figure

4.12 and 4.13 for both Nguni and Hereford livers respectively. The steady kinetics for Mg-ATP is

hyperbolic and yields a Michaelis-Menten Km of between 30.2 – 38.5mM for the Nguni livers and

15.7 – 27.5mM for the Hereford livers. The apparent Michaelis constants reported for mammalian

liver carbamoyl phosphate synthetases are in the range of 10mM (Kerson & Appel, 1968).


77

0 20 40 600

2

4

6

NLCPS1NLCPS2NLCPS3NLCPS4

Nguni

[NH4Cl]

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

g/m

in)

Figure 4.12. The effect of the concentration of NH4Cl on the specific activity of Nguni liver

carbamoyl phosphate synthetase. Specific activity as a function of ammonium chloride

concentration was measured in 20mM of NaHCO3, 2mM of ATP, 20mM of MgCl2, 100mM of KCl

and 50mM of HEPES pH 7.6 at 37oC.


78

0 20 40 600.0

0.5

1.0

1.5

2.0

HLCPS1HLCPS2HLCPS3HLCPS4

Hereford

[NH4Cl]

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

g/m

in)

Figure 4.13. The effect of the concentration of NH4Cl on the specific activity of Hereford liver

carbamoyl phosphate synthetase. Specific activity as a function of ammonium chloride

concentration was measured in 20mM of NaHCO3, 2mM of ATP, 20mM of Magnesium Chloride,

100mM of KCl and 50mM of HEPES pH 7.6 at 37oC.

The effect of the concentration of Mg-ATP on the activity of CPS is shown in Figure 4.14 and 4.15

for both Nguni and Hereford livers respectively.


79

0 2 4 6 80.0

0.2

0.4

0.6

0.8

NLCPSHLCPS

Nguni

[Mg-ATP]

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

g/m

in)

Figure 4.14. Comparison of the effect of the concentration of Mg-ATP on the specific activity of

Nguni (NLCPS) and Hereford (HLCPS) liver carbamoyl phosphate synthetase presented as the

average for each increasing Mg-ATP concentration. These assays were run in excess substrate.

.


80

0 20 40 600

2

4

6

NLCPSHLCPS

Nguni

[NH4Cl]

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

g/m

in)

Figure 4.15. Comparison of the effect of the concentration of NH4Cl on the specific activity of

Nguni (NLCPS) and Hereford (HLCPS) liver carbamoyl phosphate synthetase presented as average

of each increasing NH4Cl concentration. These assays were also run in excess substrate.


81

The effect of Mg-ATP concentration on the activity of Nguni liver and Hereford liver CPS activity

is shown in Tables 4.5 and 4.6 respectively.

Table 4.5. Comparison of Vmax and Km of Nguni liver CPS with respect to Mg-ATP (A) and also

with respect to ammonium (B) is calculated by GraphPad Prism.

Non-Linear fit NLCPS1 NLCPS2 NLCPS3 NLCPS4 Average STDEV

(A) ATP/mg

Michaelis-

Menten

Best-fit

values

Vmax 0.5031 0.4636 0.5575 0.4541 0.494575 0.040712

Km 0.5242 0.5332 0.909 0.4299 0.599075 0.183453

(B) Ammonium

Michaelis-

Menten

Best-fit

values

Vmax 9.394 8.02 8.177 8.566 8.53925 0.532015

Km 36.6 31.94 27.82 36.26 33.155 3.586346


82

Table 4.6. Comparison Vmax and Km of Hereford liver CPS with respect to Mg-ATP and

ammonium is calculated by GraphPad Prism.

Non-Linear fit HLCPS1 HLCPS2 HLCPS3 HLCPS4 Average STDEV

ATP/mg

Michaelis-

Menten

Best-fit

values

Vmax 0.7448 1.547 2.333 0.8912 1.379 0.628192

Km 0.9141 4.579 8.58 2.015 4.0221 2.948419

Ammonium

Michaelis-

Menten

Best-fit

values

Vmax 2.676 2.218 2.557 2.186 2.4091 0.21178

Km 22.74 19.21 27.54 15.68 21.2925 4.386441

The effect of Mg-ATP concentration on the activity of Nguni liver and Hereford liver carbamoyl

phosphate synthetase was also determined by a way of linear regression. The apparent Km values of

Mg-ATP were demonstrated while the concentration of ammonium chloride and sodium

bicarbonate were kept constant. At a higher concentration of Mg-ATP; double reciprocal plot

continues linear which agrees to other studies of liver carbamoyl phosphate synthetase in which

double reciprocal also continues linear (Elliot & Tipton, 1973).

4.2.2. Estimation of CPS molecular weight on SDS-PAGE

The molecular weight of CPS obtained for both bovine breeds was found to be 164,740Da, as

shown in Figure 4.16. This value agrees with the size for mitochondrial type bovine carbamoyl

phosphate synthetase reported in UniProtKB protein database (http://www.uniprot.org/uniprot),

as marked by protein molecular weight page ruler (Fermentas).

http://www.uniprot.org/uniprot


83

KDa

M 1 2 3 4 1 2 3 4

A B

Figure 4.16. SDS-PAGE (10%) analysis of partially purified liver carbamyl phosphate synthetase

M: Molecular weight standard, size in kilo-dalton is shown on the left. A: Partially purified Nguni

liver CPS indicated as 1: NLCPS1 2: NLCPS2 3: NLCPS3 4: NLCPS4 and B: Partially purified

Hereford liver CPS indicated as 1: HLCPS1 2: HLCPS2 3: HLCPS3 4: HLCPS4.

The 150KDa - 170KDa seen in different strength in SDS-PAGE (Figure 4.16 A) were eluted from

DEAE Sephacel of the Nguni liver carbamoyl phosphate synthetase. The Hereford liver carbamoyl

phosphate synthetase which is also seen as different bands (Figure 4.16 B) was also eluted from

the DEAE Sephacel. The presence of CPS in the identified bands was further confirmed using

LC/MS/MS (Section 4.2.4).

4.2.3. Trypsin digest and LC/MS/MS for CPS band Identification

Peptides were identified by QSTAR Elite ESI as gel-based ID marked by protein molecular weight

page ruler in the SDS-PAGE (Figure 4.16, Page 85). The data obtained was analysed by

ProteinPilot™ Software 3.0 (Paragon Algorithm 3.0.0.0, 113442) and reported below. Only

samples detected at a protein threshold of 1.30 (95%) indicating 2.00 Protein score were reported.

150

100

70

50

40

164.7 164.7


84

Only proteins with an unused Protein score greater than this value are shown in the Detected

Proteins table. Selected peptides of interest were recorded below as examples.

4.2.3.1. Selected peptides of interest for the identification of Nguni liver CPS

i. AVNTLNEALEFAK

ii. CLGLTEAQTR

iii. GQNQPVLNITNR

iv. IALGIPLPEIK

Figure 4.17. QSTAR Elite ESI Generated peptide profile for CPS. Peptides of interest: Precursor

MS regions analysed by ProteinPilot™ Software 3.0 (Paragon Algorithm 3.0.0.0, 113442)


85

In Figure 4.17 (Page 84), a QSTAR Elite ESI Generated peptide profile for Carbamoyl-

phosphate synthase (ammonia) (EC 6.3.4.16) I precursor– rat is displayed

Blast information for Nguni liver CPS is given in Table 4.7

Table 4.7. Blast information for Nguni liver CPS generated from NCBI database of proteins.

Accession Description Maximum

Score

Total

Score

Query

Coverage

Maximum

Identity

NP_001179187.1 Carbamoyl-

phosphate synthase

[ammonia],

mitochondrial

[Bos taurus]

34.7 99.3 80% 100%

http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome

4.2.3.2. Selected peptides for the identification of Hereford liver CPS

i. AVNTLNEALEFAK

ii. CLGLTEAQTR

iii. GQNQPVLNITNR

iv. EVEMDAVGK

Figure 4.18. A QSTAR Elite ESI Generated: Carbamoyl-phosphate synthetase I. - Homo

sapiens (Human) is displayed. Peptides of interest: Precursor MS regions analysed by

ProteinPilot™ Software 3.0 (Paragon Algorithm 3.0.0.0, 113442).

http://www.ncbi.nlm.nih.gov/protein/300795597?report=genbank&log$=prottop&blast_rank=9&RID=HUFG61SE01S



86

In Figure 4.18 (Page 85), a QSTAR Elite ESI Generated: Carbamoyl-phosphate synthetase I.

- Homo sapiens (Human) is displayed.

Blast information for Hereford liver CPS is given in Table 4.8

Table 4.8. Blast information for Hereford liver CPS generated from NCBI database of

proteins.


Score

Total

Score

Query

Coverage

Maximum

Identity

NP_001179187.1 Carbamoyl-

phosphate

synthase

[ammonia],

mitochondrial

[Bos taurus]

32.3 63.5 82% 100%


4.3. Purification of glutamine synthetase

The purification of GS from Nguni liver samples and Hereford liver samples was carried out

as described. The results obtained in the purification of GS from Nguni liver samples and

Hereford liver samples are summarized in Table 4.10. The fractional precipitation of GS with

chilled ammonium sulphate at 4 °C, resulted in equivalent yields of both Nguni and Hereford

GS.

http://www.ncbi.nlm.nih.gov/protein/300795597?report=genbank&log$=prottop&blast_rank=2&RID=HUDVCR96012



87

Table 4.9. Typicalpurification of Nguni liver (N) and Hereford liver (H) GS, indication from

both liver samples. (Units = μmoles ferric hydroximate complex/minute/mg protein). These

assays were performed in excess substrate.

4.3.1. Kinetics

The period of incubation used across the kinetic study of GS remained 90 minutes for HPLC

analysis as this was found to be optimal for the linearity of the assay (Price et al., 1978).

Table 4.10. Comparison of Vmax and Km of Nguni and Hereford liver GS with respect to Mg-

ATP is calculated by GraphPad Prism.

Non-linear

fit NLGS1 NLGS2 NLGS3 NLGS4 Average STDEV

Mg-ATP

Michaelis-

Menten

Best-fit values

Vmax 0.08269 0.1082 0.0912 0.07048 0.088143 0.013724

Km 1.953 1.204 1.048 5.635 2.46 1.864736

Non-linear

fit HLGS1 HLGS2 HLGS3 HLGS4 Average STDEV

Mg-ATP

Michaelis-

Menten

Best-fit values

Vmax 3.42 3.57 2.19 2.93 3.0275 0.538348

Km 3.2 1.2 5.7 6.3 4.1 2.038382

Purification step Volume

(ml)

Total

protein(mg)

Specific activity

(µmoles/min/mg)

N H N H

Homogenate 140 246 218 1.31 1.50

Ammonium

sulphate

50 141 106 1.21 1.13

Sephadex G-25 12 245 193 1.28 1.13

AMP Agarose 6 168 131 1.11 1.06


88

The effect of the concentration of Mg-ATP on the activity of GS is shown in Figure 4.19 and

4.20 for both Nguni and Hereford livers. The steady kinetics for Mg-ATP is hyperbolic and

yields a Michaelis-Menten Km of between 30.2 – 38.5mM (Average = 2.46mM) for the Nguni

livers and 15.7 – 27.5mM (Average = 4.1mM) for the Hereford livers.

0 2 4 60.00

0.05

0.10

0.15

NLGS1NLGS2NLGS3NLGS4

Nguni

[Mg-ATP]

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

in/m

g)

Figure 4.19. Specific activity of Nguni liver GS versus Mg-ATP concentration. Specific



89

0 2 4 6-0.1

0.0

0.1

0.2

0.3

HLGS1HLGS2HLGS3HLGS4

Hereford

[Mg-ATP]

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

in/m

g)

Figure 4.20. Specific activity of Hereford liver GS versus Mg-ATP concentration. Specific



90

0 2 4 60.0

0.1

0.2

0.3

NLGSHLGS

[Mg-ATP]

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

in/m

g)

Figure 4.21. Comparison of the specific activity of Nguni (NLGS) and Hereford (HLGS)

liver glutamine synthetase versus Mg-ATP concentration presented as average of each

increasing Mg-ATP concentration.

4.3.2. Estimation of GS molecular weight on SDS-PAGE

The molecular mass of bovine GS is 42031. The molecular weight on SDS-PAGE was was

found to be 59kDa for both Nguni and Hereford liver GS. The molecular weight obtained for

both bovine breeds GS indicated glycosylation of bovine GS. The glycosylation of

mammalian proteins significantly impacts on the quality of SDS-PAGE data.


91

KDa M 1 2 3 4

A

KDa M 1 2 3 4

B

Figure 4.22. SDS-PAGE (10%) analysis of partially purified liver glutamine synthetase, M:

Molecular weight standard, size in kDa is shown on the left of both gel A & B. A: Partially

purified Nguni liver GS indicated as 1: NLGS1 2: NLGS2 3: NLGS3 4: NLGS4. B: Partially

purified Hereford liver GS indicated as 1: HLGS1 2: HLGS2 3: HLGS3 4: HLGS4

~58kDa

150

50

100

70

40

~42kDa

~58kDa

150

50

100

70

40


92

4.4. Purification of glutamate dehydrogenase

The purification of glutamate dehydrogenase from Nguni liver samples and Hereford liver

samples was carried out as described earlier. The results from the purification of Glutamate

dehydrogenase from Nguni liver samples and Hereford liver samples are summarized in

Table 4.11. For Nguni liver, fractionation with ammonium sulphate resulted in a GDH

specific activity of about 0.58µmol/mg/ml while for Hereford liver it showed a specific

activity of about 0.61µmol/mg/ml. The specific activity of the glutamate dehydrogenase

obtained after affinity column was about 0.07µmol/mg/ml for Nguni and 0.37µmol/mg/ml for

Hereford.

Table 4.11. Typical purification of Nguni liver and Hereford liver glutamate dehydrogenase.

These assays were performed in excess substrate.

Purification

step

Volume

(ml)

Total

protein(mg)

Specific activity

(µmol/min/mg)

N H N H

Homogenate 110 232 245 1.19 0.714

Ammonium

sulphate

50 229 221 0.58 0.61

Sephadex G-25 14 257 233 3.3 1.4

DEAE Sephacel 12 167 194 1.4 0.77

GTP Sepharose 6 50.8 59.0 0.07 0.37

The presence of bovine GDH from both breeds was validated by LC-MS/MS which

identified retinal glutamate dehydrogenase amongst other bands from SDS-PAGE as the band

of interest marked with reference to protein page molecular ruler (Figure 4.29 – Figure

4.30).

4.4.1. Kinetics

The effect of varied concentrations of α-ketoglutarate on the activity of glutamate

dehydrogenase is shown in Figure 4.23 and Figure 4.24 for both Nguni and Hereford livers

respectively. The steady kinetics for α-ketoglutarate is hyperbolic and yields a Michaelis-

Menten Km of between 1.5 – 13.9mM (Average = 5.641mM) (Table 4.11) for the Nguni

livers and a Km of 2.4 – 10.53mM (Average = 6.653mM) for the Hereford livers. Nguni liver


93

GDH has a goodness of fit of 6/8 plots with the R-square of 0.9718 – 0.9945. The kinetics

for ammonium chloride was also hyperbolic yielding a Michaelis-Menten Km of between 4.9

– 15.5mM (Average = 23.79mM) for the Nguni livers and a Km of 2.3 – 17.5mM (Average =

6.474mM) for the Hereford livers. Hereford liver GDH has a goodness of fit of 6/8 plots with

the R-square of 0.9508 – 0.9841.

0 1 2 3 4 50.0

0.5

1.0

1.5

2.0

2.5

NLGDH1NLGDH2NLGDH3NLGDH4

Nguni

a-Ketoglutarate [mM]

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

in/m

g)

Figure 4.23. Effect of the variation of α-ketoglutarate concentration on the specific activity

of Nguni liver glutamate dehydrogenase at 37oC.


94

0 1 2 3 4 50.0

0.5

1.0

1.5

2.0

2.5

HLGDH1HLGDH2HLGDH3HLGDH4

Hereford


Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

in/m

g)

Figure 4.24. Effect of the variation of α-ketoglutarate concentration on the specific activity

of Hereford liver glutamate dehydrogenase at 37oC.


95

0 1 2 3 4 50.0

0.5

1.0

1.5

NLGDH1NLGDH2NLGDH3NLGDH4

Nguni

(NH4)2SO4 [mM]

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

in/m

g)



The effect of the concentration of ammonium sulphate on the activity of glutamate

dehydrogenase is shown in Figure 4.25 and Figure 4.26 for both Nguni and Hereford livers.


96

0 1 2 3 4 50.0

0.5

1.0

1.5

HLGDH1HLGDH2HLGDH3HLGDH4

Hereford

(NH4)2SO4 [mM]

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

in/m

g)




97

0 1 2 3 4 50.0

0.5

1.0

1.5

2.0

2.5

NLGDHHLGDH


Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

in/m

g)

Figure 4.27. Comparison of the effect of the variation of α-ketoglutarate concentration on the

specific activity of Nguni (NLGDH) and Hereford (HLGDH) liver glutamate dehydrogenase

presented as an average of each increasing concentration of α-ketoglutarate.


98

0 1 2 3 4 50.0

0.5

1.0

1.5

NLGDH

HLGDH

(NH4)2SO4 [mM]

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

in/m

g)

Figure 4.28. Comparison of the effect of the variation of ammonium sulphate concentration

on the specific activity of Nguni (NLGDH) and Hereford (HLGDH) liver glutamate

dehydrogenase represented as evarage of each increasing concentration of ammonium

sulphate.

The effect of α-ketoglutarate concentration on the activity of Nguni liver and Hereford liver

glutamate dehydrogenase was determined by Linear regression.


99

Table 4.12. Comparison of Vmax and Km of Nguni liver GDH with respect to substrate α-

ketoglutarate (A) and also with respect to ammonium (B) is calculated by GraphPad prism.

Non-linear fit NLGDH1 NLGDH2 NLGDH3 NLGDH4 Average STDEV

(A) a-Ketoglutarate

Michaelis-

Menten

Best-fit

values

Vmax 2.7 9.26 3.205 4.325 4.873 2.6004

Km 1.522 13.99 3.061 3.989 5.641 4.9004

(B) Ammonium

Michaelis-

Menten

Best-fit

values

Vmax 5.489 18.35 6.296 2.734 8.217 5.9973

Km 15.52 59.79 14.92 4.938 23.79 21.204

Table 4.13. Comparison of Vmax and Km of Hereford liver GDH with respect to α-

ketoglutarate (A) and also with respect to ammonium (B) is calculated by GraphPad prism.

Non-linear fit HLGDH1 HLGDH2 HLGDH3 HLGDH4 Average STDEV

(A) a-Ketoglutarate

Michaelis

-Menten

Best-fit

values

Vmax 5.232 4.397 6.038 2.52 4.547 1.3060

Km 6.747 6.929 10.53 2.405 6.653 2.8793

(B) Ammonium

Michaelis

-Menten

Best-fit

values

Vmax 5.669 1.783 2.055 2.58 3.022 1.5550

Km 17.26 2.333 2.683 3.621 6.474 6.2449


100

4.4.2. Estimation of GDH molecular weight on SDS-PAGE

KDa M 1 2

A

KDa M 3 4

B

Figure 4.29. SDS-PAGE (10%) analysis of partially purified liver glutamate dehydrogenase

M: Molecular weight standard (Fermentas), size in kilo-dalton is shown on the left. A:

Partially purified Nguni liver GDH indicated as 1: NLGDH1 2: NLGDH2 (B), 3: NLGDH3

4: NLGDH4.

~58kD

a

150

50

100

70

40

~58kD

a

150

50

100

70

40


101

KDa M 1 2

A

KDa M 3 4

B

Figure 4.30. SDS-PAGE (10%) analysis of partially purified liver glutamate dehydrogenase

M: Molecular weight standard, size in kilo-dalton is shown on the left. A: Partially purified

Hereford liver GDH indicated as 1: HLGDH1 2: HLGDH2 3: HLGDH3 4: HLGDH4.

The molecular weight on SDS-PAGE was marked at ~58 kDa for both Nguni and Hereford

liver glutamate dehydrogenase (Figure 4.29 – 4.30) with reference to protein molecular

weight page ruler. The molecular weight obtained for both bovine breeds for glutamate

dehydrogenase is slightly higher than the 56 KDa reported for mitochondrial type bovine

glutamate dehydrogenase reported in http://www.ncbi.nlm.nih.gov website of protein

~58kDa

150

50

100

70

40

~58kDa

150

50

100

70

40

http://www.ncbi.nlm.nih.gov/


102

databases. LC-MS/MS results obtained for the bovine hepatic glutamate dehydrogenase

bands cut out of the SDS-PAGE indicated the presence of bovine mitochodrial glutamate

dehydrogenase (Section 4.4).

4.4.3. Trypsin digest and LC-MS/MS for GDH band ID

Peptides were identified by QSTAR Elite ESI as gel-based ID marked by protein molecular

weight page ruler in the SDS-PAGE (Figure 4.29 - Figure 4.30, Page 100 - 101). The data

obtained was analysed by ProteinPilot™ Software 3.0 (Paragon Algorithm 3.0.0.0, 113442)

and reported in section 5.8.5.1 and 5.8.5.2 below. Only samples detected at a protein

threshold of 1.30 (95%) indicating 2.00 ProtScore. Only proteins with an unused ProtScore

greater than this value are shown in the Proteins Detected table. Peptides were recorded

below together with their precursor MS regions and as examples.

4.4.3.1. Selected peptides for the identification of Nguni liver GDH

i. IIKPCNHVLSLSFPIR

ii. DDGSWEVIEGYR

iii. GFIGPGVDVPAPDMSTGER

iv. IIAEGANGPTTPEADKIFLER


Precursor MS regions analysed by ProteinPilot™ Software 3.0.

In Figure 4.31 (Page 102) a QSTAR Elite ESI Generated data of GLUD1 proteins. - Bos

taurus (Bovine) is displayed.


103

Table 4.14. Blast information for Nguni liver GDH generated from NCBI database of

proteins. Blast Information


Score

Total Score Query

Coverage

Maximum

Identity

NP_872593.1 glutamate

dehydrogenase 1,

mitochondrial

precursor [Bos

Taurus]

47.0 172 86% 100%


4.4.3.2. Selected peptides for the identification of Hereford liver GDH

i. MYRYLGEALL

ii. GWARGQPAAA

iii. LSRAGPAALG

iv. PQPGLVPPB


Precursor MS regions analysed by ProteinPilot™ Software 3.0.

In Figure 4.32 (Page 103) a QSTAR Elite ESI generated data of GLUD1 proteins. - Bos

Taurus (Bovine) is displayed.

http://www.ncbi.nlm.nih.gov/protein/32880221?report=genbank&log$=prottop&blast_rank=2&RID=HUM7MN5Z01N



104

Table 4.15. Blast information for Hereford liver GDH generated from the NCBI blast database of

proteins. Blast Information.


Score

Total Score Query

Coverage

Maximum

Identity

NP_872593.1 glutamate

dehydrogenase 1,

mitochondrial

precursor [Bos taurus]

43.9 141 100% 100%


4.5. Purification of glutaminase

The purification of glutaminase from Nguni liver samples and Hereford liver samples was

carried out as described earlier „Purification of Bovine liver glutaminase‟. The results from

the purification of Glutaminase from Nguni liver samples and Hereford liver samples are

summarized in Table 4.16. The fractional precipitation of glutaminase with chilled

Ammonium sulphate (4 °C) at 70% saturation resulted in decreased yield measured in

specific activity of both Nguni and Hereford glutaminase. For Nguni liver, it showed a

specific activity of 1.87 µmol/mg/ml while for Hereford liver glutaminase showed a specific

activity of 0.94 µmol/mg/ml.

Table 4.16. Typical purification of Nguni liver and Hereford liver Glutaminase. These assays

were run in excess substrate.

Purification step Volume (ml) Total

protein(mg)

Specific

activity

(µmol/mg/ml)

Percentage fold

(%)

N H N H N H

Homogenate 140 215 255 3.25 0.94 100 100

Ammonium sulphate 50 107 201 1.87 0.21 58 22

Sephadex G-25 14 116 124 2.27 1.26 7 134

DEAE Sephacel 6 221 227 0.3 0.17 0.9 55

Ultrafiltrate 2 526 505 0.72 0.66 22 70

http://www.ncbi.nlm.nih.gov/protein/32880221?report=genbank&log$=prottop&blast_rank=2&RID=HUM7MN5Z01N



105

Overall protein concentration in milligrams per millilitre after precipitation with chilled

ammonium sulphate decreased substantially for both Nguni and Hereford. The presence of

bovine glutaminase from an SDS-PAGE was not validated by LC-MS/MS, the procedure

identified the band of interest expected to be glutaminase as catalase (Figure 4.28 - 4.29,

Page 98 - 100) and blasting together with aligning failed to identify the band as glutaminase.

4.5.1. Kinetics

The effect of the concentration of glutamine on the activity of glutaminase is shown in

Figure 4.34 and 4.35 for both Nguni and Hereford livers respectively.

The steady kinetics for glutamine for Nguni liver glutaminase yielded a Michaelis-Menten

Km of between 0.4607 – 0.6749mM (Average = 0.5706mM) for the Nguni livers. The kinetics

for the Hereford livers yieldeded a Michaelis-Menten Km of 3.733 – 47.11mM (Average =

22.3133mM). Both Hereford liver glutaminase and Nguni liver glutaminase showed

consistency and are not scattered. A goodness of fit of 6/8 analysed plots were achieved with

an R-square of 0.2695 – 0.4796 for Nguni breed and an R-square of 0.8336 – 0.8669 for

Hereford breed.


106

0 1 2 3 40.00

0.02

0.04

0.06

0.08NLGA1NLGA2NLGA3NLGA4

Glutamine [mM]

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

g/m

l)

Figure 4.33. Effect of glutamine concentration on the specific activity of Nguni liver



107

0 1 2 3 4 50.00

0.01

0.02

0.03

0.04HLGA1HLGA2HLGA3HLGA4

Hereford

Glutamine [mM]

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

g/m

l)

Figure 4.34. Effect of glutamine concentration on specific activity of Hereford liver


Samples were assayed for activity in quadruplicate at each glutamine concentration. The

kinetic constants Km and Vmax were determined using Graph-Pad™ prism 5.02 software

(Hearne, UK) for non-linear least-squares fit for all the data points.

The effect of glutamine concentration on the activity of Nguni liver and Hereford liver

glutaminase was determined by linear regression.


108

0 1 2 3 40.00

0.02

0.04

0.06

NLGAHLGA

Glutamine [mM]

Sp

ecif

ic A

ctiv

ity

(µ

mo

l/m

g/m

l)

Figure 4.35. Comparison of the effect of glutamine concentration on the specific activity of

Nguni (NLGA) and Hereford (HLGA) liver glutaminase at 37oC. These assays were run in

excess substrate.


109

Table 4.17. Comparison of Vmax and Km of Nguni liver glutaminase with respect to substrate

is calculated with the use of Graph-Pad™ prism 5.02.

Non-linear fit NLGA1 NLGA2 NLGA3 NLGA4 Average STDEV

[glutamine]

Michaelis-

Menten

Best-fit values

Vmax 0.05 0.07 0.06 0.07 0.052 0.00306

Km 1.022 1.699 1.804 1.605 1.532 0.08337

Allosteric

sigmoidal

Best-fit values

Vmax 0.02974 0.03602 0.03281 0.03593 0.033625 0.0025

The apparent Km values of glutamine were demonstrated while the concentration of NAD and

GDH were kept constant at 266 mM NAD and 1.9 mM GDH for both breeds

Table 4.18. Comparison of Vmax and Km of Hereford liver glutaminase with respect to

glutamine is calculated with the use of Graph-Pad™ prism 5.02.

Non-linear

fit HLGA1 HLGA2 HLGA3 HLGA4 Average STDEV

[glutamine] Michaelis-Menten

Best-fit values

Vmax 0.022 0.032 0.031 0.021 0.026 0.08861

Km 5.35 3.39 5.75 2.25 4.185 1.65125

Allosteric

sigmoidal

Best-fit values

Vmax 0.02557 0.02645 0.02371 0.0245 0.025058 0.00101


110

4.5.2. Estimation of glutaminase molecular weight on SDS-PAGE

The molecular weight on SDS-PAGE was marked at around 60 - 70 kDa for both Nguni and

Hereford liver glutaminase with the use of Fermentas molecular weight ruler and the

glutaminase reported by Perera et al., 1990 and another glutaminase reported by Kvamme et

al., 2001. This report is inconsistent with the bovine glutaminase reported in the

UniprotKB/Swiss-prot protein database. By SDS-PAGE, tissues from rat cortex and brain

stem were also found to have two bands with molecular weights of 65 and 68 kDa

respectively (Laake et al., 1999).


111

KDa M 1 2

A

KDa M 3 4

B

Figure 4.36. SDS-PAGE (10%) analysis of partially purified liver glutaminase. M:

Molecular weight standard, size in kilo-dalton is shown on the left. A & B: Partially purified

Nguni liver glutaminase indicated as 1: NLGA1 2: NLGA2 3: NLGA3 4: NLGA4.

~66kD

a

150

50

70

40

~66kD

a

150

50

70

40

~58kD

a

~58kD

a


112

~66kD

a ~58kD

a

KDa M 1 2

A

KDa M 3 4

B

Figure 4.37. SDS-PAGE (10%) analysis of partially purified liver glutaminase. M:

Molecular weight standard, size in kilo-dalton is shown on the left. A & B: Partially purified

Hereford liver glutaminase indicated as 1: HLGA1 2: HLGA2 3: HLGA3 4: HLGA4.

~66kD

a

150

50

70

40

150

50

70

40

~58kD

a

Chapter 5: Integrated Discussion and Conclusion

113

CHAPTER 5: INTERGRATED DISCUSSION AND CONCLUSION

The enzymes arginase, carbamoyl phosphate synthetase, glutamate dehydrogenase and

glutaminase, were purified from both Nguni and Hereford livers by chromatographic

separations using sephadex G-25, DEAE-sephacel and affinity columns. In some cases only

partial purification was achieved as demonstrated by SDS-PAGE homogeneity. Comparative

protein extractions were obtained. The complexities of the glycosilation of the proteins did

not allow the purity of the extracts to be assessed is in some cases. This did not however

detract from the comparison of the enzyme activities obtained for each breed. Another

compounding factor in the isolation of enzymes from liver tissue which leads to the partial

hydrolysis of enzymes by the high level of proteolytic enzymes found in the cell free extract.

Sephadex G-25 was employed for desalting and separation of small molecules from the

enzyme preparation mixture. Low molecular weight impurities, such as ammonium sulphate,

do permeate the pore volume, with a retention volume equal to the total liquid volume of the

column, because the size of the salt molecules are smaller than the pore size sephadex G-25.

Sephadex G-25 was chosen against a range of other desalting media because of its ability to

hold 25 – 30% relative sample volume and to desalt 98% salt from the sample preparation.

Sephadex G-25 was also found to have an excellent batch-to-batch performance

reproducibility.

DEAE sephacel was chosen as an anionic exchanger column for the enzyme chromatographic

separations, mainly because of several specifications which allowed for the separation of

arginase, carbamoyl phosphate synthetase, glutamate dehydrogenase, glutamine synthetase

and glutaminase from the extracted liver sample mixtures. Some specifications include the

fact that DEAE sephacel is a weak anionic exchanger with a total ionic capacity of 0.13 -

0.17 mmol/ml gel and an available capacity to hold molecules from as low as 14,3 kDa to

669 kDa. In addition, DEAE sephacel was also the column of choice because of long-term

pH stability and a wide working range of pH (from 3 – 9). All enzymes of interest in this

study ranged between 35 kDa and 170 kDa, which was well within the reliable separation

capacity of DEAE sephacel gel.


114

With the exception of a guanosine triphosphate sepharose gel used to separate glutamate

dehydrogenase, adenosine monophosphate (AMP) agarose was used to separate glutamine

synthetase and carbamoyl phosphate synthetase from the extracted liver enzyme mixtures.

The other affinity chromatographic gels were prepared and activated before use. For arginase,

an arginine affinity column was employed.

5.1. Arginase

Arginase was partially purified from both Nguni and Hereford liver by chromatographic

separations using sephadex G-25, DEAE-sephacel and arginine affinity columns. The native

molecular weight of this enzyme as reflected on SDS-PAGE, showed double bands of around

38 kDa and 52 kDa for both Nguni and Hereford liver arginase. Double bands were apparent

in Figure 4.9, Page 73, and were probably a result of the high level of glycosylation of the

enzyme that occurs in mammalian enzymes. As such, this could only be validated by

deglycosylation procedures and subsequent analysis with LC-MS/MS for band identification,

which was beyond the scope of this study. While arginase was therefore not further

deglycosylated and identified with LC-MS/MS, it was apparent that both breeds indicated

similar double bands in the SDS-PAGE (Figure 4.9, Page 73). Also evident is that the same

order of magnitude of protein was extracted at each stage of the extractions (Table 4.1, Page

62).

Arginase enzymes extracted from both Nguni and Hereford liver samples mixtures were

found to be stable at 60oC following heat treatment of the crude extract, to enhance the

coagulation of thermolabile proteins, which were subsequently removed by centrifugation.

This thermal stability of arginase enzymes has also been reported in studies by other workers,

such as that of rabbit liver (George & Harold, 1973) cotyledon seed and buffalo liver (Dabir

et al., 2005). Ammonium sulphate precipitation (70% saturation) resulted in a further

increase in the specific activity of liver arginase (Table 4.1, Page 62).

As is the case with metal requiring enzymes, arginase needs the presence of manganese to act

as a cofactor, (Angels & Legal, 1984, Helga & Greenberg, 1968). However the amount

required for optimal activity of the enzyme varies. The concentration of MnCl2 used for

optimal activitywas found to be 2 – 2.5 mM (Dabir et al., 2005), the experiment to identify


115

the divalent metal ion of choice found Mn2+

, Mg2+

, Ca2+

, Co2+

, Ni2+

and Cd2+

could all acted

as cofactors/ activators of bovine arginase (Figure 4.8, Page 72). The enzymatic activity of

arginase assayed from a number of sources has shown to be manganese-dependent (Dabir et

al., 2005).

Mn2+

was found to be the most favourable divalent cation for both the Nguni liver and.

Hereford liver arginase enzymes (Figure 4.8, Page 72).

A direct non-linear relationship was found between the products formed, in relation to the

incubation time over a 20 minute time period in the presence of excess substrate. The

maximum activity of Nguni liver arginase was observed to be at 25 minutes, while activity of

Hereford liver arginase was seen to peak at 20 minutes (Figure 4.1, Page 65) within the total

of 30 minutes allowed for the assay. The common linear period (5 - 20 minutes), of the

activity curve, was used to ensure comparative activities between arginases of the two breeds.

Arginases from both breeds indicated a decrease in activity at 30 minutes. This phenomenon

may suggest the depletion of substrate, product inhibition or enzyme inactivation.

To determine the Km and Vmax values of arginase for arginine from both Nguni and Hereford

liver extracts, arginine was used in the concentration range of 0 to 36mM (Figure 4.4, 4.5 &

4.7, Page 71). The incubation was performed under standard conditions accepted for

determination of Km which is at 37°C and pH 9.5.

The calculated Km for the substrate arginine was significantly different in each breed. For

Nguni arginase, the Km for arginine was 34.19mM and very high as compared to 1.31mM for

Hereford breed. The high Km indicated in-turn the low affinity for the substrate in the case of

the Nguni enzyme. Although the Vmax for the enzymes was essentially the same, namely

16.05 and 15.40, for the nguni and Hereford breeds respectively.

The Km values for Nguni arginase for Mn2+

ranging from 4.146 – 19.260 with one enzyme

being significantly highr than the others. Taking this into account the Km values for Mn2+

for

both breeds were similar (Table 4.2 - 4.3, Page 63 - 64). There were no substantial

differences in Vmax between Nguni & Hereford arginases, in the presence of Mn2+

(Table 4.2

- 4.3, Page 63 - 6).

From the typical extraction profile of the arginase from each breed similar enzyme

concentrations and activities were obtained (Table 4.1, Page 62).


116

The apparent Michaelis-Menten constants reported for mammalian liver arginases for

arginine are in the range of 6 – 20 mM, which are similar to that reported for yeast arginase

(Hirsch-Kolb et al., 1970). However, in this particular study, the apparent Km for the Nguni

breed exceeded the 20 mM mark for arginine substrate. This is probably significant in

understanging the urea cycling in this breed (see Conclusion). Analysis of literature data

shows that the Km value for arginase varies within rather wide limits for different animals,

ranging from 1.4 mM in rabbit liver, to 200 mM arginine in reptile liver (Cederbaum et al.,

2004; Reyero and Dorner, 1975). The mammalian Km range appears to be from 10 to 20 mM

arginine.

Although arginase is traditionally considered in terms of its biochemical role in the urea

cycle, there is a growing body of evidence that indicates that arginase may play additional

roles in mammalian metabolism (Cox et al., 1999, Mori & Gotoh, 2000). Additional

functions also include biosynthesis of ornithine, which is a precursor of polyamines and

amino acids, such as proline and glutamate (Wu & Morris, 1998). Also, absence of arginase,

or lack of activity of arginase in the bovine system, can be attributed to insufficient regulation

of arginine itself, which can lead to the toxic production of nitrogen oxide. The abundance of

arginase in the liver can therefore be considered an advantage to the animal, since arginases

have also been hypothesized to be involved in cell proliferation, differentiation and wound

healing (Mori & Gotoh, 2000).

5.2. Carbamoyl phosphate synthetase (CPS)

The CPS from Nguni and Hereford breeds was partially purified using Sephadex G-25,

DEAE-sephacel and AMP Agarose affinity columns (Table 4.4, Page 74).

As other workers have shown glycerol could act, either as a cryoprotectant, or by activating

the enzyme (Rubio et. al., 1983) 4% v/v glycerol was added to purification buffer, for

stability of CPS. DMSO (15%) was added as a cryoprotectant. In general, the method of

Pierson & Brien (1980) was used for the purification of CPS and also contained dithiothreitol

as a stabiliser.


117

Samples assayed for final kinetics were obtained from AMP agarose column with the active

eluants being collected and subsequently combined into one fraction. The AMP agarose did

give as high affinity purification as expected however, the enzyme concentration data as well

as the SDS-PAGE allowed the enzyme activities to be compared (Table 4.4, Figure 4.16).

As decreasing concentrations of protein were eluted from AMP agarose column

chromatography, faint bands were marked with a molecular weight ruler and later identified

with LC-MS/MS quality control methodology.

The effect of magnesium-ATP was determined by measuring the kinetics of both Nguni and

Hereford liver CPS for comparative purposes. The kinetic properties determined for the

partially purified liver CPS indicated different Km values for Mg-ATP between the two

breeds. For Nguni CPS, the Km for MgATP was 0.60 mM and while that for the Hereford was

4.02 mM for Hereford breed. The high Km of Hereford liver samples indicated in-turn the low

affinity for the substrate when compared with the Nguni CPS.which was also seen in the

specific activity of Hereford breed (Table 4.4, Page 74). The Vmax for Mg-ATP for the Nguni

breed CPS was found to be 0.49 while that for the Hereford breed was found to be 1.37. The

affininty for ammonium was low for Nguni breed as indicated by higher Km and Vmax.

The two curves of CPS saturation with Mg-ATP as presented in Figure 4.10 - 4.11, 4.14

(Pages 75 -76, 79) indicated a clear difference in optimum concentrations of Mg-ATP

required for each breed.

The steady state kinetics for Mg-ATP was hyperbolic and yielded a Michaelis-Menton Vmax

between 0.4541 – 0.5575 (Average = 0.4946) for the Nguni CPS and 0.744 – 1.547 (Average

= 1.379) for the Hereford CPS which indicated a higher affinity of Nguni CPS for MgATP

when compared with Hereford breed CPS (Table 4.5 – 4.6, Page 81 - 82). . The maximum

rate of reaction of the Nguni liver CPS was lower than that for the Hereford liver CPS,

The analysis of the data using the GraphPad Prism software gave a Km value for Mg-ATP

which ranged from 0.43 mM – 0.91 mM for the four Nguni livers tested, giving an average

of 0.60 (Table 4.5, Page 81; Figure 4.14, Page 79). A maximal (Vmax) rate for the reaction

was between 0.45 – 0.56 nmol of ADP/mg of protein/min, with an average of 0.50 nmol of

ADP/mg of protein/min (Table 4.7, Page 85). Similarly analysis of this data using GraphPad

Prism software gave a Km value for Mg-ATP which ranged from 0.91 mM – 8.58 mM for the

four Hereford liver extracts, with an average of 4.022 (Table 4.6, Page 82; Figure 4.14,


118

Page 79). A maximal (Vmax) rate for the reaction was between 0.75 – 1.547 nmol of ADP/mg

of protein/min, with an average of 1.379 nmol ADP/mg of protein/min (Table 4.6, Page 82).

The difference between Vmax of CPS between the two breeds was 0.88 nmol of ADP/mg of

protein/min. As such, with a P<0.0001 between samples of CPS from each breed, no

significant differences were apparent.

Other workers have reported different Km values for CPS some as high as 3 mM for Mg-ATP,

when studying rat liver enzyme (Kerson & Appel, 1968).

The effect of ammonium chloride on the activity of bovine CPS in order to compare the

kinetics of the reaction between Nguni and Hereford liver extracts is shown in Figures 4.12

and 4.13 respectively, (Pages 77 – 78). The steady state kinetics for ammonium chloride was

hyperbolic (Figure 4.12 – 4.13, Page 77 - 78) and yielded a Michaelis-Menton Km of

between 27.82 – 36.6 mM (Average = 33.115) for the Nguni liver CPS, and 15.68 – 27.54

mM (21.292) for the Hereford liver CPS (Table 4.5 and Table 4.6, Page 82). This data thus

suggests that the CPS liver extracts from the two breeds similar affinities for ammonium

chloride.

In other studies of bovine liver, rat liver and frog liver CPS kinetics, the Km values were as

low as 1 - 2 mM for ammonium chloride (Kerson & Appel, 1968). The apparent Michaelis-

Menten constants reported for mammalian liver CPSs are in the range of 10mM (Kerson &

Appel, 1968) but in this case the two breeds investigated in this study the apparent Km

exceeded the 10mM level.

The identification of Nguni and Hereford CPS was achieved with the aid of LC-MS/MS.

Peptides identified by SDS-PAGE by molecular mass (Figure 4.16, Page 83) were then

eluted and hydrolysed for LC-MS/MS analysis (Figure 4.17, Page 84). The enzymes were

found to be CPS in both cases (Tables 4.7 & 4.8, Page 85). For quality control for the

protein blast indicated that the sequence had 99% confidence level for Bos Taurus

(Appendix 4, Page 167).

5.3. Glutamine synthetase (GS)


119

The method described earlier for the isolation of glutamine synthetase (GS) from bovine

liver, has been used repeatedly by a number of investigators. Yields varied from 75% to 77%,

with the crude enzyme mixture being stable for at least a month when stored between -70oC

and -80oC (Tagaki et al., 2008).

HEPES buffer was effectively used throughout purification of GS, with the inclusion of

higher concentrations of sodium chloride and lower concentrations of magnesium chloride,

all of which proved to be the „key‟ to achieving stability and activity of GS extractions

throughout the study. To this end GS was partially purified from both Nguni and Hereford

livers by a two-step chromatographic separation protocol using Sephadex G-25 and AMP

Agarose affinity columns. A size exclusion Sephadex G-25 column was used to desalt and

concentrate the enzyme extracts. The process of desalting the ammonium sulphate from the

liver sample extract after precipitation greatly diminished GS activity.

The effect of Mg-ATP on the activity of GS was determined by measuring the kinetics of

both Nguni and Hereford liver GS extracts. The curve of GS saturation with Mg-ATP is

presented in Figure 4.19 and 4.20 (Pages 88 - 89), indicating a similarity in optimum

concentrations of Mg-ATP for each breed. Very early on in the reaction, Nguni liver GS

extracts indicated higher activity when compared to Hereford liver GS activity, which only

occurred at increased concentration of Mg-ATP around 2 mM Mg-ATP. This phenomenon

may represent the higher requirement of Mg-ATP for the Hereford GS for Mg-ATP,

compared to the Nguni GS, which was active at a lower concentration of Mg-ATP.

The steady state kinetics for Mg-ATP was hyperbolic and yielded a Michaelis-Menten Km of

between 1.048 – 5.635 mM for the Nguni liver GS extracts and 1.2 – 6.3 mM for the

Hereford GS liver extracts (Table 4.10, Page 87). The average Km of the four Nguni liver GS

extracts was lower than that for the four Hereford livers GS extracts according to Michaelis-

Menten best fit algorithms. The difference between the two Km values was almost 50%, and

may contribute to differences in physiological behaviour seen in one breed compared to the

other.

In other studies of GS activity the Km values for Mg-ATP were 2.0 mM and 2.9 mM for the

HPLC and spectrophotometric assays, respectively (Listrom et al., 1997). These values do

not deviate far from the findings of this study especially, that of the Nguni liver GS, which

had an average Km of 2.46 mM Mg-ATP (Table 4.10, Page 87).


120

In most of the studies by other workers, GS was considered only in terms of its biochemical

role in the urea cycle (Norenberg, 1979). However, there is now a growing body of evidence

that indicates that GS may play additional roles in mammalian metabolism, especially in the

removal of toxic NH4+from body fluids. In this regard, the abundance of GS in Nguni livers

can be an advantage over the Hereford breed, since GS catalyses the ligation of glutamate and

ammonia to form glutamine, with concomitant hydrolysis of ATP. The activity of bovine

liver GS may eliminate much of cytotoxic ammonia utilising lower levels of Mg-ATP, at the

same time converting neurotoxic glutamate to harmless glutamine (Krajewski et al., 2008).

The native molecular weight of GS enzyme was estimated and marked on SDS-PAGE by a

protein molecular weight Page ruler (Fermentas), indicating double bands of molecular

weight between ~42kDa and ~58kDa for Nguni liver GS, and only ~58kDa for Hereford liver

GS (Figure 4.22A & Figure 4.22B, Page 91). Double bands in Figure 4.22A may indicate

glycosylation of the Nguni liver GS, whereas Hereford liver GS did not show any indication

of being glycosylated.

5.4. Glutamate dehydrogenase (GDH)

The entire purification of glutamate dehydrogenase (GDH) was accomplished in two and a

half days, as compared to the several weeks‟ purification of Fahien et al., 1969. GDH was

purified from both Nguni and Hereford livers by chromatographic separation involving

Sephadex G-25, DEAE-Sephacel and GTP Sepharose gel columns. Purification of Nguni and

Hereford GDH is outlined in (Table 4.11, Page 92).

The kinetic studies of the reductive amination of α-ketoglutarate, which was catalysed by

GDH with NADPH as coenzyme and ammonium ions, were performed at pH 8.3 (Figures

4.23, 4.24 and 4.27, Pages 93 – 94, 97). When concentrations of both ammonium ions and α-

ketoglutarate were varied separately over a wide range, it was possible to determine the effect

of increased concentrations to specific activity of GDH. It was clear that the activity of GDH

was dependant on the concentration of both substrates (Figures 4.23, 4.24 and 4.27, Pages 93

– 94, 97).

The steady kinetics for α-ketoglutarate was hyperbolic and yielded a Michaelis-Menten Km of

between 1.522 – 13.99 mM for the Nguni liver GDH activity and 2.41 – 10.53mM for the


121

Hereford liver GDH activity (Table 4.12 and 4.13, Page 99). The Km value reported in other

studies for bovine GDH, was as low as 0.5 mM for α-ketoglutarate, and as low as 0.08 mM

for NH4+

(Engel & Dalziel, 1970). The Vmax in the presence of α-ketoglutarate for the Nguni

liver GDH was found almost identical for the two breeds as determined by Michaelis-Menten

non-linear fits. The Vmax being between 2.7 – 9.2 nmol of NAD+/mg/min for the Nguni

enzyme and 2.54 – 6.03 nmol of NAD+/mg/min (Figure 4.25 and 4.26, Pages 95 – 96; Table

4.12 and 4.13, Page 99).

The affinity for ammonium was low for Nguni breed as indicated by higher Km and Vmax.

The steady kinetics for (NH4)2SO4 was hyperbolic and yielded a Michaelis-Menten Km of

between 4.94 – 15.52 mM for the Nguni liver GDH activity and 2.33 – 17.26 mM for the

Hereford liver GDH activity, while a Vmax of between 2.73 – 18.35 mM for the Nguni liver

GDH activity and 2.01 – 5.67 mM for the Hereford liver GDH activity was obtained (Table

4.12 and 4.13, Page 99).

Both the Km values of Nguni liver GDH and Hereford liver GDH, with respect to α-

ketoglutarate, were found similar with the reported Km for human and rabbit liver GDH (6.3

mM) (Engel & Dalziel, 1970).

The native molecular weight of liver extract GDH estimated on SDS-PAGE showed a single

band in the region of ~58 kDa for both breeds (Figure 4.29 - 4.30, Pages 100 - 101) as

marked by a protein molecular weight ruler (Fermentas).

The characterization Nguni liver GDH was achieved with the aid of LC-MS/MS

chromatography. Peptides were identified (Figure 4.31 – 4.32, Page 102 - 103) by QSTAR

Elite ESI as gel-based ID as marked by a protein molecular weight page ruler in the SDS-

PAGE (Figure 4.29 – 4.30, Page 100 - 101. The NCBI protein blast (Appendix 5, Page 169)

indicated that the sequence had 99% confidence level for Bos taurus GDH.

This provided further confirmation for the existence of bovine GDH in liver sample extracts

from both breeds of animal (Table 4.14 – 4.15, Page 103 - 104).


122

5.5. Glutaminase

Glutaminase was purified in potassium phosphate buffer. Glutaminase was partially purified

from Nguni and Hereford livers via chromatographic separations, including Sephadex G-25

column chromatography, DEAE-Sephacel column chromatography and also using

ultrafiltration. A Sephadex G-25 column was used to desalt and concentrate the enzyme in

preparation for anionic binding with the DEAE sephacel chromatographic column.

Glutaminase has a strong anionic binding for DEAE sephacel according to Patel & McGivan

(1984). From the purification data, partial purified Nguni and Hereford liver glutaminase was

concentrated by ultrafiltration (Table 4.16, Page 104).

Varying degrees of success for the purification of hepatic glutaminase, has been reported by a

number of workers since the early to middle 1980‟s, with the study by Smith & Watford

(1988) being the protocol of choice used here.

The calculated Km for glutamine showed a significant difference. For Nguni glutaminase, the

Km for glutamine was 1.022 – 1.804 mM compared to 3.39 – 5.34 mM for Hereford breed.

The high Km of Hereford liver samples had indicated the low affinity for glutamine.

If Km is lower for a substrate, it maybe concluded that the affinity for such substrate for

optimal activity is high. The Km value of Nguni liver glutaminase, with respect to glutamine,

was also found to be lower than that for Hereford liver glutamine (Table 4.17 – 4.18, Page

109). The Vmax obtained for the Nguni enzyme was 0.05 – 0.07 while that for the Hereford

enzyme was 0. 022 – 0.032. The functioning of the glutaminase for the Nguni enzyme was

therefore significantly higher both when assessed by the higher affinity of the enzyme as well

as the higher Vmax. This is important as this would mean that the Nguni enzyme would

produce more NH3 which could then be used for the synthesis of carbamoyl phosphate and

subsequently urea.

The partially purified glutaminase estimated on SDS-PAGE (Figure 4.36 - 4.37, Page 111 -

112) showed a multiple bands of molecular weight between 35 kDa - 150 kDa for both Nguni

and Hereford liver glutaminase. The LC-MS/MS was unable to identify the protein of

interest. This may be due to high levels of glycosylation of the enzyme concerned.


123

The Km values of the Nguni liver glutaminase, with respect to the substrate glutamine, were

also similar when compared with that isolated from mammalian brain cells (0.8 mM

glutamine) (Nimmo & Tipton, 1981), E. coli (0.45 mM glutamine) (Hartman, 1968). Rat

kidney glutaminase (3 – 4 mM glutamine) (Kenny et al., 2003), was slightly higher than that

found in this study.

Within the range of 0 to 4.2 mM glutamine (Willemoes & Sigurskjold, 2002) concentrations

and the standard conditions accepted for determination of Km (37°C and pH 7.4), it was

possible to determine Km and Vmax values of glutaminase from Nguni and Hereford livers.

The Km values of the Nguni liver glutaminase, with respect to the substrate glutamine, were

considerably lower than the reported Km of the mammalian liver glutaminase, as reported at 6

mM glutamine (McGivan et al., 1980) and 28 mM glutamine, which was the highest

concentration reported (Huang & Knox, 1976). However the values of Hereford liver

glutaminase can be closely associated with the Km values of McGivan et al., (1980) and also

that of Huang & Knox, (1976). The Km of Nguni liver glutaminase reported in this study is

closely related to that of Lactoccocus lactis , considering the similar assaying conditions with

a coupled assay system, where NAD is reduced to NADH (1.10 mM glutamine) (Willemoes

& Sigurskjold, 2002).

Although the difference in Vmax between the two breeds for glutamine was 0.109 mmol of

NADH/mg of protein/min (Table 4.17 – 4.18, Page 109), it is imperative to note that the

behaviour of glutaminase from the two breeds could also have been similar. This is

confirmed by the non-significance of the differences in the slopes of the combined data from

both breeds.

In general, both breeds had acceptable Km values with respect to glutamine concentrations.

Results from this study are not directly comparable with data from other workers, who

reported that the behaviour of the enzyme obeys different kinetics depending on assay

conditions. In particular, soluble glutaminase from pig brain was found to follow sigmoid

kinetics according to Svenneby (1971), but not according to Kvamme & Torgner (1974).

5.6. General Conclusion

The mechanism of ammonia detoxification to form urea in mammals is outlined in Figure

5.1 and the urea cycle (Figure 5.2). The ammonia (NH4+) released by the reverse reaction of


124

glutamate dehydrogenase (GDH) is converted to carbamoyl phosphate by carbamoyl

phosphate synthase I (CPS), in the mitrochondria. Another mitrochondrial enzyme, ornithine

transcarbamoylase (OTC), then converts the carbamyl phosphate to citrulline in the presence

of L-ornithine. The citrulline synthesized in the mitrochondria is converted to arginine in the

cytosol by argininosuccinate (ASA) synthase and argininosuccinate (ASA) lyase. Arginine is

then converted to urea by arginase, regenerating ornithine and completing the urea cycle.

Glutamine synthetase (GS) in the liver also plays a role in ammonia detoxification.

Glutamine is a major blood amino acid, and may also be transported to the liver where the

amide-N is converted to urea. The amide-N of glutamine is released as ammonia,

intramitrochondrially, by the enzyme glutaminase. This ammonia may also be converted to

citrulline.

The overall regulation of these enzymes is therefore crucial in the understanding of what

separates the Nguni breed urea cycle from other less "hardy" breeds. The elevated urea levels

in the Nguni blood sera, possibly arises from increased conversion of glutamine to glutamate

in the liver by glutaminase. It is possibly that the Nguni are capable of creating glutamine

stores in the muscle as occurs in sheep.


125

Glutamate a-Ketoglutarate

NAD NADH

GDH

NH4+ + HCO3

-

Carbamylphosphate

Citrulline

Ornithine

Citrulline

Aspartate

Argininosuccinate

Arginine

Ornithine Ornithine

UREA

AMP ATP

ASA synthase

ASA lyase

Arginase

2 ATP

2 AMPCPS-

OTC

Glutamate

Transamination

Glutamine

GSGlutaminase

CytosolMytochondria

Figure 5.1. Detoxification of ammonia released via transdeamination by its conversion to urea


126

Figure 5.2. The urea cycle showing the two mitochondrial and 3 cytosolic reactions

The Nguni arginase has a lower affinity for the substrate than the Hereford enzyme (Figure

5.1). This would lead to an accumulation of arginine under equivalent conditions in the Nguni

liver, however, the Nguni CPS has a higher affinity for ATP than the Hereford enzyme and

would therefore utilise the arginine more efficiently than the Hereford enzyme creating the

necessary “pull” on the transfer of the arginine into the mitochondria of the liver. As the

arginine transport is dependent on the export of the L-citruline increase levels of citruline

would also drive the formation of arginosuccinate thereby increasing the drive for the

formation of urea. The higher affinity of the Nguni glutaminase as well as the higher Vmax

would also mean that the Nguni enzyme would produce more NH3 which could then be used

for the synthesis of carbomoyl phosphate and subsequently urea.

Chapter 6: Summary and Recommendation

127

CHAPTER 6: SUMMARY AND RECOMMENDATIONS

6.1. Summary

Protein regeneration cycle is of great significance to the survival of ruminants under

unfavourable environmental and nutritional conditions. The Nguni breed of cattle is able to

obtain optimal nutritional value from the natural vegetation available, thus enabling it to

survive under conditions that most grazers such as the European cattle breeds would find

extremely testing. This observation alone proves that the Nguni breed is an animal in

harmony with its environment despite the condition. Since animals consuming low N-diet

have been reported to have increased urea reabsorption, the Nguni can then be categorised

into the class of N-recycling ruminants.

The activity of enzymes of bovine nitrogen metabolism is hypothesized to contribute to either

the deterioration or survival of the cattle in harsh environmental conditions. Since enzymes

responsible for certain functions in bovine metabolism are known, it is important to identify

those specific enzymes by purification and characterization in bovine liver and kidney to be

able to determine the effect of such enzymes.

Partial purification and characterization of five selected enzymes of bovine nitrogen

metabolism was attempted. Crude enzymes from both Nguni and Hereford livers were

separated from most or some contaminants by sephadex G-25, DEAE sephacel, and different

affinity column chromatography depending on the size, binding, ionic and affinity strength.

Carbamoyl phosphate synthetase and GDH were successfully purified and characterized by

LC-MS/MS and the results were blasted and matched >95% with those of Bos Taurus in

NCBI protein database (Appendix 5, Page 169). Comparison of those fully characterized

enzymes was done using kinetics calculated and presented clearly in graphs. Although

partially purified and compared utilizing GraphPad™ Prism software by the use of kinetics

and graphs, enzymes such as arginase, glutamine synthetase and glutaminase failed to ionise

by LC-MS/MS during characterization.

Partial purification and characterization of selected enzymes of bovine nitrogen metabolism

was in part achieved.


128

6.2. Recommendations

For the purification of arginase, CPS, GS, GDH and glutaminase from bovine liver, more

sophisticated equipments and conditioned work environment was required since animal

tissues and cells are delicate.

The requirement of Nguni and Hereford liver arginase for higher ammonium chloride

concentration can only be explained by a further study since such trend was not completely

clear in this study. The phenomenon whereby the requirement of substrate is higher in

Hereford than in Nguni is seen in all enzymes purified in this study in varied percentages for

each individual enzyme. It is a trend which requires further investigation in a separate study.

If the Km of a substrate for an enzyme is too high, the enzyme is less likely to be active in

lower substrate concentrations.

Although partial purification was achieved for the Nguni liver and the Hereford liver by

bench-top chromatographic separations, complete purification is important to fully

characterize enzymes. There is still a need to fully and cleanly purify these enzymes to avoid

the failure of ionization of some of these enzymes by LC-MS/MS. There is also a need to

deglycosylate enzymes such as GS for the full characterization thereof. LC-MS/MS picked

human CPS for Hereford and also picked rat CPS for Nguni liver samples, analysis by

ProteinPilot™ and protein peptides blasting (Appendix 4, Page 167) indicated that the

sequence had 99% confidence Bos Taurus which was confirmatory of the presence of bovine

CPS in samples of both breeds. It is then recommended that characterization be done in fully

purified CPS to avoid confusion with contaminating bands.

GS was not characterized due to failure of ionization in LC-MS/MS. It is with this reason that

it is recommended that characterizing GS be given a sole focus in order to be able to

deglycosylate the enzyme which could not be done in this study due to scope and time

constraints. The only kinetics studied for the part of GS was that of the effect of Mg-ATP

concentration on the activity of GS. It is recommended that the effect of glutamate and NH4

be studied in order to fully characterize and compare the complete behaviour of GS from

Nguni liver with that of the Hereford liver.


129

Similarly to CPS, LC-MS/MS picked human GDH for Hereford and rat GDH for Nguni

samples. Protein peptides blast indicated that the sequence had 99% confidence Bos Taurus

which was confirmatory to prove the presence of bovine GDH in samples for both breeds in

addition to the calculated specific activity.

Estimates of the distribution of both Nguni and Hereford liver glutaminase were made

completely based on the calculated specific activity and kinetics although these were far from

satisfactory. LC-MS/MS identified the band of interest as catalase instead of glutaminase

while the calculated activity and the marking with Fermentas protein Page ruler identified the

two bands as glutaminase and GDH as a contaminant. A thorough study into the purification

and identification of hepatic glutaminase by carefully set-up chromatographic separations and

LC-MS/MS is recommended.

References

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Appendices

163

APPENDICES

Appendix 1: SDS-PAGE preparations, (Page 59)

Separating gel

This procedure was done in the fume-hood. The following tabulated reagents were added in

the tabulated sequence to make up a total volume of 15 ml 10% separating gel.

Reagents Volume (ml)

40% bis-acrylamide 3.75

1.5 M Tris-HCl, pH 8.8 3.75

Deionised water 7.418

10% SDS 0.15

10% ammonium sulphate 0.075

0.25% Tetramethylenediamine (TEMED) 0.0075

Stacking gel

This procedure was done in the fume-hood. The following tabulated reagents were added in

the tabulated sequence to make up a total volume of 5 ml 4% stacking gel.

Reagents Volume (ml)

40% bis-acrylamide 0.5

0.5 M Tris-HCl, pH 8.8 1.25

Deionised water 3.22

10% SDS 0.05

10% ammonium sulphate 0.025

0.25% Tetramethylenediamine (TEMED) 0.005

SDS-PAGE running buffer

The following reagents were weighed 3.74 mM SDS

25 mM Tris-HCl, pH 8.3

192.5 mM Glycine

Make up to final volume (1 L) with dH2O

Appendices

164

Preparation of SDS-PAGE stain

10% acetic acid, 50% methanol and 40% deionised water and 1% Sigma Brilliant Blue R'.

This procedure was done in the fume-hood. 1g of Sigma Brilliant Blue R' was dissolved in

500 ml of methanol followed by addition of 100 ml glacial acetic acid and then 400 ml of

deionised water. The total volume of stain was 1000 ml.

Preparation of SDS-PAGE de-stain

10% acetic acid, 10% methanol and 80% deionised water.

This procedure was done in the fume-hood. 100 ml of methanol was added followed by 100

ml of glacial acetic acid which was then followed by addition of deionised water to make up

a total volume of 1000 ml.

SDS-PAGE separation of bands

The purified enzyme was treated with 1% SDS and β-mercaptoethanol for 10 minutes at 90oC

and loaded in wells. Molecular weight standards used were bovine serum albumin (66,000),

Egg albumin (45,000), Glyceraldehydes triphosphate dehydrogenase (35,000), Carbonic

anhydrase (29,000) and β-lactoglobuline (18,400) (Fermentas). The gel was stained in 0.25%

w/v Coomassie brilliant blue (R-250) prepared in 50% v/v methanol and 10% v/v acetic acid.

The gels were destained by passive diffusion using a solution of 50% v/v methanol and 1%

acetic acid.

Appendices

165

Appendix 2: Invitrogen Quant-iT™ protein assay, Invitrogen cooperation, ©2007.

(Page 59)

Important: Ensure all assay reagents are at room temperature before you begin.

1. Set up your tubes: you will need two tubes for the standards (three for the protein

assay) and one tube for each of your samples.

2. Make the Quant-iT™ Working Solution by diluting the Quant-iT™ reagent 1:200 in

Quant-iT™ buffer. 200 uL of Working Solution are required for each sample and

standard.

3. Prepare Assay Tubes according to the table below.

Standard

Assay Tubes User Sample

Assay Tubes

Volume of Working Solution (from step 2) to add

190 μL

180-199 μL

Volume of Standard (from kit) to add

10 μL

-

Volume of User Sample to add

-

1–20 μL

Total Volume in each Assay Tube

200 μL

200 μL

Appendices

166

4. Vortex all tubes for 2–3 seconds.

5. Incubate the tubes for 2 minutes at room temperature (15 minutes for the Quant-iT™

protein assay).

6. Read tubes in Quant-iT™ fluorometer.

7. Multiply by the dilution factor (see Manual) to determine concentration of your

original sample. Alternatively, choose Calculate sample concentration to have the

Quant-iT™ fluorometer perform this multiplication for you.

Use only thin-wall, clear 0.5 mL PCR tubes. Acceptable tubes include Qubit™ assay

tubes. (500, Invitrogen) or Axygen PCR-05-C tubes (VWR).

Appendices

167

Appendix 3: Bio-analysis of extracted proteins with Liquid Chromatography Mass

Spectrometry (LC-MS/MS) (Page 59).

a. Tryptic digestion within cut-out bands from SDS-PAGE gels

Protein bands of interest were in-gel trypsin digested as per the protocol described in

(Shevchenko et al., 2007). In short, gel bands were destained using 50mM NH4HCO3/50%

MeOH followed by in-gel protein reduction (50mM DTT in 25 mM NH4HCO3) and

alkylation (55 mM iodoacetamide in 25 mM NH4HCO3). Proteins were digested over night at

37 °C using 5 – 50 µl, 10 ng/µl tryspin depending on the gel piece size. Peptides were

extracted using 50% acetonitrile (ACN)/5% formic acid (FA) and vacuum dried.

b. Liquid Chromatography Mass Spectrophotometer

Trypsin digested samples extracted from gel-plugs were re-suspended in 40 µl of 2%

ACN/0.2% FA and centrifuged at 20200g, 4 °C for 15 minutes. Samples were analysed on a

Dionex Ulitomate 3000 RSLC system coupled to a QSTAR®

Elite mass spectrometer.

Samples were de-salted on an Acclaim PepMap C18 trap (75 μm x 2 cm) for 8 min at 5

μl/min using 2 % ACN/0.2 % FA. Peptides were separated on Acclain PepMap C18 RSLC

column (75 μm x 15 cm, 2um particle size) connected to the trap column via 10-port

switching valve. Peptide elution was achieved using a flow-rate of 500 µl/min with a

gradient: 4-60 % B in 30 min (A: 0.1 % FA; B: 80% ACN/0.1 % FA). Nano-spray was

achieved using a MicroIonSpray head assembled with a New Objective, PicoTip emitter. An

electrospray voltage of 2.0 - 2.8 kV was applied to the emitter. The QSTAR®

Elite mass

spectrometer was operated in Information Dependant Acquisition (IDA) using an Exit Factor

of 2.0 and Maximum Accumulation Time of 2.0 sec. MS scans were acquired from m/z 400

to m/z 1500 and the three most intense ions were automatically fragmented in Q2 collision

cells using nitrogen as the collision gas. Collision energies were chosen automatically as

function of m/z and charge.

Appendices

168

Appendix 4: Carbamoyl-phosphate synthase (NCBI Blast: Generated report), (Page

59).

LOCUS NP_001179187 1500 aa linear MAM 24-OCT-

2011

DEFINITION carbamoyl-phosphate synthase[ammonia],mitochondrial

[Bos taurus].

ACCESSION NP_001179187 XP_587645

VERSION NP_001179187.1 GI:300795597

DBSOURCE REFSEQ: accession NM_001192258.1

KEYWORDS

SOURCE Bos taurus (cattle)

ORGANISM Bos taurus

Eukaryota; Metazoa; Chordata; Craniata; Vertebrata;

Euteleostomi;Mammalia; Eutheria; Laurasiatheria;

Cetartiodactyla; Ruminantia;Pecora; Bovidae; Bovinae;

Bos.

REFERENCE 1 (residues 1 to 1500)

AUTHORS Zimin,A.V.,

Delcher,A.L.,Florea,L.,Kelley,D.R.,Schatz,M.C.,

Puiu,D., Hanrahan,F., Pertea,G., Van Tassell,C.P.,

Sonstegard,T.S.,Marcais,G., Roberts,M.,

Subramanian,P., Yorke,J.A. and Salzberg,S.L.

TITLE A whole-genome assembly of the domestic cow, Bos

Taurus

JOURNAL Genome Biol. 10 (4), R42 (2009)

PUBMED 19393038

COMMENT PROVISIONAL REFSEQ: This record has not yet been

subject to final

NCBI review. The reference sequence was derived from

AAFC03081787.1, AAFC03081786.1, AAFC03081788.1,

AAFC03081789.1,AAFC03128505.1, AAFC03081790.1 and

AAFC03106155.1.On Jul 17, 2010 this sequence version

replaced gi:76659916.

Sequence Note: The RefSeq transcript and protein were

derived from genomic sequence to make the sequence

consistent with the reference genome assembly. The

genomic coordinates used for the transcript record

were based on alignments.

http://www.ncbi.nlm.nih.gov/nuccore/300795596

http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=9913

http://www.ncbi.nlm.nih.gov/pubmed/19393038

http://www.ncbi.nlm.nih.gov/RefSeq/

http://www.ncbi.nlm.nih.gov/protein/112066959








Appendices

169

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61 fghpssvage vvfntglagy pealtdpayk gqiltmanpi vgnggapdta aldelglsky

121 lesdgikvag llvlnysddy hhwlatkslg qwlreekvpa iygvdtrmlt kiirdkgtml

181 gkiefegqsv dfvdpnkqnl iaeistkdvk vygkgnpmkv vavdcgikns virllvkrga

241 evhvvpwnhd ftkmeydgll iaggpgnpal aqpliqnvkk ilesdrkepl fgistgnlit

301 glaagaqvyk msmanrgqnq pvlnitnrqa fitaqnhgya ldstlpagwk plfvnvndqt

361 negimheskp ffgvqfhpev gpgptdteyl fdsfvslikk ekgttitsvl pkpglvasrv

421 evskvlilgs gglsigqage fdysgsqavk amkeenvktv lmnpniasvq tnevglkqad

481 tvyflpitpq fvtevikaer pdglilgmgg qtalncgvel frrgvlkeyg vkvlgtsves

541 imatedrqlf sdklneinek iapsfavesi edalkaadni gypvmirsay algglgsglc

601 pnretlmdls tkafamtnqi lveksvtgwk eieyevvrda ndncvtvcnm envdamgvht

661 gdsvvvapaq tlsnaefqll rrvsinvvrh lgivgecniq falhptsmey ciievnarls

721 rssalaskat gyplafiaak ialgiplpei knvvsgktsa cfepsldymv tkiprwdldr

781 fhgtssrigs smksvgevma igrtfeesfq kalrmchpsi dgftsrlpmn kewpsdidlr

841 reladpssir iyaiakaled nmsldeimkl tsidkwflyk mrdilnmekt lkglnsesit

901 eetlkkakei gfsdkqiskc lglteaqtre lrlkknihpw vkqidtlaae ypsvtnylyv

961 tyngqehdin fddhgmmvlg cgpyhigssv efdwcavssi rtlrqlgkkt vvvncnpetv

1021 stdfdecdkl yfeelsleri ldiyhqeacg gciisvggqi pnnlavplyk ngvkimgtsp

1081 lqidraedrs ifsavldelk vaqapwkavn tlnealefak svgfpcllrp syvlsgsamn

1141 vvfsedemkk fleeatrvsq ehpvvltkfi egarevemda vgkdgrvish aisehvedag

1201 vhsgdatlml ptqtisqgai ekvkdatrki akafaisgpf nvqflvkgnd vlviecnlra

1261 srsfpfvskt lgidfidvat kvmigehide kplptlehpi ipadyvaika pmfswprlrd

1321 adpilrcema stgevacfge gihtaflkam lstgfklpqk giligiqqsf rpqflgvaeq

1381 lhnegfklfa teatsdwlna nnvpatpvaw psqegqnpsl spirklirdg sidlvinlpn

1441 nntkfvhdny virrtavdsg ialltnfqvt klfaeavkka rnvdskslfh yrqyragkel

Appendices

170

Appendix 5: Glutamate dehydrogenase (NCBI Blast: Generated report), (Page 59).

LOCUS NP_872593 558 aa linear MAM 20-NOV-

2011

DEFINITION glutamate dehydrogenase 1, mitochondrial precursor [Bos

taurus].

ACCESSION NP_872593

VERSION NP_872593.1 GI:32880221

DBSOURCE REFSEQ: accession NM_182652.1

KEYWORDS .

SOURCE Bos taurus (cattle)

ORGANISM Bos taurus

Eukaryota; Metazoa; Chordata; Craniata; Vertebrata;

Euteleostomi;

Mammalia; Eutheria; Laurasiatheria; Cetartiodactyla;

Ruminantia;

Pecora; Bovidae; Bovinae; Bos.


AUTHORS Cai,L., Xie,Y., Li,L., Li,H., Yang,X. and Liu,S.

TITLE Electrochemical and spectral study on the effects of

Al(III) and

nano-Al13 species on glutamate dehydrogenase activity

JOURNAL Colloids Surf B Biointerfaces 81 (1), 123-129 (2010)

PUBMED 20675102

REMARK GeneRIF: Data show that glutamate dehydrogenase activity

in an

enzymatic catalyzed reaction by tracing the increasing

of oxidation

current of NADH.


AUTHORS Wacker,S.A., Bradley,M.J., Marion,J. and Bell,E.

TITLE Ligand-induced changes in the conformational stability

and

flexibility of glutamate dehydrogenase and their role in

catalysis

and regulation

JOURNAL Protein Sci. 19 (10), 1820-1829 (2010)

PUBMED 20665690

REMARK GeneRIF: Data show that heat inactivation of the native

GDH enzyme

occurred with a rate constant of inactivation of 0.252

min-1.

http://www.ncbi.nlm.nih.gov/nuccore/32880220

http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=9913



Appendices

171

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241 astighydin ahacvtgkpi sqggihgris atgrgvfhgi enfineasym

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partial purification and characterization of selected enzymes of bovine nitrogen metabolism

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