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69-10,599

GOMES, Benedict, 1933-BEEF LIVER MITOCHONDRIAL AMINE OXIDASE;PURIFICATION AND STUDIES ON SOME PHYSICALAND CHEMICAL PROPERTIES.

University of Hawaii, Ph.D., 1968Biochemistry

University Microfilms, Inc., Ann Arbor, Michigan

BEEF LIVER MITOCHONDRIAL AMINE OXIDASE;

PURIFICATION AND STUDIES ON SOME

PHYSICAL AND CHEMICAL PROPERTIES

A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE

UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN BIOCHEMISTRY

SEPTEMBER 1968

by

BENEDICT GOMES

Dissertation Committee:

Kerry T. Yasunobu, ChairmanMorton MandelLawrence H. PietteRobert H. McKayJohn B. Hall

DEDICATION

TO MY MOTHER

Acknowledgements

To the East-West Center of the University of

Hawaii; the National Institute of Health; and the

Hawaii Heart Association for fellowships.

To Drs. I. Igaue and H. J. Kloepfer for their

assistance in the enzyme purification.

To Mrs. Tomi Haehnlen and Kazi Sirazul Islam

for drawing figures.

TABLE OF CONTENTS

LIST OF TABLES ••••••••••••••••••••••.••••••••••

LIST OF FIGURES •••••.•.••••••••••.••••••••••••.

ABBREVIATIONS .••••••.•.•.••••••••••.•••••••••••

ABSTRACT •..•.•.•.••.•••••••••••••••.••••••••••.

vi

viii

xi

xii

I. INTRODUCTION. • • • . • . • • • . • • • • • • • • . . • • • • • • • • • . 1

A. Historical Background of Amine 2Oxidase Studies •••..••••••••••.••••

B. Physiological Significance •.••••••• 5

C. Statement of the Problem........... 6

II. MATERIALS AND METHODS .•••••••.••••••••••.•• 8

A . Ma t e ria 1 s •••••••••••••••••.••••••.. 8

1. Materials and Reagents ObtainedComm e r cia 11 y ••••••••••••••••••• 8

2. Materials Obtained as Gifts ••.• 10

B. Methods............................ 12

1. Preparation of Adsorbents andIonexchange Materials •••••••••• 12

(a) Alum ina C/'................ 12

(b) Calcium phosphate gel..... 12

(c) Diethy1aminoethy1 (DEAE)-cellulose ••••.•••.•.•••••• 12

(d) Hydroxy1ap a t i te ••••••••••• 12

(e) Starch (for Electrophoresis) 12

(f) Sephadex G-200 ••••••••••••• 13

(g) Agarose (Bio-Gel A-1.5) gel. 13

2. Electrophoresis .••.•.••••.. 13

ii

(a)

(b)

Starch BlockElectrophoresis

Polyacrylamide GelElectrophoresis .•.••••

12

14

3. Ultracentrifuge Studies •••• lS

(a)

(b)

Sedimentation Velocity.

Sucrose DensityGradient •.......•.••.•

lS

16

4. Preparation of Mitochondria •. 17

S. Measurement of EnzymaticActivity ..•.•...•....••••.•. 17

6. Determination of HydrogenPerox ide •••••••.•.•.•••••••• 18

7. Determination of the PartialSpecific Volume, V •..•••••••• 20

8. Determination of MolecularWe igh t •....•....•...••••..•. 20

(a) Mol. Wt. by gelfiltration method 20

(b) Mol. Wt. from sedimen­tation coefficient,Stoke'~ radius, and thepartial specificvo 1 ume ...••.....••••••. 21

9. Metal Analyses ..•..•••••••.•• 23

(a) Copper ................. 23

(b) Cobalt ................. 23

(c) Iron ................... 23

(d) Manganese .............. 23

( e) Molybdenum •••• 0 •••••••• 23

10.

11.

Determination of Riboflavin •••••

Determination of Purine ••••••••

iii

23

24

12. Determination of Adenine ••••••• 25

13. Determination of Ribose ••••.••• 26

14. Determination of Phosphorus •••• 26

15. Analysis of Phospholipid .•••••• 27

16. Determination of the SulfhydrylGroups......................... 27

III. RESULTS 29

A. Purification and Purity Studies ••.•• 29

1. Purification of the Mitochon­drial Amine Oxidase •.••••••••••• 29

Calcium phosphate gelt rea tme n t. • • . • . • • • • • • • • . . . . . • • . • • 29

DEAE-cellulosechromatography

column31

Hydroxylapatite columnchromatography.................. 31

2. Studies on the Purity of theEn z ym e .•••••.••••••••••.•••••••• 40

(a)

(b)

Rechromatography on DEAE-cellulose ••.••••••••.••••••

Rechromatography onhydroxylapatite column

40

43

(c) Sephadex gel filtration 43

(d) Analytical starch blockelectrophoresis •••••••••••• 43

(e) Free boundary electrophoresis 52

(f) Polyacrylamide gelelectrophoresis •.•.•••••••. 52

(g) Ultracentrifuge studies •••• 52

B. Kinetic Properties ••••••••.•••••.••.

iv

52

1. Activity of the Enzyme •.•••••••• 52

2. Effect of Temperature on theEnzyme Activity................. 59

3. Effect of pH on the EnzymeActivity........................ 64

4.

5 •

Substrate Specificity

Inhibitor Specificity

64

64

(a) Product inhibition ..••.•.•• 64

(b) Inhibition by sulfhydrylreagents ••••••••••••.•.•••• 68

(c) Inhibition by metal chelatingagents .••.•.•.••.•••••.••.. 76

(d) Inhibition by aldehydereagents •..•••••••••••••••. 76

C. Physical Properties .••..•.•••.•••••. 85

1. Spectral Properties ••••••.••.••. 85

2. Sedimentation Coefficients •••••• 85

3. Partial Specific Volumes •.•••••• 92

4. Molecular Weights .•••••••••••••. 92

(a) Molecular weights determinedby Agarose gel filtration.. 92

(b) Molecular weights determinedfrom Stoke's radii, sedimen­tation coefficients, andpar t i a 1 s p e c if i c vol um e s ••. 9 6

(c) Molecular weights determinedfrom sedimentation-diffusioncoefficients and Stoke~s

radii •••..••••.•••..•.•••.. 102

5. Frictional Ratios .••.••••••.••••

v

102

D. Chemical Properties ••.••••••••.•••.. 106

1. Metal Content ••••••••••••.•••.•. 106

2. Phosphorus Content ••••.••••••••. 106

(a)

(b)

( c)

To ta 1 pho sphorus ••••.••••••

Phospholipid Phosphorus

Flavin dinucleotidephosphorus •••••••••.•.•••••

106

112

112

3 •

4.

Organic Prosthetic Group ••••••••

Sulfhydryl Groups .••.•••••••••••

114

118

IV. DISCUSSIONS.AND.CONCLUSION •.••••.••••.••••• 131

V. SUMMARy ••••••••••.•.•••••..••••••••••••• o. 150

VI. BIBLIOGRAPHy.............................. 153

I.

II.

LIST OF TABLES

Purification of Beef Liver MitochondrialAmine Oxidase .•.•••••••.••••••••••.••.•••

Modified Procedure for the Preparationof Amine Oxidase (FLOW SHEET) ••••••••••.•

Substrate Specificities of the two AmineOxidase Components ••••••.•••••••••.••••••

37

38- 39

67

III. A.

III. B.

III. C.

Inhibition of Amine Oxidase by Sul-fhydryl Reagents ••••••••••••••••.••••

Inhibition of Amine Oxidase by Sul-fhydryl Reagents ...•.•.••.••••...••••

Inhibition of Amine Oxidase by Sul-fhydryl Reagents •••••••••••••..•.••••

73

74

75

IV.

V.

Inhibition of Amine Oxidase by MetalChe1ating Agents •••••.•.•••••••••..••••••

The Effect of Aldehyde Reagents on theEnzyme Activity ••••••..••••.•••••.•••••••

81

84

VI.

VI.

A.

B.

Sedimentation Coefficients at DifferentProtein Concentrations of the Mito-c h 0 n dria 1 Am ine 0 x ida s e ••••....•••••• 93

Sedimentation Coefficients by SucroseDensity Gradient ••••••••••••••.•.•••• 94

VII.

VIII.

IX.

X.

XI.

Agarose Gel Filtration Data of StandardProteins, Blue Dextran 2000, and AmineOxidase Components ••.•..••••••••...•.••••

Molecular Parameters Obtained from GelFiltration Data •.•••••••••.••••••..•.••••

Physical Parameters of the MitochondrialAmine Oxidase •••.••••••••••.•.••••.••.•••

Molecular Weights of the Amine OxidaseComponents by three Methods •••••••.••••••

Frictional Ratios of the Amine OxidaseComp 0 n e n t s •..••••••••.•••..•••••...••••••

95

101

103

104

105

XII.

XIII.

Metal Content of Amine Oxidase •••••••••••

Phosphorus content of Mitochondrial Amine

vii

111

Oxidase 113

XIV. A. Riboflavin, Adenine, Ribose, andNucleotidePhosphorus Content of MitochondrialAmine Oxidase......................... 119

XIV.

XIV.

B.

C.

Riboflavin, Adenine, Ribose, andNucleotidePhosphorus Content of MitochondrialAmine Oxidase •••.•••.•••.•.••••.••.••

Pyridoxal Content of Phosphorylase aand of the Mitochondrial Amine OxidaseComponen ts •••••.•••..•••••.••••.•.•••

120

121

XV. Number of Titratab1e Sulfhydryl Groups in theMitochondrial Amine Oxidase Components 128

XVI.

XVI.

XVI.

XVI.

A.

B.

C.

D.

Properties: 1a. Kinetic Parametersof Mitochondrial Amine Oxidase •••.•••

Properties: lb. Kinetic Parametersof Mitochondrial Amine Oxidase •.•••••

Properties: 2. Molecular Parametersof Mitocnondria1 Amine Oxidase •••••••

Properties: 3. Chemical Parametersof Mitochondrial Amine Oxidase •••••••

145

146

147

148

1.

LIST OF FIGURES

Chromatography of the partially purifiedamine oxidase on the DEAE-cellulose column. 33

2. Hydroxylapatite column chromatography ofthe partially purified mitochondrial amineoxidase 0....... 36

3. Rechromatography of the purified enzymecomponent. 2 on the DEAE-cellulosecol urnn ......•. 0 • • • • • • • • • • • • • • • • • • • • • • • • • • • • 42

4. Rechromatography of purified component 2on hydroxylapatite •.•••.•.••••••••••••.•••• 45

5a. Chromatography of amine oxidase component 1on Sephadex G-200 ••••••.••••••••••••••••••• 47

5b. Chromatography of amine oxidase component 2on Sephadex G-200 •.•••••••.•••••..••.•••..• 49

6.

7.

8.

9.

Migration of the amine oxidase .components onstarch block electrophoresis •••.••••••.••••

Electrophoretic pattern of component 2

Polyacrylamide gel electrophoresis of .amine oxidase components 1 and 2 •••.•••••••

Sedimentation pattern of the amine oxidasecomponent 1 •••.•••••••••••.••••••••••••••••

51

54

56

58

lOa. Effect of temperature on the enzymaticactivity 61

lOb. Effect of temperature on the activity ofthe amine oxidase •.••••.•••.••••••••••••••• 63

11.

12.

Effect of pH variation on the activity of theenzyme components 1 and 2 ••••••.••.•••••••.

Product inhibition studies •••••••••.•••••••

66

70

l3a. Inhibition of amine oxidase by sulfhydrylreagents ....•.•.................•.......... 72

l3b. Lineweaver-Burk plot of benzylamine oxidationin the absence and presence of p-CMB •••••.• 78

ix

13c. Lineweaver-Burk plot of the benzy1amineoxidation in the presence and absence ofp - CMB ••••••••.•.••••••.••••••••••••••••••• 80

14. Lineweaver-Burk plot of benzy1amineoxidation in the presence and absence ofcupr izone ...•............... 0 • • • • • • • • • • • • • 83

15a. Absorption spectrum of the purified enzymecomponent2 ....•....•. o ••••••••••••••••••• 87

15b. Reduction of the enzyme component 2 bysubstrate and sodium dithionite .•••••••••. 89

16. Sedimentation coefficients of amine oxidasecomponent 1 at varying protein concentrations 91

17. Agarose gel fYl:tration data of variousstandard proteins and of the Dextran 2000,and amine oxidase components •••••••••••••• 98

18. Correlation of Kd with Stoke's radius ••.•• 100

19a. Copper content of the enzyme •••••••••••••• 108

19b. Iron content of the enzyme .••"............. 110

20. Flavin content of the enzyme •••••••••••••• 116

21a. p-Ch1oromercuribenzoate titration ofcomponent 1 •.••.•••••••••••••••.•••••••••• 123

21b. p-Ch1oromercuribenzoate titration of thecomponent 1 in the presence of urea •••••• 125

21c. p-Ch1oromercuribenzoate titration of thecomponent 2 ••••••••••••••••.••••••••.••.• 127

22. Activity of amine oxidase component 1during p-CMB titration ••••••••••••••••.•• 130

oA

DEAE-cellulose

M

mg

ml

o

mu

s

s(obs)

s20, w

S

TCA

cm

ug

mole

a tom (s)

mole (s)

mp.mo le (s)

K i

-SH group(s)

D

N

%

p-CMB

x·

LIST OF ABBREVIATIONS

Angstrom

Diethylaminoethyl-cellulose

Molar concentration

mill igram

milliliter

Degree(s) Centigrade

mill imicron (s)

Sedimentation coefficient

Observed sedimentation coefficient

Sedimentation coefficient correctedto water as solvent at 20 0 •

Svedberg Constant (1 S =s20,Wx 10- 13

sec) •

Trichloroacetic acid

Cen t im e t e r (s)

microgram (s)

gram molecule

microa tom (s)

micromole (s)

millimicromole(s)

Michaelies Constant

Inhibition Constant

Sulfhydryl group(s)

Diffusion coefficient

Normal concentration

Percent

parachloromercuribenzoate

ABSTRACT

Beef (steer) liver mitochondrial amine oxidase was

prepared according to the method reported earlier (Adv.

Pharmacol., ~, Part A, 43, 1968). In addition to the

usual preparation with high activity, (component 2,

specific activity of 8,000) another component of the

enzyme (component 1) with lower activity (specific

activity 3,000) was isolated (Biochem. Biophys. Res.

Commun., submitted). Studies were made on some physical

and chemical properties of these two components.

The amine oxidase components were bright yellow in

color; they were thermolabile, and unstable at room

temperature. The rate of inactivation of component 2

was faster than that of component 1. The optimum pH for

activity was found to be 9.2. Both the components were

non-competitively inhibited by p-chloromercuribenzoate.

Metal chelators like cuprizone, 8-hydroxyquinoloine,

~-phenanthroline inhibited the enzyme components. Ammonia

or aldehyde reagents did not have significant effects on

the activity. Both the components had almost the same

substrate specificity.

The molecular weights of the enzyme component 1 was

found to be 400,000 by the gel filtration technique,

396,000 ~ 10,000 on the basis of Stoke's radius,

sedimentation coefficient, and partial specific volume,

xii

and 425,000~ 10,000 on the basis of sedimentation

diffusion method. These values for component 2 were

1,300,000, 1,195,000, and 1,355,000, respectively.

The sedimentation coefficients of component 1 and

component 2 were 14.4 + 0.3 and 20.6, respectively.

Metal analyses of the enzyme yielded 1 gram atom

of copper per 400,000 grams or 3 gram atoms of the metal

per mole of component 2. Other metals, such as cobalt,

iron, manganese, and molybdenum were examined and found

to be either absent or insignificant (J. Biol. Chem.,

241, 2774, 1966).

Both the components of the mitochondrial enzyme

were found to be flavoproteins. This was amply proved

(1) from their riboflavin content as determined micro­

biologically, and spectrophotometrically (Biochem.

Biophys. Res. Commun., 23, 324, 1966), (2) from a steady

increase of riboflavin during purification processes,

and (3) from the spectrum of flavo-peptide obtained from

pronase digest of the enzyme. Besides, the prosthetic

group was found to contain ribose (Biochem. Biophys. Res.

Commun., 29, 562, 1967), adenine and phosphorus in

integral values suggesting that the "flavin prosthetic"

group was a flavin adenine dinucleotide of unknown

structure. Accordingly, component 1 contained 4 and

component 2 contained 12 FAD or FAD-like substance per

mole, respectively.

xiii

Examination of the sulfhydryl groups revealed that

components 1 and 2 of the enzyme contained 28 and 86

titratable sulfhydryl residues, respectively in their

molecules, and that they were not directly involved in

enzyme catalysis. In addition, the enzyme was found to

contain 24 and 106 moles of phospholipid in components

1 and 2, respectively.

Finally, it appeared that the high molecular weight

component was the native form from which the small

molecular component arose during the purification of

the enzyme, although no interconversions were observed

with the purified enzyme preparations.

I. INTRODUCTION

Enzymology has gained enormous popularity in a

very short time as compared to other disciplines in

biochemistry. It has also become of great importance

in other health related fields such as microbiology,

pharmacology, toxicology, pathology, medicine, etc.

However, to a biochemist, the enzyme has a very special

significance since life itself depends on a network of

complex biochemical reactions which are catalyzed by

enzymes.

One may ask questions such as how do enzymes act?

What makes them so unique as to be able to mediate such

complex biological reactions? What are their sizes and

shapes? What are they made up of? Biochemists and

physical chemists have attacked these questions with

vigour in an effort to answer these questions and con­

siderable progress has occurred. (Vide the work of

Phillips group on lysozyme, the work of numerous labora­

tories with ribonuclease, the results of Lipscombs

laboratory on carboxypeptidase, etc.). However, the

studies have all been made with enzymes which can be

readily isolated from cells. On the other hand, mito­

chondrial enzymes are in a different class because of

the fact that they are difficult, in general, to free

2

from the mitochondria or from the mitochondrial

fragments. Nevertheless, experience has shown that

it is essential to obtain the homogeneous enzyme in

order to obtain meaningful physicochemical values. Thus,

a considerable amount of time and effort must be spent

in order to work out precise isolation procedures in

the case of mitochondrial enzymes. Once the purifica~ion

procedure is developed, various properties of the pure

enzyme from the mitochondria can be investigated like

those of the more readily isolatable enzymes. The

informations derived from these investigations can thus

provide reasonable answers to those questions mentioned

above.

A. Historical Background of Amine Oxidase Studies

Amine oxidase is the common name for a group of

enzymes which catalyze the following general reaction:

R-CH2NH2 + H20 + 02 = R-CHO + NH3 + H202. The enzyme

was first described by Hare in 1928 (1) as catalyzing

the oxidative deamination of tyramine. She termed this

enzyme, tyraminase.

It was soon realized that this enzyme was widely

distributed not only in animals, but in plants, and in

bacteria. The early history and distribution of amine

oxidase have been described in numerous review articles

(2- 4) .

3

Although the general reaction shown is catalyzed by

a typical amine oxidase, it was soon observed that there

occurred certain differences among these enzymes depend­

ing on the sources they were obtained from. It was

found that those enzymes, which were mainly bound to the

mitochondria of animals had a substrate specificity

distinct from those of the animal plasma, plant, or

bacterial enzymes. Thus, the mitochondrial enzymes

were found to attack tyramine, tryptamine, catechol

amines and other "biogenic" (5) monoamines, and

benzylamine. Unlike the diamine oxidase of hog kidney

(6-8) or that of pea seedlings (9-14), the histaminase

of pig plasma (15-17), or the amine oxidase of beef

plasma (18), the mitochondrial amine oxidase did not

attack cadaverine, histamine, or putrescine; nor did it

catalyze the breakdown of spermine and spermidine.

Moreover, the aldehyde reagents which are known-to

inhibit histaminase (19,20) and related oxidases (21-23),

did not inhibit the mitochondrial amine oxidase. These

observations indicated, that the mitochondrial enzymes

were a class of enzymes distinct from those of the

kidney diamine oxidase, pig plasma histaminase, or beef

plasma enzyme which are known to be copper-pyridoxal

enzymes (24). Studies have been made on the purifica­

tion and properties of a number of mammalian plasma and

kidney enzymes. Thus, Blaschko and Bufoni purified

4

and crystallized pig plasma histaminase (25) and studied

various physical and chemical properties (26) of it.

Yamada and Yasunobu purified, crystallized, (18) and

investigated the properties of beef plasma enzyme

(27,28). McEwen reported on the purification of and

kinetic studies on human (29,30) and rabbit (31) plasma

enzymes. Purification has also been reported for amine

oxidase in insects (32) and in microorganisms (33).

On the other hand, little progress in the purifi­

cation of the mitochondrial amine oxidase has occurred

due to the particulate nature (34) and the relative

insolubility of this enzyme (24). Although the isola­

tion of partially purified mitochondrial amine oxidase

has been reported (35), a highly-purified preparation

that could be employed for studying the properties of

this enzyme was not available. It is only recently that

the purification problem has largely been overcome by

using special techniques. Thus, Barbato and Abood (36)

liberated the enzyme from the insoluble mitochondrial

structures by using a non-ionic detergent, Triton X-IOO.

Some workers used sonication (37), and sonication in the

presence of substrate (38), to release the enzyme from

particulate structures.

Recently, Erwin and Hellerman (39) purified the

amine oxidase from the bovine kidney mitochondria by

5

using digitonin as the solubilizing agent. Tipton (40)

used repeated sonication and thawing to liberate the

amine oxidase from pig brain mitochondria. These authors

also made some investigations on the properties of their

preparations.

B. Physiological Significance

Earlier literature suggested that amine oxidase

was involved in the detoxication (2) or in the oxidative

deamination of biologically active amines in animal

systems (41-45). Since certain members of the biogenic

amines are associated with hypertension (46) and hyper­

sensitivity (47), the amine oxidases were considered to

be involved in the enzymatic removal of these amines.

In other words, the amine oxidases are involved in the

"detoxication" of the biologically active amines (48).

The rich supply of this enzyme in the intestinal mucosa

indicates a protective function. The enzyme, it is

reported, thus prevents many amines formed in the gut

by bacterial decarboxylases, from entering the general

circulation. This protective role has been supported

by recent findings on the effects of monoamines when the

oxidative deamination activity was blocked by amine

oxidase inhibitors (49,50). Some workers, at the same

time reported that the products of enzymatic deamination

of monoamines alter significantly the pattern of

car~ohydrate metabolism in some tissues.

6

Barondes (51)

reported that a number of aldehydes stimulate the

glucose oxidation in beef anterior pituitary slices and

suggested that the aldehydes originating from biogenic

amines by enzymatic deamination are responsible for

this. Moreover, pep pills or psychic energizers such

as tranylcypromime, phenelzine (49), or pargyline (50)

are potent inhibitors of mitochondrial amine oxidase.

This finding suggests that mitochondrial amine oxidase

may possibly be important in maintaining the normal'

mental state of human individuals by regulating the

levels of catecholamines and other biogenic amines in

their systems.

c. Statement of the Problem

The objective of this work was to purify the beef

liver mitochondrial amine oxidase and to study some

physical and chemical properties of this enzyme. The

enzyme is a very special one since it is tightly bound

to the insoluble membrance of the mitochondrion (34,52).

Many laboratories attempted its purification without

apparent success. In this laboratory a 50-fold

purfication of the enzyme was achieved (53) for the

first time and a reasonably pure preparation was

7

available for the preliminary study of some of its

properties. Recently, our laboratory improved the

purification method to a great extent and highly purified

preparations with very high activity were obtained (55).

More recently, better yields and multiple enzyme com­

ponents with amine oxidase activity have been isolated.

The present work will describe investigations of the

highly purified enzyme components and will include the

following major aspects: (i) Purification and demon­

stration of purity (ii) effects of various physical

factors such as pH, temperature, etc., on the enzymatic

activity; (iii) effects of various inhibitors; (iv)

sedimentation behavior; (v) molecular weight determina­

tion; (vi) determination of metal components; (vii)

studies on cofactors; (viii) determination of the number

of sulfhydryl groups; and (ix) other properties of the

enzyme.

II. MATERIALS AND METHODS

A. Materials

1. Materials and Reagents Obtained Commercially

(a) J. T. Baker Chemical Co., New Jersey

2,6-Dimethy1 Pyridine (Lutidine)

(b) Bio-Rad Laboratories, California

Agarose (Bio-Ge1 A-1.S m) Beads, 100-200 mesh

(c) Ca1biochem, California

Agmatine Sulfate

Cadaverine Dihydroch1oride

Ferritin

G1ucose-6-Phosphate, Disodium Salt

n-Hepty1amine

Trimethylene diamine Dihydroch1oride

(d) Carl Schleicher & Schue1 Co., New Hampshire

Diethy1aminoethy1 (DEAE)-ce11u1ose

(e) Cyc10 Chemical Corporation, California

Di,thioerythrito1

(f) Difco Laboratories, Michigan

Yeast Extract

(g) Eastman Organic Chemicals, New York

Acry1amide

Amido Schwarz

1-Amino-2-Naphthol-4-Su1fonic Acid

Benzy1amine

9

Butane Diamine Dihydroch1oride

N,N-Methy1ene-Bis-Acry1amide

N-(1-Naphthy1)-Ethy1ene Diamine Dihydro-

chloride

N,N,N',N'-Tetramethy1ethy1ene Diamine

Tyramine Hydrochloride

(h) Fisher Scientific Company, New Jersey

Nessler's Reagent

(i) The G. Frederick Smith Chemical Company, Ohio

Bis-Cyc1ohexanone Oxa1dihydrazone (Cuprizone)

4,7-Dipheny1-1,10-Phenanthro1ine

(Bathophenanthro1ine)

Hydroxy1ammonium Chloride, 10% Solution,

Iron-Free

Sodium Acetate, 10% Solution, Iron-Free

Standard Iron Solution

(j) Hawaii Meat Co., Honolulu, Hawaii

Steer (Beef) Liver

(k) Mann Research Laboratories, Inc., New York

o-Dianisidine

Kynuramine Dihydrobromide

(1) Matheson Coleman & Bell, Ohio

Potato Starch

(m) Nutritional Biochemicals Corporation, Ohio

Bovine Serum Albumin (2 x recrystallized)

10

(n) Pharmacia Fine Chemicals, Inc., New Jersey

Blue Dextran 2000

Sephadex G-25, Coarse Grade

Sephadex G-200

(0) Pierce Chemical Company, Illinois

Cholic Acid

(p) Sigma Chemical Company, Missouri

DL-Arterenol (Norepinephrine) Hydrochloride

Catalase (6 x recrystallized)

p-Chloromercuribenzoic Acid, Sodium Salt

Cytochrome c, Type V, From Beef Heart

Flavin-5-Phosphate (FMN) Sodium Salt

Mescaline Sulfate

Spermidine Trihydrochloride

Spermine Tetrahydrochloride

Tryptamine Hydrochloride

(q) Worthington Biochemical Corporation, New Jersey

Peroxidase (from Horse Raddish)

Phosphorylase a

(r) Upjohn Research Laboratories, Michigan

5-Hydroxytryptamine (Serotonine) Sulfate

(s) Van Waters & Rogers, Inc., California

Phenol (Folin-Ciocalteau) Reagent

2. Materials Obtained as Gifts

~. coli K 12, from Dr. Morton Mandel

11

Dept. of Biochem. & Biophys. UH

E. coli C 406, from Dr. John B. Hall

Dept. of Biochem. & Biophys. UH

Triton x-lOO, from Rohm & Hass, Pennsylvania

12

B. METHODS

1. Preparation of Adsorbents and Ion Exchange

Materials

(a) Alumina Cy. was prepared according to

Willstatter and Kraut (55).

(b) Calcium phosphate gel was prepared by

the method of Keilin and Hartree (56).

(c) DEAE-cellulose, obtained commercially,

was treated according to the procedure of Peterson

and Sober (57). The dry material was allowed to

sink freely in lN NaOH and the suspension was

filtered on a sintered glass filter. Washing with

lN NaOH was repeatedly done until no more yellow

color was removed. The material was now treated with

sufficient lN HCl to make a strongly acid suspension,

which was immediately filtered and washed free of acid

with water. The filtered substance was again sus-

pended in lN NaOH, washed free of alkali with water,

and finally suspended in the selected starting buffer.

(d) Hydroxylapatite was prepared by the

method of Tiselius et al (58).

(e) Starch (for starch electrophoresis) was

treated by the procedure described by Fine and

Costello (59).

13

Commercially obtained potato starch was

suspended in approximately 3 volumes of distilled

water and allowed to settle. The supernatant was

decanted, removing suspended impurities and fine

starch particles. After it was washed 3 times with

water, the starch was washed 3 times with the buffer

in which electrophoresis was conducted. The starch,

thus treated, was kept under the same buffer in the

cold room (at 0-4 0 ) for routine use.

(f) Sephadex G - 200, obtained as a dry

powder, was added to excess water and was allowed

to stand for 3 days with occasional stirring and

decantation. The swollen gel was washed 3 times

with the starting buffer at 0_4 0 before packing the

column.

(g) Agarose (Bio-Gel A-l.5 m) beads, 100­

200 mesh, was obtained in 0.001 M tris-EDTA buffer

medium containing 0.02% sodium azide as a preserva­

tive. The agarose column was exclusively washed

free of azide and tris-buffer by running large

volume of starting buffer through the column.

2. Electrophoresis

(a) Starch block electrophoresis

Starch block electrophoresis was done

according to the method of Fine and Costello (59).

14

Blocks were prepared with starch in plastic trays

(44 x 3 x 1.5 cm). For the electrophoresis run,

potassium phosphate buffer, pH 7.4, with an ionic

strength of 0.1, was employed. Samples were

dialyzed against the same buffer for 3 hours with

2 changes and were applied in amounts of 10 to 20 mg

enzyme in 2 m1 portions. Separation was effected in

the cold room (at 0 - 4 0 ) with a voltage of about

400 volts and between 10 and 15 mA per block for 18 -

24 hours.

(b) Polyacrylamide Gel Electrophoresis

Polyacrylamide gel electrophoresis was done

according to the method described by Taber and

Sherman (60) with an alteration in solution (a). In

the present experiment, it consisted of the following

composition per 100 m1 of solution: 8 m1 1N KOH,

1.9 gm glycine, and 0.077 m1 N,N,N',N'-tetramethy1-

ethylene diamine, pH 10.3.

contained 3.75% acry1amide.

The gel system used

Gel columns (65 x 6 mm)

were prepared in 95 x 6 mm i.d. pyrex tubes. They

were soaked in solution \ .(a) wh~ch was diluted to the

same concentration that occurred in the gel, for 2

days to diffuse out any unreacted materials. Samples

containing 50 to 100 ug in 10 to 20 u1 quantities

15

were applied to the gel columns, layered on with

diluted lutidine-glycine buffer, pH 8.3, and run in

the same buffer at a potential of 410 volts and 3 mA

per tube for 2 hours. At the completion of elec-

trophoresis, gel columns were removed from the tubes

and stained by immersing them in a solution of Amido

Schwarz for 45 minutes. The gel columns were

destained by washing with 7.5% acetic acid and

stored in the same acid solution.

3. Ultracentrifuge Studies

(a) Sedimentation velocity measurements

were made in a Spinco Model E Analytical Ultracen-

trifuge equipped with a RTIC unit for controlling

the rotor temperature within + 0.1°. The conven-

tional 12 mm aluminum cell with a 4° sector shaped

centerpiece was used for all runs. The speed

employed was 35,600 rpm (73,684 x g) using a rotor

type An-D and the rotor temperature was 22.5 0 • The

sedimentation coefficient was calculated by using

the following equation:

s= 1"2w x

dxdt

(i)

where x is the distance of the boundary from the

axis of rotation in centimeters, t is the time in

seconds, and w is the angular velocity in radians

per second (211 rpm) .60

The observed sedimentation

16

coefficients (sobs) were corrected to the standard

conditions (S20,w) in terms of the density and

viscosity of water as the solvent at 20 0 according to

Svedberg and Peterson (61).

(b) Sucrose density gradient centrifugations

were carried out according to the procedure described

by Ames and Martin (62). The present method, however,

differed only in that a Beckman Model L 2-65 Ultra-

centrifuge, with a swinging bucket rotor, SW-4l in

which 14 x 89 mm cellulose nitrate tubes, were used.

A linear sucrose gradient, made from a 20% and 5%

sucrose in 0.1 M potassium phosphate buffer, pH 7.4"

containing 1 x 10- 4 M dithioerythritol, was used in

all experiments. Gradients of 11.5 ml in each tube,

prepared by using a Buchler Polystaltic Pump were

equilibrated for 4 to 8 hours at 0-4 0 in the cold

room and centrifuged at 25,000 rpm (75,000 x g) for

16 hours at 0 0 after applying samples.

4. Preparation of mitochondria

Beef liver mitochondria were prepared by the

method of Schneider and Hodgeboom (63). Select steer

livers, obtained immediately after slaughtering, were

17

brought from the Hawaii Meat Company. Membranes,

large blood vessels, and bile ducts were removed.

Weighed liver slices were homogenized in 9 volumes

of cold 0.25 M sucrose with a Waring blendor for

2 minutes at 0-4 0 • The homogenate was centrifuged in

a Model PR-2 International Refrigerated Centrifuge

at 700 x g for 10 minutes. The supernatant was

carefully decanted and re-centrifuged at 5000 x g in

a Sorval Refrigerated Centrifuge, Model RC 2-B for 10

to 15 minutes. The opalescent supernatant, together

with a pink partially sedimented layer of particles

above the firmly packed pellet of mitochondria, was

discarded. The mitochondrial pellet was washed two

times with one-third the original homogenizing volume

of 0.25 M sucrose and then centrifuged at 24,000 x g

for 10 minutes. The washing procedure waS repeated

once with 1.15% KCl solution and the mitochondria,

thus prepared, were stored frozen in 0.01 M potassium

phosphate buffer at pH 7.4.

5. Measurement of Enzymatic Activity

The enzymatic activity was determined by the

spectrophotometric method of Tabor, Tabor and

Rosenthal (64) using benzylamine as the substrate. In

this work, 2.85 ml cif 0.2 M potassium phosphate buffer,

pH, 7.4, were added to a I-cm cell containing 0.1 ml

18

of enzyme solution and the sample was mixed. To this

cell, 0.05 ml of 0.1 M benzylamine solution was added

to make a total volume of 3 ml and a final subtrate

concentration of 1.67 mM. The assay solution was

mixed by inversion. A blank was prepared likewise

except that the substrate was omitted. Readings

were made at 250 m¥ initially and then subsequently

every minute for 5 minutes.

One unit of enzymatic activity was defined

as the amount of enzyme that produced a change in

absorbance of 0.001 per minute at 250 mp at 25 0 •

Specific activity was expressed as the number of units

of activity per milligram of enzyme. The enzyme

protein was measured by the method of Lowry ~ al (65)

using bovine serum albumin as the standard. In

activity measurements, the amounts of enzyme used

showed activity in the range of 10 to 50 spectrophoto­

metric units.

6. Determination of Hydrogen Peroxide

Substrate specificity of the amine oxidase

was determined for various amines by a method developed

by McEwen (29) by coupling the normal reaction with

peroxidase in the presence of o-dianisidine (66).

In this reaction, the peroxide formed as a product of

the oxidative deamination of amines by amine oxidase

19

converts peroxidase to peroxide-peroxidase complex

(complex 2) which then converts o-dianisidine to a

reddish brown compound as shown in the following

reactions:

R-CH -NH2+ H20 + 02 Amine> R-CHO + NH3 + H202Oxidase

Peroxidase + H202--~>~p-p-Complex (Complex 2)

H3CO OC H3

p-p-Complex + H2 N -0---0-- NH2 -----.:::>~Peroxidase

o-Dianisidine H3CO OC H3

+ HN ==0==0= NH

Reddish Brown Color

For this experiment, 5 mg of horse radish peroxi-

dase and 8000 units of amine oxidase were dissolved in

99 ml of 0.1 M potassium phosphate buffer, pH 7.4. To

this enzyme mixture was added 1 ml of o-dianisidine solu-

tion made by dissolving 10 mg o-dianisidine (2 x rec~y-

stallized) in 1 ml 95% ethanol. The resulting enzyme-

chromogen (approximately 4 x 10-5M) solution was

filtered. To 2.9 ml aliquots of this solution were added

0.1 ml of the amine solutions being assayed, so that the

final concentrations of these amines were the same (3.3 x

10-3M) in all tubes. A reagent blank was prepared in the

same way except that the amines were omitted.

After 15 minutes, the reddish brown color was

measured at 450 m)l against the blank.

20

7. Determination of the Partial Specific Volume, V

Since the term (l-Vp) is contained in the

equation for molecular weight determination by the

sedimentation-diffusion method, Stoke's law, and by

the sedimentation equilibrium method, the partial

specific volume, V, has to be determined. The

measurement of this parameter was done by the

method of Schachman (67). Accordingly, the

densities of solvent and solution were measured

pycnometrica11y, and the amount of protein in

solution was determined. The apparent partial

specific volume was calculated by using the following

equation:

Vapp = 1/d o-1/x(d-d o )/do .... (ii)

where x is the concentration of protein in grams per

mi1i1iter of solution, and do and d are the densities

of solvent and solution, respectively.

8. Determination of Molecular Weight

(a) Molecular weight by the gel filtration

method

Gel filtration techniques published by

Whitaker (68) and Andrews (69) were employed for the

molecular weight determination. Agarose (Bio-Ge1

A-l.S m) gel was packed in a 120 x 1.9 (i.d.) cm

column, and the column was equilibrated with 0.05 M

potassium phosphate buffer, pH 7.4, containing 0.01 M

21

mercaptoethanol. The same buffer was used for elution

of protein standards and markers for the determination of

'elution vOlume' and 'void volume.' The void volume was

determined by passing~. coli (44) through the column and

measuring the turbidity due to these organisms. The

column was then calibrated with standard proteins of

known molecular weights before running the enzyme

sample in the column.

The elution volume is defined as the volume of

buffer eluted corresponding to the peak concentration of

the solute. Fractions of 3 ml were collected and a

standard curve was constructed by plotting the ratios of

the elution volumes to the void volume against the

logarithms of the molecular weights according to the

method of Whitaker (42).

(b) Molecular weight from sedimentation coeffici­

ent, Stoke's radius, and the partial specific volume

Elution volume is a function of the molecular

radius (or the Stoke's radius) of a protein molecule

upon chromatography on a gel column (45). A calibrated

gel filtration column can be used for the estimation of

Stoke's radius of a macromolecule present even in the

impure form, provided a method for the assay of the

macromolecule is available. The molecular or Stoke's

radius is determined from the gel filtration data

22

presented in terms of a distribution coefficient, Kd,

which is a function of the molecular size.

meter is defined as follows:

This para-

(iii)

when Ve = elution volume, Vo = void volume, and Vi=1

volume inside the gel grain. When the Kd~ values of

the standard proteins are plotted against their

molecular (Stoke's) radii, a linear curve is obtained

(72). The molecular or Stoke's radius of a macro-

molecule can easily be determined from the constructed

standard curve.

The sedimentation coefficient of a macromolecule

can be determined by the sucrose density gradient

technique or by the conventional sedimentation velocity

method (if the material is pure). The molecular weight

and the frictional ratio of the macromolecule can be

accurately determined, if the partial specific volume

is reasonably known. The molecular weight and the

frictional ratio, therefore, can be determined from the

relationship defined by the following classical equations:

M = 6:ff n Nas ( iv)(l-Vp )

1

fifo = al (3V M)3 (v)4.1T N ..

when M is the molecular weight, n is the viscosity of

the medium, a is the Stoke's radius, s is the sedimentation

23

coefficient, V is the partial specific volume, p is the

density of the medium, f/f o is the frictional ratio and

N is the Avogadro's number.

9. Metal Analyses

Purified amine oxidase components were analyzed

for their metal contents. In these experiments, 5 mg of

the purified enzyme were used for each analysis.

Copper was determined in the purified components

according to the method of Peterson and Bollier (73).

The assay solution was prepared both by extraction of

copper with 10% trichloroacetic acid and by wet ashing

(74) . Cobalt estimation was done by the ~tomic absorp-

tion spectrophotometric method of Fuwa et a1. (75) •

Iron was analyzed by the procedure of Peterson (76) on

dry or wet ashed samples of the enzyme. Manganese was

determined by atomic absorption spectrophotometry as

described by Fuwa et a1. (75) . Molybdenum was measured

by the method described by Sandell (77).

10. Determination of Riboflavin

Riboflavin was determined both microbiologically

and spectrophotometrica11y.

In the microbiological assay, the growth response

of Lactobacillus casei was measured as a function of

riboflavin concentration according to the method of Snell

and Strong (51). As these micro-organisms depend on

24

riboflavin as a growth factor, a medium containing all the

necessary growth factors except for riboflavin was prepared.

The growth of the micro-organisms in the media with varying

concentration of riboflavin was measured by titrating the

acid produced in each tube. A standard curve was constructed

by plotting the volume of acid produced and the known

concentrations of riboflavin added to each tube. The

riboflavin content in the unknown sample was determined

from the standard curve.

The spectrophotometric determination of the flavin

component of the enzyme was based on the reduction of

the 450 m~ absorptkn upon addition of sodium hydrosu1fite

(Na2S204) to the enzyme. The difference between the

absorbances at 450 m~ before and after the addition of

hydro sulfite was a measure of the flavin nucleotide

content of the enzyme. The flavin concentrations were

calculated from the molar absorbancy index of FAD at

450mr (E 450 m~ = 1.13 x 104 cm2 mo1e- 1 ).

11. Determination of Purine

Microbiological assay of the purine content of the

enzyme was made by measuring the growth response of a

special mutant of E. coli (E. coli C 406) which requires

purines in addition to other nutrients for their growth.

The medium was prepared according to the procedure of

Sedat and Sinsheimer (79), and the organisms were grown

in 100 m1 aerated cultures. After 12 hours, the organisms

25

were harvested and suspended in a sterile 0.9% NaCl

solution. A set of tubes each containing 9 ml of medium

with increasing concentrations of adenine were inoculated

with 1 ml of E. coli suspension. A blank was similarly

prepared with the exception that it did not contain any

adenine. The standard as well as the blank tubes were

incubated at 37 0 for 18 hours after which the turbidity

was measured at 650 m~. A standard curve was drawn by

plotting turbidity (O.D. at 650 m~) against adenine

concentrations.

Enzyme samples were hydrolyzed for purine deter­

mination according to the method of Vischer and Chargaff

(80). For the experiment, 4 mg of purified enzyme were

hydrolyzed in 5 ml 1 N H2S04 at 1000 for 1 hour. The

precipitated protein was filtered and washed 3 times

with 0.5 ml portions of 0.1 N H2S04. After adjusting the

pH to 6.8 with 2 N KOH and the volume to 10 ml with

water, the filtrate was employed for purine determination

~n the same way as standard.

12. Determination of Adenine

For this assay, 12 mg of pure enzyme were hydrolyzed

in 1 N H2S04 exactly in the way stated above for the purine

determination. However, the pH of the sample was

adjusted, instead, to a pH of 1 with 10 N KOH. Adenine

was determined by the colorimetric method of Koritz

et ale (81). The determination is based on a color

26

reaction of adenine with N-(1-naphthy1) ethylenediamine

hydrochloride after its reduction with zinc dust and

diazotization with NaN0 2 . The absorbance of the red

color developed is measured spectrophotometrica11y at

505 mp.

13. Determination of Ribose

The ribose content of the enzyme was measured by

the orcinol test first proposed by Bia1 (82) and later

modified by many others (83,84). In this case, 4 mg

of pure enzyme were first hydrolyzed with 0.5N KOH for

48 hours at 25 0 to liberate all the ribose quantitatively

(85) as purine nuc1eotides. The hydrolysate was then

adjusted to pH 1-2 by dropwise addition of 20% HC104.

The modified method of Dische (86) was employed for the

quantitative dete~mination of ribose.

14. Determination of Phosphorus

Phosphorus was determined by the u1tramicro­

chemical method described by Bartlett (87). In this

experiment, 5 mg enzyme were precipitated with ice cold

trich10ro-acetic acid (TCA) such that the final TCA

concentration was 7%. The precipitated enzyme was

centrifuged and the supernatant was discarded. The

enzyme precipitate was washed 3 times with 5 m1 portions

of 1% ice cold TCA. The enzyme precipitate was then wet

ashed and analyzed for phosphorus according to the method

referred to above (87).

27

15. Analysis for Phospholipid

The procedure for the extraction of lipids from

the enzyme is described by Folch ~ al. (88) . S amp 1es

containing 5 mg of enzyme in a volume of 1.5 ml were

extracted with 5 m1 of a 2:1 ~hloroform-methanol

mixture in 50 m1 glass-stoppered conical centrifuge tubes.

The chloroform extract was transferred to a fat-free

filter paper, and filtered into a 25 ml volumetric flask.

The extraction was repeated 3 more times. Occasionally,

the aqueous and ch~oroform layers were separated by

centrifugation when they did not separate clearly. The

lipid content was determined on 20 ml aliquots by the

method mentioned above (88). Phospholipid was measured

by determining the phosphorus present in the lipid

extract according to the method of Bartlett (87).

16. Determination of the Sulfhydryl Groups

The sulfhydryl groups were determined by the

spectrophotometric method of Boyer (89). In order to

pre~ent air oxidation of the sulfhydryl groups, the

0.05 M potassium phosphate buffer, pH 7.0, was equi1i-

brated with nitrogen. Since the enzyme readily preci-

pitated at the neighborhood of pH 5, determinations were

made at pH 7.0 (a) in the absence of urea, and (b) in the

presence of 8 M urea. Twice recrystallized p-ch1oromer-

curibenzoic acid (p-CMB, absorbance index, E232 mJl =

1.69 x 10 4 cm2 mo1e- 1 in 0.05 M potassium phosphate

28

buffer, pH 7.0) was used for the titration of the -SH

groups. Increasing amoun~of p-CMB (3 x 10- 4 M) solution

were added to a constant amount (0.4 mg) of enzyme in an

initial volume of 1 ml. Titration in the presence of

urea was done by adding sufficient 10 M urea (in buffer)

to the enzyme solution so that the final urea concen­

tration was 8 M. The p-chloromercuribenzoate

(3 x 10- 4 M) solution used in this titration also

contained 8 M urea. In all experiments, appropriate

blanks were used to correct absorbances due to protein

and p-CMB. Spectrophotometric readings were taken at

250 m~l hour after each addition of p-CMB.

III. RESULTS

A. Purification and Purity Studies

1. Purification of the Mitochondrial Amine Oxidase

The amine oxidase was prepared from steer (beef)

liver mitochondria according to the method described

by Yasunobu, Igaue, and Gomes (54) except for some

alterations. The method referred to above comprised

(a) homogenization of the purified mitochondria by

using Potter-Elvehjem homogenizer, (b) extraction of

the enzyme with Triton X-lOa (a non-ionic detergent),

(c) ammonium sulfate fractionation, (d) calcium

phosphate gel treatment, (e) DEAE-cellulose column

chromatography, followed by (f) hydroxylapatite

column chromatography, and finally (g) electrophore-

sis in the starch block. In the present purification

procedure, however, alterations were made in the

calcium phosphate gel and in the hydroxylapatite

column chromatography steps which are described in

some detail as follows:

(d) Calcium phosphate gel treatment

The reddish brown enzyme solution from the third

step of purification was dialyzed against 4 liters

of 0.01 M potassium phosphate buffer for 2 hours at

0-4 0 , after which the dialyzing buffer was changed

and the dialysis was continued for an additional

30

2 hour period against the same buffer. To this

dialyzed enzyme, was added enough calcium phosphate

gel to make an overall p~otein to gel ratio of 1.4.

The mixture was gently stirred while adding the gel

and the stirring was continued for 15 minutes. The

gel-enzyme mixture was centrifuged in a Model PR-2

International Refrigerated Centrifuge for 20 minutes

at 850 x g using rotor No. 276 a. The supernatant

(S) was decanted into a 2 liter beaker and was saved

for the_total absorption later.

The gel (Gl.l) was eluted with 200 ml of 0.1 M

potassium phosphate buffer, pH 7.6, and then twice

successively with 200 ml portions of 0.2 M buffer,

pH 7.6. The eluates (termed Sl.2,Sl.3, and Sl.4 in

the Flow Sheet) were combined and desalted in a

Sephadex G-25 (coarse grade) gel column (4.5 x 45 cm)

and was again treated with calcium phosphate gel such

that the protein to gel ratio was 1.1 on the basis of

the initi~l protein concentration. The mixture was

centrifuged. The gel (G c ) was discarded and the

yellow supernatant (S3) was saved.

The supernatant (S) saved above for the total

absorption was treated with calcium phosphate gel on

the initial protein to gel ratio of 1.5 and the

mixture was stirred slowly for 1 hour. The gel

obtained by centrifugation of this mixture absorbed

---------~--~. ---...._-_.------

31

almost all the enzyme activity from the supernatant

(8). The gel containing the enzyme was washed with

about 1 liter of 0.01 M potassium phosphate buffer,

pH 7.4, and was centrifuged. The supernatant (8i.l)

was discarded and the gel (G2.l) was eluted in the

same manner as described for gel (Gl.1) above. All

the eluted fractions (82.2,82.3, and 82.4) were

combined with the supernatant (83) kept aside above.

The combined supernatant (combined supernatant) had

a final volume of 1.2 liters to 1.5 liters. The

enzyme contained in this solution was concentrated,

desalted, and passed through a DEAE-cellulose column.

(e) DEAE-cellulose column chromatography

The DEAE-cellulose column chromatography was

carried out in the same way as described earlier (54).

The present work, however, differed from the earlier

(54) one in that, the enzyme fractions having

specific activities of 1,500 to 4,000 were collected

(Figure 1). The procedure for concentrating and

desalting the enzyme before applying it to the

hydroxylapatite column was similar to that reported

previously (54).

(f) Hydroxylapatite column chromatography

The hydroxylapatite column (2.9 x 18 cm) was

equilibrated with 0.01 M potassium phosphate buffer,

Figure 1. Chromatography of the partially purified mitochondrial

amine oxidase on the DEA~-c~llulose column. Protein (about 340 mg),

containing 7 x 105 units of activity was applied to a column (45 x 2.2 cm)

which was equilibrated with 0.01 M potassium phosphate buffer, pH 7.4.

Gradient elution (900 ml of 0.1 M potassium phosphate buffer in mixing

flask and 900 ml of 0.2% Triton X-lOO in the same buffer in the

reservoir) was used to elute the enzyme. Fractions of 12 ml were col-

1ected at a flow rate of 0.75 m1 per minute. The symbols used are:

-0-0-, enzyme activity (units per milliliter); -e-e-, protein concen-

tration (milligram per milliliter); -~-~-, specific activity (units per

milligram of protein), and -x-x-, Triton X-lOO concentration (%).

V>r->

£_01 X A11J\ 1.1::>'1 ::>1.~1::>3dS0 10

(IWI OW) N13.l0~d q 00 to V (\J

0 0 0 0

001 -x NO.lIH.l 010

\ 0

\\ -\ 0 .&;

ux 0 0\ Q)\\ E

10. 0en -

a::0

wm .-Ien en :i!:c

0QJ

:::> l-<- Z :l(,)0 eo... 10 -.-I

u.. CD Z J::.<

-,::, 0~ I-00 0 U0- CD «

a::LL

10r-

or-

\L----L --L ----'I 0

33

oN

o

(IWI s~!un) A.1IJ\I.1~'1

34

pH 7.4 and the desalted enzyme concentrate (80 ml to

100 ml) from the DEAE-cellulose step was applied to

it. The column containing the absorbed enzyme was

washed with about 200 ml of equilibrating buffer, and

the enzyme fractions were collected by the stepwise

elution with 200 ml each of 0.1 M, 0.2 M and 0.2 M

buffer containing 0.15% potassium cholate.

used for elution was of pH 7.6 (Figure 2).

The buffer

Two bright yellow fractions of enzyme were

obtained--one eluted with 0.1 M or 0.2 M buffer

(component 1), and the other with 0.2 M buffer con-

taining cholate (component 2). The two fractions,

components 1 and 2 had specific activities in the

order of 2,000 to 4,000, and 6,000 to 7,500,

respectively.

The final step of purification of these fractions

was achieved by the starch zone electrophoresis under

the same conditions published earlier (54). The

enzyme components 1 and 2 after starch zone

electrophoresis attained specific activities of

3,000 to 4,000 and 7,000 to 9,000, respectively.

Table I summarizes all the steps involved in this

purification as developed by Yasunobu ~ ale (54)

and the attached FLOW SHEET briefly describes the

newly modified procedure.

Figure 2. Hydroxylapatite column chromatography of the partially

purified mitochondrial amine oxidase. One hundred and twenty milligrams

of protein containing 3.6 x 10 5 units of enzyme activity was applied to a

column (2.9 x 15 cm) which had been equilibrated with 0.01 M potassium

phosphate buffer, pH 7.4. Fractionation of the enzyme was made by

stepwise elution with, A, 0.01 M potassium phosphate buffer, pH 7.4;

B, 0.1 M fo1~owed by 0.2 M potassium phosphate buffer, pH 7.4; and C,

0.2 M potassium phosphate buffer, pH 7.4 plus 0.15% potassium cho1ate.

Curve -6-6- indicates enzyme activity, and curve -0-0- indicates protein

concentration.

wVI

A ·I~ B ~ C ,--E.....If) 0'I E0 Component 2 --X 4 Z-- lJJ- l-E..... 0." 0::... 0-0-c=' I I T .., 1.0-

>- 2t:>-l- I L~ II~ IT \~ .., 0.50«

Pooled - J 60 I~ Pooled ~l I 00Fractions Fractions

FRACTION NUMBER (6 ml each)

Figure 2W0\

TABLE I

Purification of Beef Liver Mitochondrial Amine Oxidase*

Purification Step Volume

(ml)

Totalprotein

(mg)

Totalunits**xlO- 3

Specificactivity

(units/mg)

Yield%

Purifi­cation

Solubility

1. Mitochondrial 1,800homogenate

2. Triton X-100 plus 0.15 720saturated (NH4)2S04

3. 0.25-0.40 saturated 515( NH4)2 S04

4. Calcium phosphate 80gel eluate

5. DEAE-cellulose eluate 54

6. Hydroxylapatite eluate 5

7. Starch block 16electrophoresis

37,080

14,976

7,151

432

133

45

31

4,860

4,392

2,987

1,088

631

356

249

131

293

417

2,519

4,750

7,900

8,050

100

90

62

22

13

7.3

5.1

1

2

3

19

37

60

61

Insoluble

Insoluble

Insoluble

Insoluble

Insoluble

Soluble

Soluble

* From 39 gm (dry weight) of purified mitochondria.**A unit of enzyme activity is the amount of enzyme required to change the absorbance 0.001

per minute at 250 mp using the spectrophotometric assay of Tabor ~~. in which benzyl­amine is used as the substrate. Insoluble means that detergent is required for solubility.Soluble means that no detergent is needed and that the enzyme precipitates instead offloats when ammonium sulfate is added to precipitate the enzyme.

VJ......

38

FLOW SHEET

Modified Procedure for the Preparation of Amine Oxidase

STEP IV. Calcium Phosphate Gel Treatment (54)

Reddish Brown Enzyme SOlutionlfrom STEP III C54)

• I(1) Dialyzed for 4 hr.

I .(2) Ca-ph gel (1:4, prote~n:ge1) added

I(3) Centr ifuged

Supernatant

S2.1

Ca- ph gel (1: 5 ,protein: gel) added

ICentrifuged(2)

(1)Eluted with 0.1 M

cenLifUged

(1)

(1) Eluted 2 x with 0.2K-ph buffer, pH 7.6I

(2) Centrifuged

(1)

(2)

Discarded

Washed with 0.01 MK-ih buffer, pH 7.4

Centrifuged.

SupernatantS1. 3 and S1. 4

Gel, G2.2 SupernaiantS2.2

Discarded Discarded

Combined S2.2,and S2.3, and S2.4

(1)

(2) Ca-ph gel (1: 1, protein:ge1) ~added

ce+trifugedCombined

(3) Supernatants

-t. ,j, !'

Gel, Gc SupernatantS3

Discarded

39

Zoneis

!combined Supernatants II

Concentrated by Amm-S04 (0.4 satd )

Desalted in SrPhadex G-25 Column

Eluate(100 to 150 ml)

DEAE-cellulosl Column Chromatography (54)I

Hydroxylapatite Column Chromatography (54).j,

~ ~

Component 1 Component 21VII. !Starch Zone STEP VII. StarchlectrrPhOreSiS Elect ophores

\

Purified PurifiedComponent 1 Component 2

(1)

(2)

STEPE

STEP VI.

STEP V.

40

2. Studies on the Purity of the Enzyme

It is a necessary condition to ascertain that

the enzyme is reasonably pure, before studying its

properties. Numerous tests, therefore, should be

employed to prove that the enzyme consists of only

one protein. Since each test establishes a certain

degree of purity, all the tests together will confirm

whether the enzyme is very pure or not.

Tests employed to study the homogeneity of a

protein or an enzyme are based on the measurement of

certain physical properties of the macromolecule.

In order to determine the degree of purity of the

mitochondrial amine oxidase, the following experiments

were done:

(a) Rechromatography on DEAE-ce11u1ose

After the final step of purification (starch

block electrophoresis step), component 2 was rechro­

matographed on the DEAE-ce11ulose column (25 x 1.6

cm), which was equilibrated with 0.01 M potassium

phosphate buffer, pH 7.4. Gradient elution,

using 250 m1 of 0.01 M buffer containing 0.5 M

sodium chloride in the reservoir and 250 m1 of 0.01

M potassium phosphate, pH 7.4, in the mixing chamber,

was made. As shown in Figure 3, a single component

was observed.

Figure 3. Rechromatography of the purified enzyme component 2

on the DEAE-ce11u10se column. Protein, 15.1 mg, containing 1.18 x 105 units

of activity, was applied to a column (25 x 1.6 cm) which had been equi1i-

brated with 0.01 M potassium phosphate buffer pH 7.4. Gradient elution was

used to elute the enzyme (250 ml of 0.01 M buffer in the mixing chamber

and 250 m1 of 0.5 M sodium chloride in 0.01 M buffer in the reservoir).

Fractions of 5.4 m1 were collected at a flow rate of 0.3 m1 per minute.

The symbols indicate: -0-0-0-, enzyme activity; -0-0-, protein concentration;

and -6-6-, sodium chloride concentration.

.p­I-'

42

(lW/OW) NI3J.0~d dI

v(W) I:>DN 0

_ 0eN

Il\.\

•\ •

\ \ !J\ ,/ /., ~..o 0

....0.... /~_o........ /0

__-0----0- , /0..0-- \ "~-o • O~---0 ---- ~~ ~Lr--"'"i',-1.. -'. ----o_~

\\

•\

•~

\ •

-0-U) E

VIt)-0:IJJm~

C""')

::::;) <I)

Z l-I

0 :sbOv Z -.-I

0 J::.l....(.)

«0:LL.

\•\

•\

•\~--1.-_--_---J._--__...L- --IO

eN

£_01 X(lW / SI!Un) A..1IAIJ.:>\1

43

(b) Rechromatography ~ hydroxylapatite column

After the starch block electrophoresis step,

component 2 was subjected to hydroxylapatite column

chromatography. It was applied on a hydroxylapatite column

(10 x 1.6 cm), pre-equilibrated with 0.01 M potassium

phosphate buffer, pH 7.4. Fraction 2 was eluted as

one peak with 0.2 M buffer, when stepwise elution

with 0.1 M, and 0.2 M buffer, was used. The result is

shown in Figure 4.

(c) Sephadex ~ filtration

A Sephadex G-200 gel column (120 x 1.9 cm)

chromatography of the fractions 1 and 2 are shown in

Figures 5a and 5b. A single peak was observed for

each component.

(d) Analytical starch block ~lectrophoresis

Starch block electrophoresis was performed as

described by Fine and Costello (59) in the section,

Materials and Methods. At the close of the experiment,

1/2 cm transverse sections were cut and eluted separately

with 2 ml of 0.1 M potassium phosphate buffer, pH 7.4,

containing 1 x 10-4 M dithioerythritol. Protein and

activity determinations were made on the eluates. Both

components moved as single bands as shown in Figure 6.

The first component moved a distance of 4.8 cm and the

second, a distance of 6.8 cm from the point of origin

towards the anode.

Figure 4. Rechromatography of purified component 2 on hydroxylapatite.

Protein, 8.75 mg, containing 6.9 x 104 units of activity, was applied to

a column (10.5 x 1.6 cm) which was equilibrated with 0.01 M potassium

phosphate buffer, pH 7.4. Stepwise elution was used. Fractions of 2.2 m1

were collected at a flow rate of 0.2 ml per minute. The symbols represent:

A, 0.1 M potassium phosphate buffer, pH 7.4; B, 0.2 M potassium phosphate

bufferi pH 7.4; -~-~- is enzyme activity, and -o-o-is protein concentration.

.p­

.p-

\,

45

It)

(lWI f»W) N131.0~d d ~.--.....----------~--__r--__,~

......<1..".,

m _<1----- <J ------<1 ---­.......-­"'1--------

ov

-E

C\I

ONrt) -

a:LLJm --r::E::> <1l

z l-l::l

0 00

Z"..I

N r=..0-i-0«0::LL

0

en v£_01 X (IWI Sl!un) Al.IAll.:l'1

I--..L..-.....!-------_L...-. ---JO

46

Figure Sa. Chromatography of amine oxidase

components 1 on Sephadex G-200. Three milligrams of

enzyme component 1 in 1.5 ml of 0.05 M potassium

phosphate buffer, pH 7.4 containing 0.01 M mercapto­

ethanol were applied to the column and eluted with

the same buffer. Fractions of 3 ml were collected

Curve -~-~- represents protein, and curve

-0-0- represents activity.

8

(\I 710 1\- 6X 0-E 5

.......II)- 4·c~- 3~.-- 2>-.-(,) I«

o 4 20 24 28 32 36 40

FRACTION NUMBER (3 ml each)

Figure Sa

47

-E

.......C'E-Z

0.20 W

b0.15 0::a..

0.10

0.05

48

Figure 5b. Chromatography of amine oxidase

component 2 on Sephadex G-200. Two milligrams of

enzyme component 2 in 1.5,ml of 0.05 M potassium

phosphate buffer, pH 7.4, containing 0.01 M

mercaptoethanol were applied to the column and eluted

with the same buffer. Fractions of 3 ml were

collected at 0_4 0 • Curve -0-0- indicates activity

and curve -~-~- indicates protein concentration.

49

oFRACTION NUMBER (3 ml

rt)

I 8.00-X-E"" 6.0 2.0 -=en

E-,c "":::J C'- E>- 4.0 -~ Z- -> 1.0 IJJ- ~~ 00« 2.0 0::

Q.

Figure Sb

Figure 6. Migration of the amine oxidase components in starch

block electrophoresis. About 10 mg of each component in 2 rol buffer,

pH 7.4 with ionic strength of 0.1, were used. Electrophoresis

continued for l7~ hours at 410 volts and 15 mA per block. Charging

of protein in the block and electrophoresis run was carried out in the

cold room (0-4 0 ). Curve-o-o- indicates activity and curve -t:.-b.- indicates

protein concentration.

VIa

10. i j

024

t STARTING ZONE

If)

I 8

0-X-- 6E

"en-.-C::J 4->-t: 2>-I-U«

Component

6'\

1 7 If~ ,~/ \.l

Figure 6

Component 2

6,~I ,

d\I ,I ,

I ', 'I

6 8

( centimeter)

+10

~

E"1.2 e-z-ILl

.8 I­oa::0.

.4

Vt....

52

(e) Free boundary electrophoresis

Purity check of component 2 was made in the Perkin-

Elmer electrophoresis apparatus. The Fraction 2 was

dissolved and dialyzed against potassium phosphate buffer

of 0.1 ionic strength, pH 7.4, containing 0.2 M mercapto­

ethanol for 4 hours with 2 changes of buffer. Electro­

phoresis was carried out in the same buffer at 4 0• A

single peak was observed (Figure 7).

(f) Polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis was carried out as

described in the Methods section (39). Component 1 moved

a distance of 2.2 cm and component 2 moved 1.2 cm from the

starting zone as shown in Figure 8.

(g) Ultracentrifuge studies

The sedimentation behavior of the enzyme component 1

was studied in phosphate buffer, pH 7.4. The sedimentation

pattern shown in Figure 9, indicates homogeneity of the

component.

B. Kinetic Properties

1. Activity of the Enzyme

The mitochondrial amine oxidase components 1 and 2

have specific activities of 3000 to 4000 and 7000 to 9000,

respectively. Although both the components are unstable,

component 1 shows relatively high stability as compared to

component 2. In 0.1 M potassium phosphate buffer, pH 7.4

containing 1 x 10- 4 M dithioerythritol at 0 - 4 0 , component

---_.._-_ _-_..__ .

53

Figure 7. Electrophoretic pattern of component 2.

A 0.5% solution of the enzyme (specific activity, 8000)

dissolved in 0.1 ionic strength of potassium phosphate

buffer, pH 7.4, containing 0.02 M mercaptoethanol, was

used. The electrophoretic pattern of the ascending

limb was photographed after 75 minutes. The run was

made at 4 0 and the field strength was 9.28 volts per

cm 2 . The mobility was calculated to be -5.21 x 10- 5

cm2 volt- l sec-I.

'" ;

Figure 7

54

Figure 8. Polyacrylamide gel electrophoresis of amine oxidase

VIVI

components 1 and 2. (a) separation of component 1 from component 2

when a mixture of both was applied; (b) a single band of component 1;

and (c) single bands of component 2. Electrophoresis runs were

carried out under the conditions described in the Materials and

Methods Section.

00

56

Figure 9. Sedimentation pattern of amine oxidase component 1.

Sedimentation studies of a 3.6 mg enzyme dissolved in 0.1 M potassium

phosphate buffer, pH 7.4, were made at 22.5°, and at 35,600 rpm.

Photogralphs we re taken at 8 minu te in terva 1 s a f te rat ta ining top

speed.

I.n

"

58

59

was found to be stable at least for a week, after which the

activity declined gradually. Component 2 deteriorated

faster than component 1 under the same conditions. Stability,

moreover, was found to be a function of the enzyme concen-

tration. The more concentrated the enzyme components in

solution were, the more unstable they were. Freezing

destroyed component 2 in a day and component 1, in a few

days.

2. Effect of Temperature ~ the Enzyme Activity

The effects of temperature on the partially purified

enzyme after the DEAE-cellulose step, as well as on the

purified enzyme components 1 and 2 were studied and the

results are shown in Figures 10aand lab. The partially

purified enzyme from the DEAE-ce11ulose column incubated

for 15 minutes at various temperatures, and was found to be

unstable at any temperature above 30 0 • The purified enzyme

component 2, under the same conditions, was found to be

more stable than the partially purified enzyme. Component 2

retained the initial activity up to 40 0 whereafter the

activity dropped progressively with increasing temperatures.

The partially purified enzyme, on the other hand, retained

only 65% of the initial activity at 40 0• Furthermore, the

purified enzyme component 2 was more heat stable in the

presence of 1 x 10-4 M dithioerythrito1. Similar findings

were obtained with the enzyme component 1.

60

Figure lOa. Effect of temperature on the

enzymatic activity. For the experiments, 0.5 ml

samples of the enzyme (1.94 mg per ml, specific

activity, 3920) after the DEAE-cellulose step were

diluted in 4.5 ml of 0.1 M potassium phosphate

buffer, pH 7.4, and preinculated for 15 minutes at

temperatures from 25-60°. The solutions were cooled

to 25°, and the activity of 0.05 ml aliquots was

determined at 250. The standard assay method was

used.

0.4.-----------------.,

61

-c.-E

"'~ 0.3EoanN

C; 0.2

oo<1->- 0.1~->-~o«

20 30 40 50 60

TEMPERATURE (OC)

Figure lOa

62

Figure lOb. Effect of temperature on the

activity of the amine oxidase. For this experiment,

720 units of enzyme component 2 (specific activity,

7,650) in 2 m1 of 0.1 M potassium phosphate buffer,

pH 7.4, were incubated for 15 minutes at various

temperatures ranging from 25 to 60°. The solutions

were cooled to 25°, and the activity of 0.1 m1

a1iquots was determined under the usual assay

conditions a t2 5 ° . "

150

>-....->- 100....0<t

~0 50

25 30 35 40 45 50 55 60 65

TEMPERATURE (OC)

Figure lOb

63

64

3. Effect of ~ ~ ~ Enzyme Activity

The effect of variations of pH on the activity of the

enzyme components 1 and 2 were studied. As found with the

partially purified enzyme (53) the pH optima of the enzyme

components 1 and 2 were pH 9.1-9.2 which are in close

agreement with the value reported earlier by Hare (1).

Both these values, however, are different from those

reported by others (39, 40). The buffer systems used were

0.2 M potassium phosphate-pyrophosphate buffers. Figure 11

illustrates the eff~ of pH variation on the activity of the

enzyme components.

4. Substrate Specificity

The substrate specificities of the two components were

determined with various amines by a method developed by

McEwen (12) in which the usual amine oxidase assay was

coupled with the peroxidase-o-dianisidine color reaction (41)

Table II summarizes the results on various amines including

an amino acid.

5. Inhibitor Specificity

Inhibition studies provide good tools to track down

dertain specific groups or moieties which might be involved

in the cataytic function of an enzyme. Accordingly, the

following inhibition studies were made.

(a) Product inhibition

As many enzymes are inhibited by a product of the

reaction they catalyze (feed back inhibicion), the effect

Figure 11. Effect of pH variation on the activity of the enzyme

components 1 and 2. Potassium pyrophosphate buffer (0.2 M) was used.

Each reaction mixture contained 0.05 ml of the enzyme component 1 (0.12

mg per milliliter, specific activity 3,200), 1.67 mM of benzylamine in

a total volume of 3 mI. For component 2, the reaction mixture contained

0.05 ml of the enzyme solution (0.07 mg per milliliter, specific activity,

7,100). Other conditions were like those of component 1. Activity at

various pH values was measured by the usual method.

CJ\VI

C\I-cCDco0-Eou

orr>

oC\I

-cCDco0-Eou

o

o

66

( U!W/ OW OSZ aov ) A.LIAll.~'f

Sub s tra te

67

TABLE II

Substrate Specificities of the Two AmineOxidase Components*

Relative Specificity of Components

Component 1 Component 2

MonoamineBenzylamine 100 100Heptylamine 82 79Tryptamine 18 32Tyramine 13 30Mescaline 0 0Serotonin 3 5

DiamineKynuramine 43 52Agmatine 0 0Butanediamine 0 0Cadaverine 0 0Histamine 0 0Trimethy1enediamine 0 0

CatecholamineNorepinephrine 41 46Epinephrine 21 25

PolyamineSpermidine 0 0Spermine 0 0

Basic amino acidLysine 0 0

*Each reaction mixture contained 45 units of enzyme (specificactivity 5,600, and 7,800 for first and second componentsrespectively), and 3-3 mM of substrate in 0-06 M potassiumphosphate buffer, pH 7-0_ The table shows relativesubstrate specificity with different amines at 25 0 _

68

of NH 3 on the re~ion of the enzymeoomponents was investigated.

The activity was examined in the presence of various concen­

trations of (NH4)ZS04' A 50% inhibition of the enzyme

(after the DEAE-cellulose step) was observed at an (NH4)ZS04

concentration of 0'4 M. The results are shown in Figure lZ.

(b) Inhibition ~ sulfhydryl ±eagents

Since there are many reports on the inhibition of

mitochondrial amine oxidase by sulfhydryl reagents, and

reports that the enzyme is a sulfhydryl enzyme (90-93),

effects of various sulfhydryl reagents on the enzymatic

activity of the partially purified enzyme and that of the

two purified components were investigated.

Mercuric chloride, silver nitrate, p-chlormercuribenzoate

(p-CMB), cadmium sulfate, and sodium arsenite were studied

from amongvarious mercaptide forming reagents. All but

arsenite had inhibitory effects. Cadmium showed 60%

inhibition in the activity of the partially purified enzyme

only when its concentration was prohibitively high (Figure l3a,

Tables III A, III B, and III C). However, these results

did not show conclusively whether the inhibition was

indicative of the participation of sulfhydryl group(s) in

the enzyme activity or was due to a general effect of heavy

metals on enzyme.

As p-CMB is a most widely used thiol reagent, it was

employed to examine the type of inhibition produced by a

thiol reagent. Accordingly, activities of the two components

Figure 12. Product inhibition studies. The reaction mixture

contained 35 units of amine oxidase component 1 (specific activity,

3,250). The ordinate shows the percentage of inhibition and the

abscissa shows the molar concentrations of ammonium-sulfate.

0\\0

oU)

ov

oC\I

70

10o

~IJJt-

O <tl.L...J::>(/)

ro0 I

~

~ .-I

:::> Q)- ,..2 ::l

0 be

~-r-!

~r:..

0~<[

LLJ

d ...J0~

NOIJ.18IHNI J.N30~3d

Figure l3a. Inhibition of amine oxidase by sufhydryl reagents.

Each reaction mixture contained 46 units of amine oxidase (specific

activity, 4,200), plus 1.7 mM benzylamine as substrate and various

concentrations of inhibitors shown in the plot. The ordinate(s)

indicates percent of activity and the abscissa gives the molar

concentrations of the inhibitors used.

-..J....

100

90AgN03

>-eOr \ \ \f

t- 70->-60tHgCI2t-

O

50 ~<t

t-ZL1J 4000::L1J 30Q. . , . x

20 \- \ \ \10 \- \ \

~. I v-7 -6 -5 -4 -3 -2

LOG MOLE INHIBITOR.......N

Figure 13a

73

TABLE III A

Inhibition of Amine Oxidase by Sulfhydryl Reagents*

Inhibitor ConcentrationpM

Inhibition%

p-Chloromercuribenzoate 5'00 37'5

2'50 22'7

Silver nitrate 5'00 37·5

2'50 10' 9

Mercuric chloride 1'00 100'0

0"25 0"0

Cadmium sulfate 1000'00 60·0

Sodium arsenite 1000'00 0'0

* Standard assay conditions were maintained, and eachreaction mixture contained 46 units of enzyme with aspecific activity of 4,200.

74

TABLE III B

Inhibition of Amine Oxidase by Sulfhydryl Reagents*

Inhibitor

Mercuric chloride

Silver nitrate

p-Chloromercuribenzoate

Cadmium sulfate

Concentration for 50% InhibitionpM

0-56

3-31

4-27

513-00

*The table shows the inhibitor concentrations at which 50%inhibition occurs in the enzyme_ Standard conditions wereused in each determination and each reaction mixturecontained 46 units of enzyme having a specific activity of4,200_

75

TABLE III C

Inhibition of Amine Oxidase a by Sulfhydryl Reagents

Inhibitor Inhibitor % InhibitionConc.pM Component 1 Component 2

p-Chloromercuribenzoate 2.5 26 24

Silver nitrate 5.0 91 96

Mercuric chloride 1.0 96 91

0.5 72 80

Sodium arsenite 1000 0 0

Cadmium chloride 330 45

Iodoacetic acid 50 0 0

Iodoacetamide 1000 Ob_1Oc

N-ethylmaleimide 1000 Ob_ 34c

aThirty-two units of compon~ 1 (specific activity 3290) and35 units of component 2 (specific activity 7,050) were usedfor these experiments. Mole ratios of the inhibitors toenzyme components varied from 200 (for HgC1 2 ) to 4,000,000(for alkylating reagents) considering the molecular weightsof 400,000 and 1,280,000 for components 1 and 2, respective­ly. Activity was measured by the usual benzylamine assaymethod.

blnhibitions after incubating for 2 hours.

clnhibitions after incubating for 24 hours.

76

at various substrate concentrations were determined at a

fixed concentration of p-CMB (5 x 10- 6 M). A Lineweaver-Burk

plot, as shown in Figure l3b demonstrates that p-CMB is a

non-competitne inhibitor of the component 1. The same type

of inhibition was also observed with component 2 (Figure l3c).

(c) Inhibition Ex. Metal Chelating Agents

Substances like cyanide, azide, phenanthroline, and

some other metal chelating reagents inactivate the enzymes

which contain heavy metal(s) (e.g. iron, copper, molybdenum,

manganese, etc.) as prosthetic group(s). In order to

determine whether a metal was involved in the activity of

the mitochondrial enzyme, the effect of some metal chelators

on the activities of the enzyme was investigated. Table IV

summarizes the results of these studies. In addition, the

type of inhibition produced by cuprizone is illustrated in

Figure 14.

(d) Inhibition Ex. Aldehyde Reagents

Aldehyde or carbonyl reagents may in some cases act

like inhibitors by combining with a carbonyl group in the

enzyme itself, or with a cofactor or prosthetic group

(e.g. pyridoxal phosphate) as in the case of the plasma

amine oxidase (28). Studies were made with both enzyme

components to determine whether or not they are inhibited

by carbonyl reagents. The results show the effect of some

of the well known carbonyl reagents (Table V).

Figure l3b. Lineweaver-Burk plot of benzylamine oxidation in

the absence and presence of p-CMB. Each reaction mixture contained

45.5 units of enzyme component 1, specific activity 3,550. The

ordinate gives the reciprocal of the activity in terms of the change

in absorbance at 250 mpper minute and the abscissa gives the reciprocal

of the molarity of the benzylamine. I is the concentration of p-CMB

used.

,

""

78

-I

N0..

" \ 00

en

CO

to-X

10 <DII

H 10It') ..c

V '0<"")

.....Ql

It')l-<

X ::leo

-I(/) .~

N ~

NI

~

VI

'-------------------_--J 10I

Figure l3c. Lineweaver"Burk plot of benzylamine oxidation in

the presence and absence of p"CMB. Each reaction mixture contained

52 units of enzyme component 2, specific activity 7,600. The ordinate

gives the reciprocal of the activity in terms of the change in absorbance

at 250 mll per minute and the abscissa gives the reciprocal of the molarity

of benzylamine. I is the concentration of p"CMB used.

,

.....\0

80

--0

0 0'" 0 en

II0)

X ~

&0

.. (D

.....&0

0'\\ q-", 0

'0l:'"l......

rt> - QJ

~

X ;:lbO

\;\ -len -.-I(\J ~

000

-

"I

~Ol X T (\JI

rt>I

q-I

&0I

81

TABLE IV

Inhibition of Amine Oxidase by Metal Che1ating Agents*

Chela tors

None

Bis-cyc1ohexanone oxa1­dihydrazone (Cuprizone)

Neo cupro ine

8-Hydroxyquiono1ine

Sodium diethy1dithio­carbamate

o-Phenanthro1ine

a"a -Dipyridine

Ethy1enediaminetetraacetate

Sodium azide

NaCN

Final Inhibition

Concentration %

mM

0 0

0.3 76

0.3 33

3.0 90

3.0 24

0.3 19

3.0 10

3.0 0

30.0 0

30.0 0

*The enzyme after the DEAE-ce11u1ose column chromatographywas assayed llsing the kynuramine assay of Weissbach et a1.(94). For the experiments, 0.1 m1 of partially purifiedenzyme, specific activity 3360, was preincubated with theche1ating agent mentioned in the table for 15 minutes at26 0 and" then assayed.

Figure 14. Lineweaver-Burk plot of benzy1amine oxidation in

the presence and absence of cuprizone. Each reaction mixture contained

22 units of enzyme, specific activity 3,500. The ordinate gives the

reciprocal of the activity in terms of the change in absorbance at

250 mp per minute and the abscissa gives the reciprocal of the molarity

of benzylamine. I is the concentration of the cuprizone.

(Xl

N

83

~<X>

LOI0 rt>

CDI

X 0

LO X..v -1(1)

-::t~

C\IOJl-l

=='be.~

r:..

84

TABLE V

The Effect of Aldehyde Reagents on the Enzyme

Activity*

Inhib itor Final Inhibition

Concentration %}lM Component 1 Component 2

Hydroxylamine 330 0 0

Phenylhydrazine 3.3 31 30

p-Nitropheny1hydrazine 3.3 53 44

Semicarbazide 33 1 0

Hydrazine 33 2 2

Potassium benzoate 3.3 10 2

*The reaction mixture contained 35 units of enzyme. Themole ratio of inhibitor to enzyme components 1 (M.W.407,000) and 2 (M. W. 1,280,000) varied between 1200(forphenylhydrazine, etc.) and 120,000 (for hydroxylamine), and2440 (for p-nitropheny1, etc.) and 240,400 (for hydroxylamine),respectively. The results did not change on preincubationof the enzyme with the inhibitors for 10 minutes at 25 0 •

85

C. Physical Properties

1. Spectral Properties

The enzyme components are bright yellow in color at the

final step of purification in contrast to the plasma enzyme

which is pink in color. The absorption spectrum of

component 2 of the mitochondrial amine oxidase is mown in

Figure 15a. The spectrum of the enzyme in 0.1 M potassium

phosphate buffer, pH 7.4, was taken in Beckman DK-2 Ratio

Recording SpectrophotomSEr. The absorption spectrum differs

from that of a typical f1avoenzyme. However, the 450 mp

peak is indicative of the presence of flavin. There are, in

addition, an absorption maximum at 410 m~ and a shoulder

at 480 mp. When the enzyme was treated with substrate

(benzy1amine) or sodium hydrosu1fite (Na2S204), the 450 m~

shoulder disappeared (Figure 15b). The peak at 450 mp and

the 480 m~ shoulder were partially restored when air was

carefully admitted. Component 1 showed similar spectral

properties.

2. Sedimentation Coefficients

A sedimentation pattern of component 1 has been shown

in Figure 9. Runs were made in 0.1 M potassium phosphate

buffer, pH 7.4, in the Spinco analytical ultracentrifuge.

Also, sedimentation coefficients were determined in the

preparative ultracentrifuge. Figure 16 summarizes the

sedimentation coefficients determined at various protein

concen tra t ions. The values obtained for component 1 was

Figure 15a. Absorption spectrum of the purified enzyme component 2.

The enzyme with specific activity of 7800, was used. The concentration

of the enzyme was 0.56 mg/m1 and the enzyme was dissolved in 0.1 M

potassium phosphate buffer, pH 7.4.

00(J\

1.0

UJoz«en0::oCJ)

~ 0.5

89

0.4

0.1

480

240 280 320 360 400 450 500WAVE LENGTH m)J

Figure lSa

550ex>.....

Figure l5b. Reduction of the spectrum of enzyme component 2

by substrate and sodium dithionite. Spectra of the purified enzyme

(2.15 mg/ml; specific activity, 7950), ; the benzylamine (150

mole per mo~ of enzyme) reduced enzyme, -.-.-; and the sodium

dithionite (5 mole per mole enzyme) reduced enzyme, ----. Spectra

were taken in 0.1 M potassium phosphate buffer, pH 7.4 aerobically.

0000

289

1.0

180

0.1

0.3

0.4LLI()Z<tm0:oCJ)m<t 0.5

240 280 320 360 400 450 500 550WAVE LENGTH m)J

Figure 15b00\0

Figure 16. Sedimentation coefficients of amine oxidase component 1

at varying protein concentrations, Sedimentain behavior was studied in

0.1 M potassium phosphate buffer, pH 7.4 at 22.5°, Runs were made at 35,600

rpm (73,684 x g) using the rotor type An-D.

1.0o

I 10- Z00

00 -l-v <t- - 0::0 I-

ZIJJ

rt') 0-0 z

0 0 ""0 ...-l

(\JQ)

0 l-<- . z ;::l

I 0 - bll

IJJ-,-I

r:«t-

O 0- 0 0::a.~0

I I I I I

..... eD 10 V rt') (\J

~3S £1°1X M'O~S

91

92

found to be independent of concentration (Table VI A). When

corrected to the standard conditions (i.e., at 20 0 in water

as solvent), the sedimentation coefficient of component 1

was found to be 14.4 + 0.3. Values obtained in sucrose

density gradient were 14,5 ± 0.2 and 20.6 + 0.4 for

components 1 and 2, respectively (Table VI B).

3 . Par t i a 1 SP e c i f i c Vo 1 um e s

Table VII shows the results of the determination of

partial specific volumes of components 1 and 2. For these

experiments, the enzyme components were dialyzed for 4

hours in 0.05 M potassium phosphate buffer, pH 7.4 with

three changes.

as the solvent.

The buffer from the last dialyzate was used

The relative densities of the enzyme

solution and solvent were determined pycnometrically at

The partial specific volumes of the enzyme

components 1 and 2 were calculated as described by

Schachman (67) and were found to be 0.782 cm 3 jg and

0.805 c m3Jg respectively.

4. Molecular Weights

(a) Molecular weights detemined by Agarose (Bio Gel

A-~ m) gel filtration

Molecular weights for the two components of the

mitochondrial enzyme were estimated by the gel filtration

technique using agarose gel columns. Table VII lists the

elution volumes of the standard (or marker) proteins, and

Blue Dextran 2000. The void volume was found to be 93 ml

by using E. coli which is excluded by the agarose gel used (7Q).

TABLE VI A

Sedimentation Coefficients at Different Protein

Concentrations of the Mitochondrial Amine Oxidase*

93

No °

1.

2.

3 °

4o

5 °

Proteinconceno

%

0°45

0°27

0~20

0°14

0°44

s20 w x 10 13 sec,

14°3

14°6

14·3

14°5

14°1

Average

s20 w x 10 13 sec,

14°4 + 0°3

*Runs were made in 0°1 M potassium phosphate buffer,

pH 7°4, at 35,600 rpm, using analytical rotor, type

An-D a t 22 ° 5 0 °

94

TABLE VI B

Sedimentation Coefficients by Sucrose Density Gradient*

Species

Component 1

Component 2

'>'(Runs were made

s20 w x 10 13 sec,

14·5 + 0·2

20"6 + 0"4

in 20% - 5% sucrose gradients in 0"1 M

potassium phosphate buffer containing 1 x 10- 4 M

dithioerythrito1 at 75,000 x g for 16 hours at 0 0• The

values are averages from 2 runs.

95

TABLE VII

Agarose Gel Filtration Data of Standard Proteins, BlueDextran 2000, and Amine Oxidase Components a

Species

Standard

Cyt. c

BSA (monomer)

Catalase

Ferritin

Blue Dextran

Amine Oxidase

Component 1

Component 2

Mol. Wt.

x 10- 3

12.4

65- 70

250

747

2,000

V be

(ml)

207

174

149

130

105

141

114

V· Iv ce 0

2.23

1. 87

1. 61

1. 39

1.13

1. 52

1. 23

log Mol. Wt.

4.09

4.845

5.398

5.874

6.310

5.61

6.114

aEquilibration and elution were done with 0.05 M potassiumphosphate buffer, pH 7.4 containing 0.01 M mercaptoethano1.

b Ve = Elution volume.

cVe/vo = Ratio of elution volume to void volume (V o)' Vovolume was determined by using E. coli K 12 which isex c 1 u de d by a 11 kin d s 0 f s e p h a dex and a gar 0 s e gel s ( 70) .

96

Figure 17 shows the linear relationship of the logarithm

of molecular weights to the ratios of elution volumes of

the proteins to void volume (Ve/Vo). The ratios Ve/Vo

were found to be 1.52 and 1.23 for component 1 and component 2

corresponding to logarithms of molecular weights of 5.61 and

6.114 respectively. These values correspond to molecular

weights of 400,000 for component 1 and 1,300,000 for

component 2.

(b) Molecular weights determined from Stoke's Radii,

Sedimentation coefficients, and Partial Specific Volumes

Stoke's radii for components 1 and 2 were estimated

from the known linear relationship of the distribution

coefficient (Kd) to the molecular (or Stoke~s) radius

(Figure 18) (from agarose gel filtration data). The

Stoke's radii of various proteins and the two amine oxidase

components are illustrated in Table VIII. Stoke's radii

for components 1 and 2 were found to be 60 ~ and 106 ~,

respectively. Sedimentation coefficients as mentioned in

section 7 above were 14.4 + 0.3 and 20.6 for the two

components. Partial specific volumes were determined by

the method of Schachman (67) and were found to be

0.782 cm3 /g for component 1 and 0.805 cm3 /g for component 2.

Placing these values in equation 3 (page 15), molecular

weights were calculated as 396,000 + 10,000 and 1,195,000

for component 1 and component 2 respective1y~

97

Figure 17. Agarose gel filtration data of various

standard proteins and Blue Dextran 2000, and amine oxidase

components. Agarose gel was equilibrated in a 1.9 x 120 cm

column with 0.05 M potassium phosphate buffer, pH 7.4,

containing 0.01 M mercaptoethano1. Fractions of 3 m1 were

collected. The work was conducted at 0_4 0 •

o>.......Q)

>

2.0

1.0

'0

98

O~~:~:::aseMAO~",

Ferritino~

MA02~

"-Blue De xtran 0,

4 5 6

LOG MOLECULAR WEIGHT

Figure 17

6.5

99

Figure 18. Correlation of Kd with Stoke's

radius. Agarose gel filtration data were plotted

as described by Porath (72).

0.9 .---------------------.

100

0.8 -

0.7 ­I

K 3d

0.6 -

0.5 -

"o~

O~ (monomerl

O~atalase

~~o Ferritin

0 11 I I I.4 '--__.J...-__-'--__...&.-__...L--__--'--__---'

20 40 60 80 100 120o

STOKE'S RADIUS, A

Figure 18

101

TABLE VIII

Molecular Parameters Obtained from Gel Filtration Data

Species Mol. Wt. Stoke's Ve Kdx 10- 3 radius (ml)

0A

Standard

Cyt. c 12"4 10 207 0·675

BSA (monomer) 65- 70 35 174 0·480

Catalase 250 52 149 0·335

Ferritin 747 79 130 0"220

Amine OxidaseComponents

Component 1 60 141 0·32

Component 2 106 114 0·124

•102

(c) Molecular weights determined from sedim~ation-

diffusion coefficients and Stoke's radii

If Stoke's radius of a macromolecule is known, the

diffusion coefficient can be calculated from the equation

Dk T

6 na

(vi)

where T absolute temperature, n = viscosity of the

medium, and k = Boltzmann's constant. Diffusion coefficients

Placing

for components 1 and 2 were calculated to be 3.8 x 10- 7

cm 2 sec- l and 2.03 x 10- 7 cm 2 sec- l , respectively.

these values in Svedberg's well known equation

MR T.s

D(l-Vp)(vii)

molecular weights obtained were 423,000 and 1,355,000

respectively for components 1 and 2. Various molecular

constants and the molecular weights of the two components

determined by three different methods, are summarized in

Tables IX and X, respectively.

5. Frictional Ratios

The frictional ratios of the amine oxidase components are

shown in Table XI • These values were calculated from

the molecular weights of the two components by using the

equation (v). Values obtained for components 1 and 2 were

1.17 and 1.46, respectively, indicating that the component 1

is more ~herical than the component 2.

103

TABLE IX

Physical Parameters of the Mitochondrial Amine Oxidase

Species Stoke'saRadius

oA

Component 1 60

Component 2 106

bs20, W

(X 1013 sec)

14.4 + 0.3

20.6 + 0.4

3.8

2.0

Vd

cm 3 Jg

0.78

0.80

aStoke's (molecular) radii were determined from the KdlJ3 vs

Stoke's radius standard plot (Figure 18).

b The sedimentation coefficient of component 1 is the averageof five values (Table VI A), and that of component 2 is theaverage of 2 values determined by sucrose density gradienttechnique.

cDiffusion coefficients of the two components were determinedfrom the equation, D = k T

6 na

dV is the partial specific volume determined by the methodof Schachman (67).

104

TABLE X

Molecular Weights of the Amine Oxidase Components by threeMethods

Method

Gel Fi1trationa

6 nNas bM =

(l-"Vp)

Component 1

408,000 + 9,000

396,000 + 17,000

Component 2

1,300,000 + 70,500

1,195,000 + 90,500

M = RTs cD(l-Vp)

423,000 + 10,000 1,355,000 + 27,500

Average 406,000 + 14,700 1,280,000 + 91,500

aThe molecular weight determinations are based on threegel filtration runs.

bMo1ecu1ar weights are estimated from five sedimentationcoefficient values for component 1 and two from two valuesfor component 2.

cS ame as that for molecular weight determinations fromStoke's 1awb .

105

TABLE XI

Frictional Ratios of the Amine Oxidase Components

Species

Component 1

Component 2

Stoke'sRadius

oA

60

106

FrictionalRatiof/f o *

1. 17

1. 46

*Frictiona1 ratios for the two enzyme components

were calculated by using the following equation:

f/ f o= a/(3VM)1/3

(4 N)

106

D. Chemical Properties

1. Metal Content

Metal analyses were made according to the methods

mentioned earlier in Section 8 under Materials and Methods.

The copper content was determined in a number of purified

preparations of component 2 which yielded values ranging

from 0.15 to 0.17 pg/mg protein. In addition, copper content

was measured in each step of purification (Figure 19a).

Determinations were also made for other metals such as

cobalt, iron, manganese, and molybdenum. Iron was present

in insignificant amount (0.02 pm/mg protein) and was

considered to be a contaminant as determined by measuring

its content in each purification step (Figure 19b). Other

metals examined were found to be absent. The results of

purified components 1

Total(87) .

these determinations are summarized in Table XII.

2. Phosphorus Content

(a) Total phosphorus

Total phosphorus was determined in

and 2 by the method referred to earlier

phosphorus content determined in a number of preparations

yielded average values of 2.56 + 0.05 pg and 3.37 + 0.03 pg

per milligram of component 1 and component 2, respectively.

These values correspond to total phosphorus contents of

0.0815 + 0.0015 pmole and 0.1095 + 0.0008 p mole per

milligram of protein, respectively, for component 1 and

component 2.

Figure 19a. Copper content of the enzyme. The copper content

and specific activity of the enzyme were determined at each step of

the purification procedure. The copper contents were determined by the

method of Peterson and Bollier (73).

t-'o

"

.)

108

00 _0

\ 0ex>

\, 00, - 0

\ .....\ >-

0 I-\ - 0 -\ 0 >(0

\ -\ 0 I-

- 0 u, 0 <{0

10

\ 0U, 0 ell

- 0 0-

\ v LL .-l

\ UCI.l

0 l-<,W ::s, 0 bO- 0 CL ..-l

\ rt> C/)~

00\ 0

\ - 0 W\ C\J

~\I >-J 0 NI

_ 0Z0

/ W_/o _----0

0------;--- -I I I

<0 10 V rt> C\J -. .0 0 0 0 0 0

'NI3J.OHd OWl H3ddO~ orl

Figure 19b. Iron content of the enzyme. The iron content and

the specific activity were determined at each step of the purification

procedure. Iron was determined according to the method of peterson (76).

I-'o\0

0.6

0.5

0« 0.4::E

0-E

....... 0.30-::J..

0.2

0.1

110

o 1000 2000 3000 7000 8000

SPECIFIC ACTIVITYFigure 19b

111

TABLE XII

Metal Content of the Amine Oxidase

Metals rg/mg Protein Method

Copper a 0'15- O' 17 Microchemical (46)

Ironb 0'02 Micro chemica 1 (49)

Cobaltb 0'00 Atomic Absorption (48)

Manganeseb 0'00 Atomic Absorption (48)

Molybdenumb 0'00 Micro chemica 1 (50)

aCopper was determined in purified component 2 only.

bThese determinations were made on the enzyme after theDEAE-cellulose step. As these metals were either absentor in insignificant amounts, they were not investigatedin purified enzyme.

112

(b) Phospholipid phosphorus

Lipid was extracted from a number of purified

preparations of component 1 and component 2 by the method of

Fo1ch et al. (88). Phosphorus determinations were made on

the extracted lipid according to the method of Bartlett (87).

From a number of determinations. average values obtained

were 1.84 + 0.01 pg and 2.71 + 0.05 pg per milligram of

protein for component 1 and 2. respectively. These values

correspond to phospholipid content of 0.059 + 0.0005 ~M and

0.086 + 0.02 ~M per milligram of protein (considering

1 g-atom phosphorus per mole of phospholipid).

(c) Flavin dinucleotide phosphorus

These values were calculated by subtracting phospholipid

phosphorus from total phosphorus which yielded values of

0.67 pg and 0.679 pg per milligram of protein. respectively.

for components 1 and 2.

Tab 1 e XIII.

All these values are summarized in

113

TABLE XIII

Phosphorus Content of Mitochondrial Amine Oxidase

Species

Component 1

Component 2

Total aPhosphorus

p.atom/mgProtein

0-0814

0-1094

Phospho1ipid b

Phosphorus

p.atom/mgProtein

0-059

0-086

Nucleotide cPhosphorus

}latom/mgProtein

0-0216

0-0219

aTotal phosp~orus was determined in three differentpreparations and figures shown for the two components arethe average values of these determinations_ The phosphoruscontents were determined by the method of Bartlett (87).

bphospholipid phosphorus was determined on the total lipidextracted according to the method described by Fo1chetal_ (88).

cNucleotide phosphorus was estimated by subtracting thephospholipid phosphorus from the total phosphorus.

114

3~ Organic Prosthetic Group

Riboflavin determinations were made on the enzyme

during the various steps of purification of component 2. The

results are shown in Figure 20. In the first step, the

riboflavin content was high due to the presence of other

flavo-enzymes and free-riboflavin. But in the two subsequent

steps, there was marked decrease in riboflavin resulting

from the removal of contaminating flavo-proteins. Thereafter,

the riboflavin content increased proportionately with the

specific activity of the enzyme. In addition, the riboflavin

content was determined microbiologically in a number of

purified preparations and a value of 1.2 ~g or 3.3 mrmo~

riboflavin per milligram of protein was obtained corresponding

to a value of 0.33 moles riboflavin per 100,000 grams of

protein.

The most accurate result for the determination of

riboflavin was obtained spectrophotometrically. By this

method, an average value of 10.3 mpmoles and 10.1 mpmoles

flavin per milligram of protein were obtained for the

purified components 1 and 2, respectively. These values

correspond to 1.03 moles and 1.01 moles of riboflavin per

100,000 grams of protein for the two components.

Adenine determination was made both microbiologically

and micro chemically. Microbiological assay yielded a value

of 0.513 ~g or 3.8 m?moles adenine per milligram of protein

indicating an adenine content of 0.38 moles per 100,000 grams

of component 2. Precise values, however, were obtained from

Figure 20. Flavin content of the enzyme. The flavin content and

specific activity of the enzyme were determined at each step of the

purification procedure according to the method described by Snell and

Strong (78).

t-'t-'V1

., ,,

o CD

116

000CD

000to-

o00 >-U) I--0 >-0 I-0 U10 « 0

N

Cll0 0 l-I

0 ;:l- co0 LL -,-I

V - ~

0

0wa..0 CJ)

0rt)

000C\I

000

oC\I

Nf3~O~d Ow / NIA\fl.:1081~ On

117

microchemical (spectrophotometric) determination which

yielded values of 1.42 ~g and 1.38 ~g or 10.5 m~ moles

and 10.2 m~ moles adenine per milligram of enzyme

components 1 and 2, respectively. These values correspond

to 1.05 moles and 1.02 moles respectively of adenine per

100,000 grams of component 1 and 2.

The microchemical determination of ribose gave a value

of 1.55 fg or 10.3 m~ moles of ribose per milligram of

protein indicating a ribose content of 1.04 moles per

100,000 grams of enzyme component 2.

The nucleotide phosphorus contents in the enzyme

components were determined by subtracting the phospholipid

phosphorus from total phosphorus contents in the enzyme

components 1 and 2 as shown in Table XIIlin the preceeding

section. The values calculated were 0.67 pg and 0.679 pg

corresponding to 21.6 m~atoms and 21.9 m~atoms of phosphorus

per mi11~am of protein of the enzyme components 1 and 2,

respectively. These values suggest that there are 2.16 gram

atoms and 2.19 gram atoms of phosphorus per 100,000 grams

of enzyme components 1 and 2, respectively.

In addition to the investigation of the "flavin

prosthetic" group in the amine oxidase components, examinations

were made of the pyridoxal content of the enzyme by the

microbiological procedure described by Miyazawa (95). In

these experiments, phosphorylase a was used as a standard.

In MAO, there was 0.03 ~g pyridoxal per milligram of

118

purified enzyme component 2 as compared to 1.4 pg per

miligram of phosphorylase a. These results ~ield values

of 0.07 moles per mole of enzyme component 2 (MW 1,280,000)

as compared to 4.3 moles of pyridoxal per mole of phosphory­

lase a (MW 500,000). All these results are summaried in

Tables XIV A, XIV B, and XIV C.

4. Sulfhydryl Groups

Sulfhydryl groups were determined for enzyme component 1

as well as component 2. The results are shown in Figures 2la,

b, and c. Enzyme component 1 was titrated with increasing

amounts of p-CMB solution. The break point indicated that

there were 6.95 titrable sulfhydryl groups per 100,000 grams

of component 1 (Figure 2la). This value did not change

when the p-CMB titration was done in the presence of 8 M

urea. The value obtained in the latter case was found to

be 7 sulfhydryl groups per 100,000 grams of protein

(Figure 2lb). When the p-CMB titration experiment was

performed on enzyme component 2, a value of 7.15 sulfhydryl

residues per 100,000 gram of protein were obtained

(Figure 2lc). These results are summarized in Table XV.

In a separate experiment, the activity of the enzyme

component 1, during the p-CMB titration experiments, was

simultaneously measured. About 86% of the activity was

retained when all the titrable sulfhydryl groups in enzyme

component 1 had reacted with p-CMB as shown in Figure 22.

A similar result was obtained with component 2.

TABLE XIV A

Riboflavin, Adenine, Ribose, and Nucleotide Phosphorus

Content of Mitochondrial Amine Oxidase

Material mumole/mg Protein Method Reference

Component 1 Component 2

Riboflavin - - 3.3 Microb io log ica 1 (57)

10.3 10.1 Spectrophotometric (69)

Adenine -- 3.8 Microb iolog ica 1 (52)

10.5 10.2 Microchemical (54)

Ribose -- 10.3 Microchemical (59)

Nucleotide Phosphorus 21. 6 21. 8 Ultramicrochemical (68)

.....

.....\0

120

TABLE XIV B

Riboflavin, Adenine, Ribose, and Nucleotide Phosphorus

Content. of Mitochondrial Amine Oxidase

Material Mole/100,000g Enzyme

Component 1 Component 2

Riboflavin 1"03 1"01

Adenine 1"05 1"02

Ribose 1"03

Nucleotide Phosphorus 2"16 2"18

121

TABLE XIV C

Pyridoxal Content of Phosphorylase aa and of the Mito­

chondrial Amine Oxidase Components b

Species

Component 1

Component 2

Phosphorylase a

Pyridoxal Content C

Mole/mole of Enzyme

0'07

aHighly purified rabbit muscle phosphorylase was usedfor these analyses.

bPurified enzyme component ~ with specific activity of8000 was used for these analyses.

cLactobacillus casei ATCC NO 7469 was used for thedetermination of pyridoxal as described by Miyazawa(95) •

Figure 2la. p-Chloromercuribenzoate titration of component 1.

The preparation had a specific activity of 3560. The initial solution

contained 0.4 mg of enzyme in 1 ml of 0.05 M potassium phosphate buffer

at pH 7.0, which had been flushed with nitrogen. To this solution was

added increasing amount of p-CMB (3 x 10-4 M) solution in the same

buffer. The mixture of enzyme and p-CMB was incubated for 1 hour at

room temperature (25 ) after each addition of p-CMB and optical

density measured subsequently in a Beckman DU spectrophotometer for

a total period of 12 hours.

....l'-ll'-l

0.20 /0-0 0

0.16

0-

::s-E0

/0It) 0.12N

0

0

0<J 0.08

0.04

o 2 4 6 8 10 12

No. OF - SH GROUPS (moles p CMB)/I050 PROTEINFigure 21a

....NW

Figure 2lb. p-Chloromeruribenzoate titration of the component 1

in the presence of urea. The reaction mixture contained 0.399 mg of

component 1 (specific activity of 3,560) in 1 ml of 8 M urea prepared

in 0.05 M potassium phosphate buffer pH 7.0, which had been saturated

with nitrogen. The enzyme was allowed to stand in the above mixture

for 150 minutes before titration with p-CMB. Other conditions were

identical to that described in Figure 2la.

t-'N~

:-J

t-~

z-LaJI-0

I a:0 Q.

N0-

U)

00-"-

0 -OJ:E

0 u

IC-

eo .0f/) ..-l

Q)N

0 - Q)

0 l-<

"'-0;:l

E bOor!

U) - ~

~oenQ.::>

~v 0a:

(.!)

:x:0, N en0

Iu.

0 010 V rt) C\l .0 0 q 0 0 00 0 0 0 0 z

rfw OgZ aov

125

Figure 21c. p-Ch10romercuribenzoate titration of the component 2.

The initial reaction mixture contained 0.4 mg enzyme protein (specific

activity 7850) in 0.05 M potassium phosphate buffer, pH 7.0. The

buffer was flushed with nitrogen before using in this experiment.

The titration was made by adding increasing amounts of p-CMB (of initial

concentration of 3 x 10- 4 M) to the enzyme solution. Absorbances were

measured under identical conditions described in Figure 21a.

t-'N0\

0.3

0-00 0-0-

0.2 l- I:l.E /0

10C\J /0

0.10 00 /<J /0

0,I I I I I I I

0 2 4 6 8 10 12 14 16

No. OF - SH GROUPS (moles p CMB)lI059 PROTEINt-'N

Figure 21c -....I

TABLE XV

Number of Titrab1e Sufhydry1 Groups in the

Mitochondrial Amine Oxidase Components*

128

Species Number of SH/100,OOO g EnzymeNo Urea 8 M Urea

Component 1

Component 2

6 .95

7 ".10

7: 0

*The number of sulfhydryl groups was determinedby p-CMB titration according to the method ofBoyer (89).

129

Figure 22. Activity of amine oxidase component 1

during p-CMB titration. For this experiment 0.4 mg enzyme

(specific activity of 4,020) in 1 ml of 0.05 M potassium

phosphate buffer, pH 7.0, flushed with nitrogen was used.

To this enzyme solution were added increasing quantities of

p-CMB and 0.01 ml samples were withdrawn one hour after each

addition of p-CMB, and the activity measured at 25°.

>- 110t: 100>.... 900« 80....

70Zl.LJ0 60a:l.LJ 50a..

4010 20 30 40 50

130

60

MOLE RATIO (pCMB :ENZ)Figure 22

IV. DISCUSSION AND CONCLUSIONS

Since Cotzias and Dole (96) reported that rat liver

amine oxidase is predominantly associated with the

mitochondrial fraction, many attempts have been made to

localize this enzyme in the mitochondria. The problem,

however, of subcellular localization of amine oxidase is

complicated by the fact that the so called mitochondrial

fraction is biochemically and morphologically heterogeneous

(97-100). Extensive studies of the subcellular fractions

from tissue homogenates have shown that many enzymes or

enzyme systems concerned with respiration and intermediary

metabolism are associated with the mitochondria.

Investigations have been carried out in recent years

to separate and characterize the mitochondrial membranes

and localize the enzymes and the chemical components, whose

location has been somewhat uncertain for a long time. Thus,

Parsons et al. (101, 102) have reported the separation of

the "inner" and the "outer" membranes of the rat liver

mitochondria. Levy et al. (103) have used digitonin to

remove the outer membranes of rat liver mitochondria to

investigate the structure of the "inner" membrane. Advantage

has been taken of the use of digitonin to remove the "outer"

mitochondrial membrane by Schnaitman, Erwin, and Greenwalt

(104), who reported that the mitochondrial amine oxidase is

localized in the "outer" membrane of the rat liver mitochondria.

132

Recently, DeRobertis et a1. (105) subfractionated the

mitochondrial fraction of the rat brain into five sub-

fractions which consisted of (i) myelin, (ii) membranes

and fragmented cholinergic endings, (iii) cholinergic

nerve endings, (iv) non-cholinergic nerve endings, and

(v) the free mitochondria. Subfractions (iv) and (v)

accounted for about 37% and 61%, respectively, (comprising

together, 98%) of the total amine oxidase activity. The

localization of the amine oxidase in the non-cholinergic

synapses led to suggestions that this enzyme plays a role

similar to that of cholinesterase in the cholinergic ones

(106). In fact, amine oxidase has been shown to control

the levels of neural hormones (e.g., epinephrine, nor-

epinephrine) by catalytically removing them when they are

present in excess (2, 107).

These possibilities give considerable interest to the

investigation of the various integral properties of the

mitochondrial amine oxidase, the physiological role of

which is not completely known. However, the complex

structure of the mitochondrion itself, (104), the presence

of multiple enzyme complexes of the electron transport

system and the citric acid cycle (108-110) the similar

distribution of the amine oxidase and the succinate

dehydrogenase (111), and above all, the firm attachment of

these enzymes or enzyme systems to the mitochondrial

133

structural protein or to the lipid, or both, made it

difficult to purify the mitochondrial amine oxidase, and

many attempts, as mentioned earlier, have led only to the

partial purification of the enzyme (35, 36).

Initial investigations in this laboratory to purify

the amine oxidase from beef liver mitochondria, however,

resulted in a fifty-fold purification of the enzyme (53).

In these preparations, the enzyme was eluted from the DEAE­

cellulose column as a final step of purification and the

specific activity was 4,000 to 4,500. Later, the highly

purified preparations with very high activity were obtained

by extending the purification proc_edure to include

hydroxylapatite column chromatography and starch block

electrophoresis steps. The outcome was the separation

of two major fractions with specific activities of 3,000

to 4,000 (112), and 7,000 to 9,000 (112, 113). They are

termed component 1 and component 2, respectively, throughout

this presentation. Preliminary studies done on a few

properties of the earlier preparations of mitochondrial

enzyme agreed well with later findings and a brief

discussion of these results will be made.

The substrate specificity, behavior towards various

inhibitors, and pH optima, indicated that the beef liver

mitochondrial enzyme was the well known, classical

mitochondrial amine oxidase. The results of the earlier

investigations on the substrate specificity agreed well with

134

the findings on purified enzyme components. In contrast to

the observation made by Gorkin (35) a few years ago, that

there were two amine oxidases present in rat liver mitochon­

dria with different substrate specificities, the beef liver

mitochondrial enzyme components were found to possess

the same substrate specificity (11 2). The degree of

deamination in case of tryptamine and tyramine differed a

little, but both the components showed similar activities

on all the amines investigated (Table II). This finding

also differs from that recently reported by Ragland (114).

However, it should be realized that different methods of

purification were used by the various investigators as

well as different analytical methods to detect the multiple

forms of the enzyme.

Both the components were unaffected by aldehyde

reagents indicating that a pyridoxal prosthetic group was

not present in them (Table V). The slight inhibition

shown by p-nitrophenylhydrazine or phenylhydrazine was

possibly due to the benzene rings rather than the hydrazine

groups that these compounds contain. This was evident from

the fact that sodium benzoate inhibited the enzyme components

whereas hydrazine, semicarbazide, or hydroxylamine were

without effect. Metal chelators, such as cuprizone,

neocuproine, 8-hydroxyquinoline, o-phenanthroline,

diethyldithiocarbamate, etc. (Table IV) produced significant

inhibition. These findings suggested the presence of a

135

metal in the mitochondrial enzyme components.

Ethylenediaminetetraacetate (EDTA), sodium azide, or sodium

cyanide, on the other hand, did not show any inhibition.

These differential effects, however, need not be considered

as contradictory since EDTA and a few other chelating

agents which are known to form highly stable complexes with

metal ions in aqueous solutions were found to be relatively

weak chelators of protein bound metals (115).

Preliminary results suggested that the beef liver

mitochondrial amine oxidase was sensitive to sulfhydryl

reagents. The various sulfhydryl reagents investigated

required concentrations of 1 x 10- 3 M to 1 x 10- 6 M to

produce a 50% inhibition of the enzyme. In the presence of

4.3 x 10- 6 M p-CMB, the oxidation of benzylamine was inhi-

bited by 50%. Similar observations were made by Lagnado and

Sourkes (92) with the rat liver mitochondrial enzyme.

When p-CMB inhibition was examined with purified

enzyme components 1 and 2, very interesting results were

obtained. With a 370-fold molar excess of p-CMB component 1

showed a 26% inhibition in the enzyme activity (Table IIIC).

When a 800-fold molar excess of p-CMB was used, enzyme

component 2 was inhibited only by 24% (Table III C). Line-

weaver-Burk plots (Figures 13 b anc c) showed that p-CMB

was a non-competitive inhibitor of both the enzyme components.

The Michaelis-Menten constants, Km, for the purified enzyme

components 1 and 2 were 3.1 x 10- 4 M and 2.9 x 10- 4 M at

25 0 , and the inhibition constants, Ki , under the same

136

conditions were 1.9 x 10- 5 M and 1.6 x 10- 5 M, respectively,

with p-CMB.

The sedimentation coefficient was determined for a

number of purified preparations of component 1. The values

obtained ranged from 14 S to 14.7S. The average of 5

such determinations yielded a sedimentation coefficient

of 14.4 + 0.3 when corrected to standard conditions at 20 0

in water and extrapolated to zero protein concentration.

When the sedimentation coefficient was determined by

sucrose density method, a value of 14.7 + 0.3 S was

estimated for component 1, and 20.6 + 0.4 S for component 2.

The Stoke's (molecular) radii estimated from gel filtrationo 0

data were 60 A and 106 A for components 1 and 2, respectively.

Partial specific volumes determined for components 1 and 2

were 0.78 cm3 jg and 0.80 cm3 /g, respectively. These values

of the partial specific volumes determined are markedly

higher than those usually obtained for most proteins which

yield values in the range of 0.7 to 0.75 cm3 jg. These

components

differences in partial specific volumes of the two components

can be explained by the fact that these enzyme components

contain significant amounts of phospholipid, and proteins

containing lipid materials in their molecules yield higher

partial specific volumes in the hydrated form (71).

Diffusion coefficients calculated from Stoke's radii of

1 and 2 were found to be 3.8 x--10- 7 cm2 sec- 1 and

sec~l, respectively.

137

When the molecular weight was determined from the gel

filtration data by the method of Whitaker (68), a value of

400,000 was obtained for component 1. This value was

found to be 396,000 when calculated from Stoke's radius,

sedimentation coefficient, and partial specific volume

according to the equation (iv) of the Materials and Methods

Section. The molecular weight calculated for component 1

from the sedimentation and diffusion coefficients were

423,000. The molecular weights determined by these three

methods were 1,300,000 1,195,000, and 1,355,000,respective1y,

for component 2. These molecular weights for the two beef

liver mitochondrial amine oxidase components differ from

those reported by Erwin and Hellerman (39) for bovine

kidney mitochondrial monoamine oxidase. These authors

reported a molecular weight of 290,000 calculated from a

sedimentation coefficient of 10.6 S, an assumed partial

specific volume of 0.75, and an apparent diffusion constant

(D20) of 3.5 x 10- 7 cm2 sec- l calculated from Sephadex G-200

gel filtration data by the method of Ackers (116). Same

molecular weight (290,000) was reported by Youdim and

Sourkes (38) for rat liver monoamine oxidase. These authors,

however, showed a sedimentation coefficient of 6.3 S for

their enzyme in contrast to 10.6 S for that of Erwin and

Hellerman (39). On the other hand, the molecular weight

reported by Tipton (40) for pig brain mitochondrial enzyme

was 102,000 as determined by Sephadex G-200 gel filtration.

138

In addition to his major fraction with the above molecular

weight, he eluted another small fraction which corresponded

to a molecular weight of 435,000, which, he considered

might be a tetrameric form of the lower molecular weight

fraction. In these regards, the low molecular weight

component or component 1 of beef liver enzyme corresponds

to the tetrameric form of Tipton's enzyme, and the high

molecular weight fraction or component 2 represents a

trimer of component 1. There are many reasons to support

this contention which will be discussed later.

Of all the metals analyzed (Table XII) only copper

was found to be present in significant amounts. A number

of highly purified fractions of the enzyme component 2

yielded values ranging from 0.15 pg to 0.17 ~g of copper per

milligram of protein. On the basis of molecular weight of

1,300,000, the enzyme contains 3 gram atoms of copper per

mole of component 2. The preliminary assumption that a

metal participated in the catalytic activity of the enzyme

could not be conclusively proved. However, bis-cyc1ohexanone

oxa1dihydrazone (cuprizone), 'a specific che1ating agent for

copper, produced mixed type of inhibition of the DEAE­

cellulose eluted enzyme (Figure 14). In addition, sodium

diethy1dithiocarbamate, a-hydroxyquinoline, a,a -bipyridy1,

etc, inhibited the enzyme, indicating the presence of a metal

in it. The determination of other metals demonstrated that

they are either absent or present in negligible amounts

(Table XII).

139

The copper content, nevertheless, showed an

initial drop in the first two steps of purification, and

indicated slight but steady increase in the subsequent

steps of purification (Figure 19a). Erwin and Hellerman,

in this respect, reported similar findings with beef

kidney mitochondrial monoamine oxidase. They estimated a

copper content of 0.15 pg to 0.19 pg per milligram of

their enzyme. Youdim and Sourkes (38) on the other hand,

found that the rat:1iver mitochondrial enzyme contains

0.12% iron and 0.034% copper, corresponding to 1.2 pg iron

and 0.34 pg copper per milligram of their enzyme protein.

In beef liver mitochondrial enzyme, however, iron occurred

only in the crude enzyme and the largest amount found was

0.07 pg/mg of protein in the second step of purification.

The iron content dropped sharply in the subsequent steps

as shown in Figure 19b, and is considered to be an impurity.

The result obtained in this work suggests that copper

is the only metal present in significant amount. The

reason it did not respond to cyanide, or EDTA, is that

many chelating agents which chelate metals in aqueous

solutions fail to do so when the metal is bound to a

protein (115). Copper ions, in addition, react with amino

acids or proteins more strongly than do any other metal

ions (117). When copper is a prosthetic group of an enzyme,

the copper ion cannot be separated by any amount of

dialysis. A drastic treatment is necessary to remove it

140

from the protein (117).

rhe beef liver mitochondrial amine oxidase contains a

flavin which is covalently linked to the enzyme protein.

Due to this tenacious attachment to the enzyme molecule,

it was not easy to remove it from the enzyme and

characterize it. Despite this difficulty, however, the

enzyme has conclusively been shown to be a flavoenzyme by

physical, chemical, and microbiological methods. The

spectrum, however, does not resemble that of a typical

flavoenzyme (118), but has an absorption peak at 4S0 mp

and a shoulder at 480 mp(Figure lSa) which are reducible

by the substrate"benzylamine and by sodium hydrosulfite

(Figure lSb). This property of the enzyme has been used

for the spectrophotometric determination of the flavin

dinucleotide cofactor in both the components of the enzyme.

That the enzyme was a flavoprotein was inferred from the

following observations: (i) The yellow color of the enzyme

was intensified with each step of purifica~ion. (ii) The

purified yellow-peptide of the pronase digest of the enzyme

exhibited spectral properties characteristic of riboflavin,

or flavin nucleotides. ~iii) The yellow colored material

promoted the growth of L. casei which cannot thrive without

riboflavin. (iv) The riboflavin content increased

steadily in the subsequent steps of purification and was

proportional to the specific activity of the enzyme

(Figure 20). About 1.3 moles of riboflavin per 400,000 g of

component 2 were determined by the microbiological method.

141

The most accurate results were obtained by the spectrophoto­

metric method which yielded a value of 4.2 moles of flavin

nucleotide per 400,000 g of component 1 and 4.05 moles per

400,000 g of component 2.

Once it was confirmed that the beef liver enzyme

contained riboflavin, it was necessary to determine if it

were a flavin di- or mono-nucleotide.

does not contain ribose or adenine.

Flavin mononucleotide

It was, therefore,

decided to investigate the adenine and ribose content of

the enzyme. Microbiological assay of the enzyme hydrolyzate

yielded a value of 1.48 moles of purine (probably adenine)

for 400,000 g of enzyme component 2. A microchemical

method (81), however, demonstrated conclusively that the

enzyme contained 4.3 moles of adenine per 400,000 g of

component 1 and 4.02 moles per 400,000 g of component 2.

The ribose content of the enzyme was then determined to

be about 4.1 moles of ribose per 400,000 g of enzyme

component 2. Next the phosphorus content of the purified

enzyme was then measured. Since the mitochondria are rich

in phospholipids, the determination of the nucleotide

phosphorus was not easy. Total phosphorus and the

phospholipid phosphorus were estimated for both the enzyme

components and the difference between total phospherous and

phospholipid phosphorus yielded the values for the

nucleotide phosphorus per 400,000 grams of enzyme component,

1 and 2, respectively. These results suggest that the

flavin nucleotide is flavin adenine dinucleotide or FAD.

142

Since 1 mole of FAD contains 1 mole each of riboflavin,

ribose, and adenine, and 2 gram atoms of phosphorus, these

results further suggest that the beef liver mitochondrial

amine oxidase contains 4 moles of FAD per mole of component 1,

and 12 moles of FAD per mole of component 2. Microbiological

determination of riboflavin yielded low values which cannot

be explained satisfactorily. The value obtained by this

method accounts for only 33% of that determined spectro­

photometrically. The low value estimated by microbiological

method was also obser~ed in the case of succinate dehydro­

genase (119) where a value, 29% of the exact flavin content

of that enzyme, was estimated.

That the FAD was catalytically involved in the enzyme

was suggested by the observation that the 450 mp peak and

the 480 mp shoulder were bleached by the substrate

benzylamine and by sodium hydrosulfite. Specific inhibitors

of the enzyme were found to prevent the reduction of the

visible maximum at 450 mp. Those which were not substrates,

did not bleach the 450 mp absorption band (113).

Determination of total phosphorus yielded a value of

132 gram atoms of phosphorus per mole of enzyme component 2.

When phosphorus determinations were made on the lipid

extracts of the enzyme, 24 moles ~nd 106 moles of phospho­

lipids per mole of enzyme were found in components 1 and 2,

respectively. These values correspond to 0.059 mp mole and

0.086 mp mole of phospholipid, respectively, per milligram

of component 1 and component 2. These values are in

143

agreement with the result of Erwin and Hellerman (39) who

estimated a value of 0.06 mp mole of phospholipid for their

enzyme. As already mentioned, this high phospholipid

content in components 1 and 2 is responsible for the high

partial specific volumes of these proteins. The partial

specific volumes determined for component 1 and component 2,

did not agree, therefore, with the assumed partial specific

volume of 0.75 reported by Erwin and Hellerman (39).

Determination of the sulfhydryl residues revealed that

100,000 grams each of component 1 and component 2 contained

7 and 7.1 titratable -SH groups, respectively. These values

correspond to 28 -SH groups per mole of component 1 and

86 per mole of component 2. Erwin and Hellerman (39)

found 8 titrable thiol residues per 100,000 grams of

their enzyme. They have reported further, that the activity

of their enzyme declined with increasing p-CMB concentrations

and was completely inhibited when 7.3 moles of p-CMB per

100,000 grams of protein were added. This indicated that

the inhibition was complete when all the detectable thiol

groups were titrated.

In the present work, however, contrary results were

obtained. The residual activity was as much as 86% in

component 1 and around 30% in component 2 when all the -SH

groups of these components were titrated with p-CMB. Both

the components of the enzyme exhibited the non-competitive

144

type of inhibition (Figures l3b and c). Alkylating agents

such as iodoacetic acid, iodoacetamide, N-ethylmaleimide,

etc., which are potent inhibitors of certain sulfhydryl

enzymes (120-122) were found to be apparently ineffictive

in inhibiting the enzyme (Table III P). Further, the

inhibition due to p-CMB was observed to be reversed on

dialysis. From these findings, it appears that the

sulfhydryl groups are not involved in the catalytic

function of the enzyme. They are possibly associated

with the conformational requirement of the enzyme.

Finally, the various properties of the beef liver

mitochondrial amine oxidase are summarized in Tables XVI A,

XVI B, XVI C, and XVI D. It seems that there are at least

2 components of the enzyme, one possibly being the polymeric

form of the other. The similarities in their substrate

specificities (Table II), inhibition by various chelating

agents (Table IV), non-competitive inhibition with p-CMB

etc. (Figures l3b and c), support this contention.

Evidence derived from chemical characterization of the two

components demonstrates that they have the same FAD and

sulfhydryl group contents, relative to their molecular

weights. The most convincing evidence is the fact that the

two components have approximately the same amino acid

composition (123). It is also a reasonable assumption that

the higher molecular weight fraction or component 2 is the

parent enzyme and that the lower molecular weight fraction

Properties:

TABLE XVI A

1 a. Kinetic Parameters of Mitochondrial Amine Oxidase

Species Substrate a Specific Stability Optimal c R'om

Component 1

Component 2

Benzylamine

Benzylamine

Activityeub / mg

Protein

3000-4000

7000- 9000

Thermolabile

Thermolabile

pH

9.2

9.2

(X 10 4 M)

3.1

2.9

aAmong 17 mono-, di-, and poly-amines examined (Table II), benzylamine provedto be the best substrate and was used in all enzyme determinations.

beu = enzyme unit; one unit is defined as the amount of enzyme which producesa change in absorbance of 0.001 per minute at 250 mu at 25 0 •

cOptimal pH was determined on the enzyme after DEAE-cellulose step and was notdone after final purification of the two components.

dMichaelis-Menten constant, Km, was determined at pH 7.4, using 0.2 M potassiumphosphate buffer, and benzylamine substrate at 250.

t-'.poVI

TABLE XVI B

Properties: 1 b.' Kinetic Parameters of Mitochondrial Amine Oxidase

InhibitionSpecies

Component 1

Component 2

ProductInhibitiona

(NH 4+)

Sulfhydrylbreagents

+

+

Metal C

Chela tors

+

+

Aldehyde d

reagents

Ki e

(X 10 5 M)

1.9

1.6

a(NH4)2S04 was used as for product inhibition.

bThiol reagents used inhibited in the order Hg+-S Ag+· = p-CMB > Cd++.

cCuprizone, Neocuproine, and 8-hydroxyquiloline (Table IV) inhibited, whereascyanide, azide, and EDTA, did not.

dAmong aldehyde reagents, only phenylhydrazine, and p-nitrophenylhydrazine showedsome inhibition (Table V).

eInhibition constant, Ki' was determined by using p-CMB which inhibitednon-competitively. ....

.I:­0\

TABLE XVI C

Properties: 2. Molecular Parameters of Mitochondrial Amine Oxidase

Species Stoke's V s20,Wc n d Mol. Wt. e f/ f o

Radius a Partial (X 1013 sec) (X 107cm2sec- l )Specific

A Volume

cm 3/g b

Component 1 60 0.78 14.4 3.8 407,000 1. 17

Component 2 106 0.80 20.6 2.0 1,280,000 1. 46

aStoke's radii for components 1 and 2 were determined from gel filtration dataaccording to Siegel and Monty (45).

bpartial specific volumes were determined pycnometrically (67).

cSedimentation coefficient for component 1 was determined by sedimentation velocitymethod (61) and that of component 2 by sucrose density gradient centrifugation(37) .

dniffusion coeffkients were calculated from Stoke's radii and the use of thefollowing equation, n = kT/6J1Na.

eMolecular weights of the two amine oxidase components are the average values ofthose determined by three methods--gel filtration, the method based on Stoke'slaw, and the sedimentation-diffusion method.

.....

.po.

"

TABLE XVI D

Properties: 3. Chemical Parameters of Mitochondrial Amine Oxidase

Species

Component 1

Component 2

Copper a

( g- a tom )( per )(mo Ie Enzymf!)

3.1

Phospholipidb

( mole )( per )(mole Enzyme)

24

106

FAD c

( mole )( per )(mole Enzyme)

4

12

Pyridoxal d

( mo 1e )( per )(mole Enzyme)

0.07

-SH residue e

( number )( per )(mo Ie Enzyme)

28

86

aCopper was determined by the microchemical method of Peterson and Bollier (76).

bphospholipid was extracted by the method of Folch et al. (88) and quantitated byphosphorus determination (87). -- --

cRiboflavin, ribose, adenine, and nucleotide phosphorus were in the proportion of1:1:1:2 as found in FAD.

dPyridoxal was determined by the method of Miyazawa (94).

eSu~fhydryl groups were determined by the method of Boyer (89).

I-'-I:'00

149

or component 1 is derived from component 2. Results of the

phospholipid determinations show that component 2 has a

higher phospholipid content than that of component 1,

suggesting a release of component 1 from the lipid-enzyme

complex of the parent molecule or component 2, since it is

unlikely that component 1 with a proportionately lower

phospholipid content will aggregate to yield a trimer with a

proportionately higher phospholipid content. The frictional

ratios of component 1 and component 2 were calculated to be

1.17 and 1.46, respectively, suggesting that component I is

more spherical than component 2.

The question whether two or more than two components

are present in the beef liver mitochondrial enzyme as

separate components or whether they are artifacts of the

purification procedure merits further investigation.

Moreover, since Tipton's (92) investigations with the pig

brain mitochondrial enzyme demonstrated a major component

and a minor component with molecular weights of 102,000 and

435,000, respectively, another form with a molecular weight

in the order of 100,000 may be isolated by the sonication

procedure. Sonication procedure was not used in the present

study. However, it is hoped that the properties of the two

enzyme components outlined in this dissertation will provide

some insight to the other workers who intend to study this

aspect as well as other properties of this enzyme.

V. SUMMARY

Beef liver mitochondrial amine oxidase was purified

in this laboratory by extraction with a nonionic detergent

Triton X-lOO, ammonium sulfate fractionation, column

chromatography, and electrophoresis. Two fractions, which

are described as component 1 and component 2 in the text,

were isolated. Various purity studies were made on these

purified enzyme components.

The enzyme components 1 and 2 were thermolabile and

lost 25% and 40%, respectively of their activity on

standing at room temperature for 7-8 hours. Freezing

resulted in prompt loss in activity due to denaturation

of both the components.

When the substrate specificity of the components

were examined, they showed similar specificities towards

the amines tested. Lysine and diamines except kynuramine,

and all polyamines, were not deaminated by either enzyme

component.

Certain metal chelators like cuprizone, 8-hydroxy­

quinoline,a ,a -bipyridine, and neocuproine inhibited the

enzyme, whereas other chelating agents like EDTA, cyanide,

or azide did not. Aldehyde reagents did not show signifi­

cant inhibition of either component. Certain thiol

reagents like p-CMB, HgC12, AgN03, etc., which form metal

151

mercaptides with the enzyme sulfhydryl groups moderately

inhibited the enzyme at high concentrations. p-Chloro-

mercuribenzoate was a noncompetitive inhibitor of both

components. The Km for components 1 and 2 were 3.1 x

10- 4 M and 2.9 x 10- 4 M, respectively. The corresponding

Ki values in the presence of p-CMB were 1.9 x 10- 5 M and

The product, NH3 (in the ionized form,

NH4+ ), did not have an inhibitory effect.

Sedimentation studies showed that the sedimentation

coefficients of components 1 and 2 were 14.4 + 0.3 Sand

20.6 + 0.4 S, respectively. Frictional ratios of 1.17 and

1.46, respectively, for components 1 and 2 were calculated,

indicating that component 1 is more spherical than

component 2. Molecular (or Stoke's) radii calculated fromo 0

gel filtration data were 60 A for component 1 and 106 A

for component 2. Diffusion coefficients for components 1

and 2 were calculated to be 3.8 x 10- 7 cm2 sec-land

2.0 x 10- 7 cm2 sec- 1 , respectively. The partial specific

volumes were estimated pycnometrica1ly and were found to

be 0.78 cm3 jg for component 1 and 0.80 cm3 }g for com-

ponent 2. Molecular weights as determined by the gel

filtration method were 400,000 and 1,300,000 for components

1 and 2, respectively. Stoke's Law yielded molecular

weights of 396,000 + 10,000 and 1,195,000 and Svedberg's

152

equation (S and D), values 425,000 ~ 10,000 and 1,355,000,

respectively. Averages of the molecular weights determined

by these three methods were 406,000 ~ 14,700 and 1,280,000

+ 91,500, respectively, for components 1 and 2.

Metal analyses of the enzyme yielded values of 0.15

to 0.17 pg copper per milligram of the enzyme protein.

This value corresponded to 1 gram atom of copper per mole

of component 1 or 3 gram atoms of copper per mole of

enzyme component 2. The presence of iron was insignificant

and was considered to be an impurity. Cobalt, manganese,

and molybdenum were found to be absent.

The prosthetic group , FAD, is covalently linked to

the enzyme. There is 1 FAD per 100,000 grams of either

component suggesting the presence of 4 moles of the

dinucleotide in component 1 and 12 moles in component 2.

Phospholipid is present in markedly large amounts in

the enzyme to the extent of 24 moles per mole of component

1 and 106 moles per mole of component 2.

There are 28 -SH groups in a molecule of the compo­

nent 1 and as many as 86 such residues in component 2.

The substrate specificity, the inhibitor specificity,

amino acid composition, and other properties of component

1 and component 2 are remarkably similar suggesting that

one is the polymeric form of the other. The ratio of the

molecular weights, FAD contents, and numbers of -SH groups

suggest that component 2 may be a trimer of component 1.

Zeller, E.A., in "The Enzymes" ed. by Sumner, J.B.,

and Myrback, K., 1st. Ed., Vol. II, Part 1, p. 536.

Academic Press, New York (1951).

B1aschko, H., Brit. Med. Bull. ~, 46 (1953).

Buffoni, F., Pharmaco1. Rev., 18, 1163 (1966).

Gorkin, V.Z., Pharmaco1. Rev., 18, 115 (1966).

Tabor, H., J. BioI. Chem., 188, 125 (1951).

Mondovi, B., Roti1io, G., Finazzi-Agro, A., and

1.

2 •

3 •

4.

5 •

6 •

7 •

VI. BIBLIOGRAPHY

Hare, M.L.C., Biochem. J., 22,968 (1928).

Seioscia-Santoro, A., Biochem. 3"., ~...!.' 408 (1964).

8. Mondovi, B., Roti1io, G., Costa, M.T., Finazzi-Agro,

A., Chiancone, E., Hansen, R.E., and Beinert, H.,

J. Bi 0 1. Chem., 242, 1160 ( 1967) .

9. Werle, E~, and Pechmann, E., Leibigs Ann., 562, 44

(1949).

10. Kenten, R.H., and Mann, P.J.G., Biochem. J., 50, 360

(1952).

11. Mann, P.J .G., Biochem. J., 59, 609 (1955).

12. Clarke, A.J., and Mann, P.J.G., Biochem. J., 71, 596

13. Hill, J.M., and Mann, P.J.G., Biochem. J., 85, 198

(1962).

14. Hill, J.M., and Mann, P.J.G., Biochem. J., 91, 171

(1964).

154

15. Buffoni, F., and B1aschko, H., Proc. Roy. Soc. B,

161, 153 (1964).

16. Buffoni, F., J. Physio1., 169, 121 p (1963).

17. B1~~chko, H., Friedman, P.J., Howes, R., and Nilsson,

K. T., J. Physiol., 145, 384 (1959).

18. Yamada, H., and Yasunobu, K.T., J. BioI. Chem., 237,

1511 (1962).

19. Buffoni, F., Pharmaco1. Rev., 18, 1163 (1966).

20. Zeller, E.A., Adv. Enzymol., ~, 93 (1942).

21. Arun1akshana, 0., Mongar, J.L., and Schild, H.O.,

J. Physiol. (London), 123,32 (1954).

22. Schuler, W., Experi"entia, ~, 230 (1952).

23. Sinclair, H.M., Biochem. J. 51, x-xi (1952).

24. B1aschko, H., in "The Enzymes", ed. by Boyer, P.D.,

Lardy, H., and Myrback, K., 2nd. Ed., Vol. 8, p. 337.

Academic Press, New York (1963):

25. Buffoni, F., and B1aschko, H., Proc. Roy. Soc. B,

161, 153 (1964).

26. B1aschko, H., and Buffoni, F., Proc. Roy, Soc. B, 163,

45 (1965).

27. Yamada, H., and Yasunobu, K.T., J. BioI. Chem., 237,

3077 (1962).

28. Yamada, H., and Yasunobu, K.T., J. BioI. Chem., 238,

2669 (1963).

29. McEwen, C.M., Jr., J. Biol. Chem., 240, 2003 (1965).

155

156

157

58. Tise1ius, A.~ Hjerten, S., and Levin, 0., Arch.

Biochem. Biophys., 65, 132 (1956).

59. Fine, I.H., and Costello, L.A., in the "Methods in

Enzymology", ed. by Co1owick, S.P., and Kaplan, N.D.,

Vol. VI, p. 958. Academic Press, New York (1963).

60. Taber, H., and Sherman, F., Ann. N.Y. Acad. Sci.,

121, 600 (1964).

61. Svedberg, T., and Pederson, K.O., "The Ultracen­

trifuge", Oxford University Press, (Johnson Reprint

Corporation, New York), London (1940).

62. Martin, R.G., and Ames, B.N., J. Biol. Chem., 236,

1372 (1961).

63. Schneider, W.C., and Hogeboom, G.H., J. Biol. Chem.,

183, 123 (1950).

64. Tabor, C.W., Tabor, H., and Rosenthal, S.M., J. Biol.

Chem., 208,645 (1954).

65. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and

Randall, R.J., J. Bio1. Chem., 193, 265 (1951).

66. Schachman, H.K., in "Methods in Enzymology" ed. by

Colowick, S.P., and Kaplan, N.D., Vol. IV, p. 32,

Academic Press, New York (1957).

68. Whitaker, J.R., Anal. Chem., 12,1950 (1963 ).

69. Andrews, P., Biochem. J., 96, 595 (1965).

70. Representative from Bio-Rad Laboratories, Technical

Information Division: by personal communication.

===--------------,--:c:::======~~---.--- -- - - .

158

71. Siegel, L.M. and Monty, K.J., Biochim. Biophys.

Acta, 112, 346 (1966).

72. Porath, J., Pure Appl. Chem.,~, 233 (1963).

73. Peterson, R.E., and Bollier, M.E., Anal. Chem., 27,

1195 (1955).

83. Dische, Z., and Schwartz, K., Mikrochim. Acta, ~'

13 (1937).

--,,------;:---- - - -- ---- --------------------

159

160

98. Whittaker, V.P., Biochem. J., 72, 694 (1959).

99. Gray, E.G., and Whittaker, V.P., J. Physio1. 153,

2 (1960).

100.

101.

102.

De Robertis, E., Pellegrino De Iraldi, A.,

Rodriquez, G., and Gomez, C.J., J. Biophys.

Biochim. Cytol., ~, 229 (1961).

Parsons, D.R., and Verboon, J.G., J. Appl. Phys.,

36, 2615 (1965).

Parsons, D.R., William, G.R., and Chance, B.,

Ann. N.Y. Acad. Sci., 137, Art 2, 643 (1966).

103. Levy, M., Toury, R., and Andrew, J., Compt. Rend.

Soc. Bio1. Ser. D., 262, 1593 (1966).

104.

105.

106.

107.

108.

109.

Schnaitman, C., Erwin, V.G., and Greenwalt, J.W.,

J. Cell Bio1., 32, 719 (1967).

De Robertis, E., Pellegrino De Ira1di, A.,

Rodriquez De Lores Arnaiz, G., and Sa1ganicoff, L.,

J. Neurochem.,~, 23 (1962).

Rodriquez de Lorez Arnaiz, G., and De Robertis,

E., J. Neurochem. ~, 503 (1962).

Davison, A.N., Physio1. Rev., 38, 729 (1958).

Green, D.E., Bachmann, E., A11mann, D.W., and

Perdue, J.F., Arch. Biochem. Biophys. 115, 172

(1966).

Bachmann, E., A11mann, D.W., and Green, D.E.,

Arch. Biochem. Biophys., 115, 153 (1966).

112.

113.

114.

161

110. A11mann, D.W., Bachman, E., and Green, D.E.,

Arch. Biochem. Biophys., 115, 165 (1966).

111. Oswald, E., and Strittmatter, C., Proc. Expt1.

BioI. Med., 114, 664 (1963).

Gomes, B., Kloepfer, H.G., and Yasunobu, K.T.,

submit ted.

Igaue, I., Gomes, B., and Yasunobu, K.T., Biochem.

Biophys. Res. Commun., 29, 562 (1967).

Ragland, J.B., Biochem. Biophys. Res. Commun., 31,

203 (1968).

115. Vallee, B.L., in "The Enzymes" ed. by Boyer, P.D.,

Lardy, H.A., and Myrback, K., Vol. 3, p.225,

Academic Press, Inc., New York (1960).

116.

117.

118.

119.

120.

Aeke r s, G. K., Bi 0 c hem i s try, ~' 723 (1964).

Frieden, E., Scientific American, 218, 102 (1968).

Yagi, K., Ozawa, T., and Harada, M., Nature, 184,

1938 (1959); 188, 745 (1960).

Kearney, E.B., J. BioI. Chem., 235, 865 (1960).

Sund, H., and Theore11, H., in "The Enzymes" ed. by

Boyer, P.D.-, -Lardy, H., and Myrback, K., Vol. 7,

p. 25. Academic Press, New York (1963).

121. Li, T., K., and Vallee, B.L., Biochem. Biophys.

Res. Commun., 12, 44 (1963).

122.

123.

162

Ve1ick, S.F., and Furfine, C., in "The Enzymes" ed.

by Boyer, P.D., Lardy, H., and Myrback, K., Vol. 7,

p. 243. Academic Press, New York (1963).

Dr. Kloepfer, H.J., in this laboratory has deter­

mined the amino acid composition of the two enzyme

components.