REGULATORY ENZYMES OF

FILARIAL PARASITES

A DISSERTATION SUBMITTED TO THE

ALIGARH MUSLIM UNIVERSITY, ALIGARH FOR THE DEGREE OF

Master of Philosophtf IN

Blochemfstrq

BY HUNEZA HU5SAIN

M. Sc. (Biochem.)

DIVISION OF BIOCHEMISTRY CENTRAL DRUG RESEARCH INSTITUTE

LUCKNOW-226001 December, 1988

DEDICATED

TO

APAJAN

AND

PAPAJI

Dr. s. N. ^mm EtiS^tU* Sct«ftti«^ H'-^^P ( Fvmef D»puiy timASt)

^ , /Vo.

CENTRAi DRUG RESEARCH INSTITUTE ( A Constftuont Establishment o( CSIfi)

Chattar Manzil, Post Box No. 173 LUCKNOW-226 001 ( I N D I A )

Date VQ-C. 29,19SS

CERTIFICATE

Thi^ 16 to (iQ.fiti{\q that the. wofik (imbodizd in thii, thd-il^i

eMt-iti&d "Regulators/ Enzifme.^ o^ FiiaJiiai Parasite.^" ha-6 been ca^^iad

out bij M-t4A Humza Hu4.Aain, M.Sc. [Biochzm] andzK mq ^ap^^vi^ion.

S/ie ha-i {sui{^illtd thz KzquiKe.rmnt6 oi thz . kliQaih Mu -f m

linivZK^^tij izqaKding thz pKZ^CKibzd pzfiiod o{) invz^t-tgationat wo fe

{\0>i thz avoaxd o^ H.Phil, dzgizz.

Thz woAk includzd in thii, thz-^i^ i-i, original unlzi6 -itatzd

uthzfiu)i.iz, and ha-^ not bzzn i>ubmittzd (s,oK anij othzn. dzgizz.

(S.W. GHATAIC)

,.^,„.^ ^ilut 1 aiM....9iW Tslssriim : CEWORUG Phone

Off. :32411-T8PABX Res. : 73490

ACKNOWLEVGEUENTS

It kai bo-zn my pfioad pfilvH^g^ io havz wo feed undzK tht lni,pi>i-

ing_guidance. _oi__my fL^^pgctzd tzacke.^, P^OI^ZMOH. A.M. SiddiQui, Hzad,

Vzpaitmznt Oj$ Bioch(iml-{>tfiy, MiQaih Muslim Unlve-fi^lty, Aliga^h, whoAZ

gzntfio-iity and anilagging intzud^t hai, hzZpdd me in achieving my

objZctiv&-i>.

I gmtzially acknouilQ-dgQ. Vi. S.N. Ghatak, Emznitai, Sciznti^t

0($ CSIR (J'ome.^ Hdad and VO-paty Vifizctofi), division o^ Biochzmi^tiy,

Czntn.al Vliig Rd^za^ck Jn^titutd (CVRI) iofi hii> kind i>apQ.>ivii>ion, untie­

ing hzlp and valuablz ^uggz^tion^ in bfiinging up thd thu^i^ to iti

piz^iint ihapz.

I al-60 take, thii, opportunity to e.xpfie^^ mt/ de.ep ^zn^e o(, grati­

tude. Vr(MA.^] hluzhat A. Kau^hal, Scizntiit, division 0)$ Biodi2.miUry

{^oK her untiring heZp, kind co-operation and vaiuabiz -i>ugge..6tion.6

(^or conducting my e.xperiment^ wihotLt which thi-i> dissertation would

not have been completed.

Words will not su^ice to e.xpre.ss my hzarty j ee- -cng-A and great-

{iUlne.ss to Vr. O.P. Shukla, Assistant Vire.ctor ior his valuable. he.lp

and suggestions and ^ruit^^ul criticism as well as interpretation oi

the data generated during the course o{^ these studie.s.

J am grateful to Vr. M.M. Vhar [Ex-Virzctor], Proizssor B.N.

Vhawan, director, CVRI and Vr. K.C. Saxzna, Head, Vivision o{^ Bio­

chemistry ^or providing the neci-^bsary laboratory {facilities and to

the. Council o^ Scientific and Industrial Research, New Velhi ior {fin­

ancial support.

I wouid like, to thank my collQ.agaQ.^, i,pzdially di. Bzchan Sha^ma,

Shalini, PooKnima, Hamta and {/ibha ion. tk2.lfi anc2.a-iing h^lp and co-

opziation. My ^-twce^e thanks to Shfii Shyam Sand^fi and Sii Uatadln

{,on. th^AK technical aMLitancz and SkAi K.L. Gupta ^ofi ^xcdlle^nt typing

oi my the.6lA.

HUNEZA HUSSAW

C O N T E N T S

Page No.

Abbreviat ions i

CHAPTER I

Introduction and Review of L i t e r a t u r e . . . , 1-17

CHAPTER II

Materials and Methods 18-25

CHAPTER III

Results and Discussion 26-61

CHAPTER IV

Summary and Conclusion. '. . 62-65

BIBLIOGRAPHY 66-72

* * * * *

• * •

*

A B B R E V I A T I O N S

A DP

BSA

DTT

GDP

LDH

MDH

I^ME

NAD

NADH

NADP

NADPH

OAA

PEP

PMSF

PFK

PK

SDH

Adenosine d iphospha t e

Bovine serum albumin

Di th io th r i to l

Guanidine d iphospha t e

Lactate dehydrogenase

Malate dehydrogenase

/ -mercaptoethanol

Nicotinamide adenine dinucleot ide

Reduced NAD

Nicotinamide adenine d inucleot ide

Reduced NADP

Oxaloacetate

Phosphoenol py ruva t e

Phenylmethyl su lphonyl f luor ide

Phosphofructokinase

Pyruva te kinase

Succinate dehydrogenase

phospha te

CHAPTER I

INTRODUCTION AND REVIEW OF LITERATURE

In recent years there has been a resurgence of interest

in research into various aspects of parasitic and other infectious

diseases with special emphasis on the search for new and improved

tools to combat those_ diseases for which the available techTiology

is inadequate. Helminthic infections like f i lar ias is , ascar iaasis ,

hookworm disease are the major health hazards to the mankind

in developing and under developed countries affecting millions of

human population. Although they do not cause acute mortality,

they sap the vi tal i ty of the nation already plagued by over popu­

lation, food shortage, poor sanitation and lack of personal hygiene.

As compared to their hosts, these organisms generally have

primitive level of structural and biochemical refinement and some

of them survive under very selective ecological conditions, and

have to adapt thei r life cycle with the functioning of the a l te r ­

nate host system. They have developed intricate molecular mechan­

isms for counteracting host defence system and to exploit their

metabolic machinery and regulatory molecules for thei r own survival .

F i la r ias i s , a disease caused by "thread like" worms of

the family Filaroidae of Phylum Nematoda is a chronic arthropod-

transmitted disease of great public health importance. Over 900

million people around the world are living in areas where the disease

is endemic. In India, the population exposed to the r isk of f i la r i ­

asis has increased from 25 million in 1955 to 304 million in 1982

and an estimated 22 million people are known to harbour circulating

microfilaria (mf) and another 16 million suffer from clinical mani­

festations of filariasis (Ghatak et_ al^., 1987). Although the disease

is not fatal, it causes considerable morbidity, disability and social

stigma and the terminal stage of the disease is popularly known

as elephantiasis. The magnitude of suffering due to filariasis is

so enormous that world health organisation has included it in i ts

special programme of research and training for the control of tropical

diseases.

Filariasis is endemic all over India except few western,

northern and eastern s tates . It is prevalent in Assam, Kerala,

Andhra Pradesh, Uttar Pradesh, Madhya Pradesh and West Bengal.

Rapid industrialization and consequent migration of people has helped

in the spreading of the disease in areas where it did not exist

before. Hence filariasis has now been recognised as a disease

of national importance in this country.

Causative organisms

Several species of filarial parasi tes are known, each re la­

tively specific for i ts host. Important pathogenic species of man

are Wuchereria bancrofti, Brugia malayi. Onchocerca volvulus and

Loa loa; while in animals and birds the disease is caused by

Setaria cervi (cat t le) , Dirofilaria immitis (dog), D.uniformis ( rabb i t ) ,

Litomosoides carinii (cotton r a t ) , Acanthocheilonema viteae (gerbil/

mastomys) and Chandlerella hawkingi (jungle crow). There are

three distinct phases in the development of f i larial parasites v i z . ,

microfilaria! (mf), infective larvae ( L , ) , and adult . The adult (male/

female) parasite resides in lymphatic/connective tissues and cavities

of the host and releases mf (sheath/unsheathed) in the peripheral

blood. These mf have a life span of 14-17 days and exhibit noctur­

nal or diurnal periodici ty. The filarial parasite requires an al ter­

nate host (mosquito/mite) for completing i t s life cycle. In India

Mansonella species of mosquito transmits B.malayi whereas Culex

quinquefasciatus spreads W.bancrofti. Adult worms have host speci­

ficity and survive for 10-18 years . B.malayi and W.bancrofti are

lymph dwelling parasites whereas (Acanthocheilonema) and Onchocerca

species live in the tissues under the skin of the host .

Treatment of filariasis

Though filariasis was recognised as far back as 6th century

B.C. yet no effective chemical or immunological remedies could

be discovered for eradicating th is parasi t ic scourge. Few drugs

with limited efficacy and considerable side effects (sometimes fatal)

are available (Wang, 1982). Diethylcarbamazine (DEC) discovered

in mid forties, is essentially microfilaricidal and acts rather slowly

on adult worms. Hence it is not suitable for eradicating filarial

infections. Thus better drugs specially macrofilaricides are needed

for curing the disease. According to the existing l i terature some

compounds have been used as antifilarials l ike quinoline, metrifonate,

nitrofurantoin, levamisole, suramin, stibophen, DEC, centperazine,

flubendazole, mebendazole e tc .

The brief account presented above indicates the seriousness

of the disease and points towards making coordinated efforts to

control f i lar ias is . This could be achieved either by developing

an effective, nontoxic or least toxic chemotherapeutic agent which

can act both on microfilaria and adult worms or a suitable vaccine.

In either case it is necessary to have a sound knowledge of the

morphology, physiology and anatomy of the parasite especially i ts

biochemical machinery at the different life stages.

In recent years two main approaches have been made for

elucidating the mechanisms of action of anthelmintics. The first

approach is derived from the observation that several anthelmintics

have effect on neuromuscular system of the parasite leading to either

increase or decrease in their motil i ty. The other approach is

based on the knowledge that some compounds affect the energy generat­

ing system of the parasite by selectively interfering with the funct­

ional integrity of the parasite enzyme(s) or the uptake of carbo­

hydrates (WHO, 1983). However, only meagre information is available

about the neuromuscular system as well as the energy metabolism

in filarial v/orms. Studies regarding these two systems may lead

to the development of specific drugs which can selectively inhibit

neurotransmission process or the enzymes of the parasite without

having adverse effect on the host .

The available information regarding the metabolic pattern

of filarial parasites in relation to drug action with special reference

to neurotransmission and energy metabolism published during the

last two decades are summarised below.

Biochemical analysis of filariids and their developmental stages

Glycoproteins have been identified as a structural component

of the cuticle in A.immitis, B.pahangi, and B.malayi (Cherlan^ et

al_., 1980; Furman and Ash, 1983; Kaushal et_ a l . , 1984). The sheath

present in the mf of W.bancrofti, Loa loa and B.malayi but absent

in A.perstans and 0.volvulus acts as a barr ier between the parasite

and the host defences. A better understanding of the chemistry

of filarial sheath would facilitate the development of procedure

for disruption of i ts biological function. Srivastava (1985) achieved

exsheathment of B.malayi mf by treatment with endopeptidases and

papain. Lectin binding studies revealed the presence of N-acetyl

glucosamine, glucose and mannose on the mature mf of B.pahangi,

while sheath of immature mf contain sialic acid, galactose, and

N-acetyl glucosamine (Furman and Ash, 1983).

Comparative chemical analysis of mf and adult have indicated

that dry matter accounts for 10 and 25% respectively of fresh body

weight in these two stages of S.cervi . The dry matter consisted

of carbohydrates, l i p ids , protein and traces of nucleic acids (Rathaur

et a l . , 1980). Filarial parasites do not store glycogen, the main

reserve food for providing energy under adverse conditions. Mf

of S.cervi contain very l i t t le glycogen as compared to adult, suggest­

ing that this developmental stage of the parasite does not have

sufficient polysaccharide reserve (Rathaur et a l . , 1980). D.immitis

mf have, however, been reported to possess glycogen (Jaffe and

Doremus, 1970).

Reducing sugars account for the major portion of the total

carbohydrate in both mf (52%) and adult (67%) of S.cervi . Glucose

was the main constituent of the reducing sugars while concentration

of fructose was very low in adults and undetectable in mf (Rathaur

13 et a l . , 1980). Using C-glucose and NMR spectroscopy, Powell

et a l . (1986) showed the presence of trehalose in B.pahangi and

B.malayi. Glucose, galactose, mannose, myoinositol, ribose and

xylose were found in D.immitis (Ueda and Sawada, 1968), while

significant amounts of glucosamine and glucuronic acid were detected

both in adult and mf of S.cervi (Rathaur et_ al_., 1980).

Neuromuscular transmission

Neurotransmitter system is fairly well developed in various

helminths including filarial worms. They have underneath the cuticle

a single layer of muscles consisting of longitudinal f ibres . The

movement of these muscles are controlled by nervous system consist­

ing of nerve f ibres , which operate through release of neurotrans­

mit ters . The presence of a number of putative neurotransmitters

v i z . , catecholamines (norepinephrine, dopamine ) 5-hydroxytryptartiine

(5-HT) and acetylcholine with excitatory and inhibitory propert ies

have been established in filarial worms (Saxena et_ al^., 1977; Rathaur

et a l . , 1985; Singhal et_ a]_., 1975). In addition to thei r role in

neurotransmission and behavioural coordination these neurotransmitters

have been shown to be involved in regulating many metablic r e ­

actions like stimulation of phosphofructokinase and phosphorylase

by 5-HT resulting in enhancement of carbohydrate metabolism. Neuro­

transmitters execute their physiological responses through specific

membrane bound receptors , the properties and significance of which

had been well documented in vertebrate t issues . Although thei r

presence have been speculated in filarial worms, no reports are

available regarding the nature of the receptors.

Energy metabolism

Considerable evidence has accumulated showing that carbo­

hydrate is the primary source for the energy production in para­

sitic helminths. These organisms assimilate carbohydrates, but

the processes involved in carbohydrate utilization are not identical

with those established in vertebrate tissues as none of these para­

sites oxidise carbohydrate completely to C0_ and H_0. The in­

complete oxidation leads to the accumulation and excretion of a

variety of part ial ly oxidised metabolic end products (Saz, 1970;

von Brand, 1973). The biological significance of incomplete oxidation

of carbohydrates is not properly understood. McManus (1986) while

reviewing part ial oxidation of carbohydrates reempKasized the out­

standing feature of carbohydrate catabolism in these organisms v i z . ,

production of reduced organic end products even under aerobic condi­

t ions.

Utilization of hexoses

The fi larial parasites derive their energy mainly through

carbohydrates and remain motile in a mineral medium fortified with

glucose or mannose. However, when the sugar concentration goes

down the worms start degrading their polysaccharide reserves for

remaining a l ive . The nature of the metabolic processes by which

filarial parasites obtain energy have been examined by several

investigators (von Brand, 1973). Incubation of L.carinii and S.cervi

adults with glucose, mannose, fructose or galactose produced lactic

and pyruvic acid. Glucose and mannose were utilized at a rate

much faster than that with galactose and fructose (Anwar et a l . ,

1975). L.carinii adult was found to be aerobic but l ike all other

parasit ic helminths it accumulates the end products of carbohydrate

utilization consisting of acetate or lactate and C0_ (Saz, 1981).

A.viteae and B.pahangi, however, differ markedly from L.carinii

as former two parasites survive for extended periods anaerobically

whereas L.carinii loses motility rapidly in absence of a i r . In

addition, isotope and aerobic fermentation balance studies indicate

that A.viteae and B.pahangi are homolactate fermenting anaerobes.

L.carinii on the other hand accumulate acetate in addition to lactate

and tr icarboxylic acid (TCA) cycle is not operative as an energy

generating pathway in this paras i te . Acomplete carbon balance

was not obtained in L.carinii and other products are presumed to

be present which must be identified before more definite statements

on i ts 0- requirement can be made (Dunn et_ a]^., 1988).

13 Recent studies using C glucose and NMR have detected

1-2% and 2-5% succinate among the fermentation products in B.pahangi

and A.viteae suggesting minor involvement of alternate pattern of

glucose catabolism (Powell et a l . , 1986).

Enzymes of carbohydrate metabolism

Evidence for the functioning of different metabolic pathways

in filarial worms has been adduced mainly by the demonstration

of the enzymatic steps or the identification of the intermediates

of the pathway. The carbohydrate metabolism of a few filarial

parasites (C.hawkingi, L.carinii and S.cervi) have been studied

in detail in this Institute. These filarial worms have been shown

to be equipped with many of the enzymes of anaerobic glycolysis,

PEP-succinate pathway and TCA cycle (Ghatak et al •, 1987). Distri­

bution studies on m.any of the glycolytic enzymes and the demons­

tration of phosphorylated glycolytic intermediates in filarial worms

clearly indicate the functioning of typical glycolytic sequence until

phosphoenol pyruvate (PEP) or pyruvate is reached (Barret, 1983;

McManus, 1986). The operation of a full glycolytic pathway for

conversion of glucose to lactic acid has been demonstrated

in C.hawkingi (Srivastava et_ a l . , 1968; Srivastava and Ghatak,

1971), L.carinii (Srivastava et_ al_. , 1970) and D.immitis (Hutchison

and McNeill, 1970). The enzymes of Embden Meyerhof scheme were

localized in the cytosol showing a resemblance with the mammalian

system. The activity of some of the key enzymes were higher

than the corresponding values reported for other f i l a r i ids .

Although most of the enzymes of the TCA cycle have been

documented in a few filarial worms (McNeill and Hutchison, 1971;

10

Agarwal et a l . , 1986; Dunn et_ a l . , 1988), the functioning of a true

TCA cycle in filarial parasi tes is st i l l a debatable point. Similarly

the presence of low or negligible activit ies of glucose 6-phosphate

dehydrogenase and 6-phosphogluconate dehydrogenase in S.cervi

(Anwar et_ aJ_., 1977), O.gutturosa (Dunn et_ a]_., 1988), C.hawking!

(Srivastava, 1969) cast a doubt about the functioning of a pentose

phosphate pathway in fi larial paras i tes . So it is presumed that

S.cervi mainly gains energy from the glycolytic pathway for i ts

survival . Presence of comparatively low activity of phosphofructo-

kinase (PFK) in S.cervi suggests that th is key enzyme may be play-,

ing a regulatory role in controlling the, operation of glycolytic path­

way. Under aerobic conditions L.carinii produces acetate which

is derived from decarboxylation of pyruvate, probably via pyruvate

dehydrogenase. This enzyme is present in significant amount in

L.carinii but in lower amount in B.pahangi and A .viteae (Wang and

Saz, 1974; Meddulin and Saz, 1979).

The balance between pyruvate kinase (PK) and PEP-carboxy-

kinase (PEPCK) and their affinity for their common substrate (PEP)

are the factors that determine whether metabolic products are

channeled to succinate or to lactate (von Brand, 1973). S.cervi

which converts 25% of metabolised glucose to lactic acid and possesses

low level of PK and lactate dehydrogenase (LDH) but high activities

of PEPCK and malate dehydrogenase (MDH) has a functional PEP-

succinate pathway. On the other hand, typical lactic acid producers,

like C.hawkingi (Srivastava et a l . , 1968; Srivastava and Ghatak

1971) which convert 80-90% glucose into lactic acid, resemble

11

v e r t e b r a t e t i s sues in possess ing high l eve l s of PK and LDH and

low l eve l s of PEPCK and MDH. According to Bueding and Saz (1968)

PK/PEPCK ra t io g r e a t e r than one indicate tha t the metabolic pathway-

leads to lac ta te accumulation while the values l e s s than one suggest

the operat ion of C0_ fixing pathway and probable product ion of

succ ina te . Both succinate and lac ta te have been found to accumulate

in ra t io dependent on the age of the worms (Watts and F a i r b a i r n ,

1974) and the amount of 0- in the medium (von Brand, 1973). Both

mf and adu l t s of S . ce rv i have a PK/PEPCK ra t io l e s s than one (Anwar

et a l . , 1977; Rathaur et_ al_., 1982). However, in adul t D. immit is

(Braz ie r and Jaffe , 1973), C.hawkingi (Sr ivas tava and Ghatak, 1971)

and L . ca r in i i (S r ivas tava iet_ a l . , 1970a) the ra t io was h ighe r than

one, suppor t ing the p re fe ren t i a l formation of l a c t a t e . Among t h e

o the r carbon d iox ide fixing enzymes, malic enzyme seems to be

of some importance in a few he lmin ths . Malate en te r s the mi to ­

chondrion and undergoes a dismutation to pyruva te (under the influence

of NADP l inked malic enzyme) and to succinate (under the action

of fumarase and t h e subsequent reduction of fumarate by a fumarate

reduc tase c o m p l e x ) . The nature of terminal ox idase in f i l a r i a l

worms i s uncer ta in . F a i r l y normal looking c r i s t a t e mitochondria

have been r e p o r t e d in adu l t s of B.pahangi , L . ca r i n i i and D.immitif;

(Lee and Mi l l e r , 1969; Johnson and Bemrick, 1969; Middleton and

Saz, 1969). However, ne i t he r cytochrome-C nor cy tochrome-C-oxidase

could be de t ec t ed in adu l t s of L . c a r i n i i , B.pahangi or A .v i t eae

(Wang and Saz, 1974). In con t ras t , Rew and Saz (1977) de tec ted

cytochrome ox idase in mf of B .pahangi . S . ce rv i adu l t s contained

12

GUYCOGEH

GLUCOSE;

LACTATE

OXALOACETATE

NADH^

NAD MALATE

Id < a: U S D

• U . -HOH

FUMARATE

FADH,

SUCCINATE

Fig. 1: Phosphoenol pyruvate-succinate pathway in bovine f i larial

parasi te Setaria ce rv i .

13

cytochrome b , and in spite of several attempts presence of cytochrome

P.j.^ could not be detected in this parasite (Saxena e^ al_. in p re s s ) .

Lack of cytochrome P^cn in O.gibsoni and A, viteae was reported

by Mendis (1985).

Succinate is excreted in exceeding amounts under conditions

of low glucose availability (Powell et_ al_., 1986) and provided that

succinate formation is important or v i ta l , under these conditions

inhibition of PEP carboxykinase would provide a novel target for

filarial chemotherapy. PEPCK has been shown to be very active

and i ts properties have been determined in several paras i tes .

The enzyme occurs both in cytosol and mitochondria of l iver although

the distribution of the enzyme between these two cell compartments

varies from one species to another. Another important branch point

enzyme of PEP-succinate pathway is pyruvate kinase. Two types

of pyruvate kinase (L and M type) have been reported in mammals

as well as in paras i tes . L-type enzyme seems to be predominant

in fi larial worms - D.immitis and L.carini i . The other important

enzymes are fumarase, malic enzyme, lactate dehydrogenase, malate

dehydrogenase, succinate dehydrogenase and fumarate reductase (a

flavoprotein containing covalently bound FAD and iron sulphur groups).

(Fig. 1).

Metabolic targets for chemotherapeutic attack

The aim of specific chemotherapy is the removal of invading

organism without causing injury to the host. In order to achieve

14

t h i s , we must define the biochemical structure and metabolic pathways

of the parasite and its various developmental stages and synthesize

a selective agent which can knock off the parasite witKout showing

adverse effect on the host harbouring them. The identification

of sensitive molecular targets can provide a more rational approach

for the chemotherapy of f i lar ias is .

As mentioned ear l ier , most anthelmintic agents act directly

or indirectly by inhibiting ei ther neuromuscular transmission or

energy generation. The general sites of action of two well known

antifilarial compounds, DEC and suramin and antinematodal compound,

levamisole have been discussed by van den Bossche (1981).

DEC has been found to be an effective filaricide ir]_ vivo

for W.bancrofti, 0.volvulus, A. viteae and B.malayi. It acts p r i ­

marily as a microfilaricide, although some action against adults

of W.bancrofti, B.malayi (Otteson, 1984; Goodwin, 1984) and L

larvae of B.malayi (Ewert and Emerson, 1975) has also been demons­

t ra ted . DEC, however, has no action against L_ larvae of

W.bancrofti (Jorden, 1958) and exhibi ts poor and impredictable

response in A. viteae and B.malayi infection in j i rds (Tanaka e^

a l . , 1981; Nogami et al_., 1984). In the studies by Langham et_

a l . (1980) all 0 . volvulus died at 0.1 mg per cent concentration

of DEC which is in the plasma concentration range of DEC found

in patients infected with onchocerca (Ree et_ a l . , 1977). Thus,

it appears that death of 0.volvulus mf may be related to an effect

15

on the neuromuscular sys tem. DEC also affects mot i l i ty in A .v i t eae

(Cavier et a l . , 1971) and Breinlia sergent i (Natarajan et a l . , 1973).

Levamisole at 100 ng/ml concentration completely s tops moti­

l i t y of f i l a r i a l p a r a s i t e s such as L . c a r i n i i , A.vi teae and B.pahangi

(Wang and Saz, 1974; Rew and Saz, 1977). Levamisole ac ts by s t imu­

lation of ganglion s t ruc tu res followed by a depolar i s ing type of

neuromuscular inhibi t ion (van Nueten, 1972; Coles et_ a l . , 1974).

Energy metabolism is a lso affected with decreased uti l izat ion of

glucose and shift to homolactate fermentation (Saz, 1981).

Suramin, an ant i t rypanosomal compound was found to act

upon 0 .vo lvu lus (van Hoof et_ al_., 1947). Due to the tendency of

suramin to combine with p r o t e i n s , i t i n h i b i t s enzymes involved

in energy metabolism, calcium t r a n s p o r t and var ious enzymes con­

cerned with phospho ry l a t i on /dephosphory l a t i on r e a c t i o n s .

Centperazine , CDRI compound 72/70, levamisole and DEC

signif icant ly inh ib i t glucose u t i l iza t ion and syn the s i s of glycogen

in S .ce rv i (Anwar et_ a]_., 1978).

Avermectins are effect ive in des t roy ing mf of D. immit i s .

The drug b locks t ransmiss ion of command from vent ra l interneurons

to motor neurons in Ascaris and enhances Y-aminobutyr ic ac id (GABA)

re l ease and i t s binding to post synap t i c GABA recep to r (Pong et^

a l . , 1980; Pong and Wang, 1982).

16

SCOPE AND PLAN OF WORK

The information on the metabolic aspects of filarial parasites

presented above reveals fascinating mosaic of biochemical reactions

employed by the organisms for the i r survival and for their adaptat­

ions to different hos ts . The filarial worms differ widely from

the respective hosts with regard to different metabolic reactions

and biochemical proper t ies . Further , the parasite differs consider­

ably from each other reaffirming the suggestion of Saz (1981) that

each organism must be examined as a biochemical entity before

any reasonable understanding of i t s metabolism can be attained.

The filarial worms studied so far employ predominantly

anaerobic metabolism of carbohydrate as the major energy yielding

pathway and utilize limited quantity of 0^ (if avai lable) . However,

they do not possess the abi l i ty of bringing about complete oxidation

of substrates to C0_. Electron transport chain is rudimentary and

the parasites catalyse only limited terminal oxidation with generation

of l i t t le energy. Since PEP-succinate pathway has a crucial regu­

latory function in channelling of carbon, regeneration of pyridine

nucleotides as well as generation of energy in filarial parasi tes ,

a study of the status of the enzymes of this pathway with special

reference to the effect of anthelmintic/antifilarial drugs would provide

an attractive area of research . The basic problem encountered

in studying f i lariasis is the non-availability of real human pathogen

(W.bancrofti/B.malayi) in sufficient amounts. S.cervi a bovine

filarial worm resembles human parasite in microfilarial periodicity,

17

antigenic pattern (Malhotra e^ a]_. , 1986; Kaushal e^ aj^., 1987) and

sensitivity towards known antif i lar ials . The parasite is available

in sufficient quantity^ from local slaughter house and hence it i s

ideal for conducting biochemical investigation.

Attempts have been made for studying the following aspects

in the present dissertat ion.

1. Subcellular localization of various enzymes of PEP-succinate

pathway and effect of ant if i lar ials .

2. Isolation and propert ies of PEP-carboxykinase from S.cervi .

CHAPTER II

MATERIALS AND METHODS

18

Biochemicals and enzymes

The biochemicals obtained from Sigma Chemical Company,

St . Louis , USA were ADP, BSA, DTT, GDP, L D H . ' M D H , ^-UE, OAk,

PMSF, PEP and sodium p y r u v a t e . Malic acid from E.Merck, West

Germany; sodium fumarate and sodium succinate from BDH, UK were

used for t h i s s t u d y . Sephadex G-25 and Blue Sepharose CL-6B

used for the pur i f icat ion s tud ies were procured from Pharmac ia ,

Uppsala , Sweden. NADH and NADP were suppl ied by CSIR Centre

for Biochemicals , Delhi , Ind ia . Among the anthelmint ics u sed ,

suramin was from Hoechst, AG, West Germany; levamisole was a

gift from Late Prof. G. Lammler, FRG and DEC c i t r a t e were p u r ­

chased from Cyanamide Ind ia . Pure base of DEC was p r e p a r e d

in Medicinal Chemis t ry Division of t h i s Inst i tute by Column Chromato-

'g raphy of DEC c i t r a t e . Centperazine and compound 72/70 were

supp l ied from Medicinal Chemis t ry Division of CDRI.

All o the r chemicals used were of ana ly t ica l grade and t r i p l e

g lass d i s t i l l e d water was used for a l l biochemical e x p e r i m e n t s .

Parasite

Motile adul t females (average length 6.0 ± 1.0 cm, average

weight 35 ± 6 mg) of Setar ia c e r v i , the bovine f i l a r i a l p a r a s i t e

were col lec ted from the per i tonea l cavi ty of f r e sh ly s l augh te red

, buffaloes (Bubalus buba l i s L inn) . The motile worms were brought

to the l a b o r a t o r y in Ringers solution (Singhal et aJ_, , 1973) wi th in

19

2 hours of s laughter ing from the local a b a t t o i r . The worms were

thoroughly washed with luke warm isotonic sa l ine for removing

t h e adher ing contaminants and used for enzymic s tud i e s within

2-3 hours of t h e i r removal from the c a d a v e r s , or immediately ch i l l ed

in ice and homogenized.

Preparation of medium

Ringers solution : 9 g of sodium c h l o r i d e , 420 mg of potassium

c h l o r i d e and 500 mg of glucose were weighed and d i s s o l v e d in mini ­

mum amount of w a t e r . After ra i s ing the volume to approx imate ly

900 ml , s e p a r a t e l y d i s so lved 250 mg sodium bicarbonate and 420

mg of calcium ch lo r ide were added and the volume made up to

one l i t r e .

Buffers; The buffers used for the s tudies were made with Tr i s

and t h e pH was adjusted with HCl ( l .ON). Sodium-potassium buffer

was made by adding the two sa l t s at such a concentrat ion tha t

r e q u i r e d pH was ob ta ined .

Preparation of homogenate

The homogenate of the f i l a r i a l pa r a s i t e Se tar ia c e r v i used

for t h e enzymic s tud ies was made in KCl (150 mM; 1:10 w / v ) .

F r e sh intact worms p rev ious ly ch i l l ed in ice were homogenised

using a high speed t ight f i t t ing Pot ter -Elvehjem homogeniser . The

homogenate t hus obtained was centrifuged under cold condit ions

at 800xg for 10 min at 12,000xg for 30 min and at 105,000xg for

20

60 min for the separat ion of cell d e b r i s , mi tochondr ia l , post mi to­

chondr ia l and microsomal f rac t ions ,

I . Enzyme assay:

Details of the reaction mixtures used for the measurement

of PK, PEPCK, LDH, MDH, FRD, SDH, fumarase and malic enzyme

a r e d e s c r i b e d under each assay sy s t em. Spect rophotometr ic measure ­

ments were made in a final volume of 3 ml using a Beckman DU

spec t rophotometer or spec t ronic-21 UVD model with s i l i ca cuvet tes

of 1 cm l ight p a t h . All assay mix tures were p re incuba ted for

3 min at room tempera ture (28±2 C) before s t a r t ing the react ion

by t h e addi t ion of the s u b s t r a t e s . Change in absorbance at 340

nm was noted p r i o r to the addi t ion of the s u b s t r a t e s as well as

for t h e pe r iod of actual enzymic react ion following the addi t ion

of the s u b s t r a t e .

Pyruvate kinase (ATP, Pyruva te phospho t r ans fe ra se EC 2 . 7 . 1 . 4 0 ) :

Assay was based on Bucher and P f l e i d e r e r (1955) modified by the

use of Tr i s -buf fe r ins tead of Triethanolamine buffer . Assay mixture

contained: Tr i s HCl (pH 7.4) 200 mM; KCl, 100 mM; MgCl2, 10 mM;

ADP, 5 mM; PEP, 5 mM; LDH, 4 un i t s ; NADH-; 0,24 mM and enzymic

pro te in 100-200 ug.

Phosphoenol pyruvate carboxykinase (GTP: oxaloaceta te ca rboxy lase

(Phosphory la t ing ) EC 4.1.1 .32) was a s sayed according to Ward et^

a l . (1969) , as modified by the use of Tr i s -buf fe r (pH 6.5) ins tead

of T r i s buffer pH 7.4 200 mM, MnCl2lO mM, NaHCO^ 50 mM, GDP,

21

1 mM; PEP, 5 mM; MDH 4 uni t s ; NADH^ 0.24 mM and enzyme protein

20-50 ug.

Lactate dehydrogenase (L-lactate:NAD oxido r e d u c t a s e , EC 1.1.1.27)

-was assayed according to Kornberg (1955). Assay mixture contained

Tr i s HCl (pH 7.4) 200 mM; KCl, 100 mM; sodium p y r u v a t e , 5 mM,

NADH_ 0.24 mM and enzyme protein 5-15 ug.

Malate dehydrogenase (L-malate:NAD oxido r educ ta se EC 1.1.1.37)

was assayed according to Shonk and Boxer (1964) . Assay mixture

contained Tr i s HCl (pH 8.0) 200 mM; MgCl_ 10 mM; oxa loace ta te ,

10 mM, NADH_, 0.24 mM and enzyme prote in 0 .6-1 .5 ug.

Malic enzyme (L-mala te : NADP oxido r educ tase (deca rboxy la t ing)

EC 1.1.1.40) was assayed according to Ochoa (1955) . Assay mixture

contained Tr i s HCl (pH 7.4) 20D mM, MnCl- 10 mM; L malate 10

mM; NADP 0.24 mM and enzyme pro te in 0 .5-1 .5 mg.

Fumarase (L-malate hyd ro lyase EC 4 .2 .1 .2 ) was a s sayed according

to the method of Racker (1950). The react ion mixture consis ted

of potassium phospha te buffer (pH 7 . 4 ) , 150 mM; L-mala te , 30 mM

and enzyme protein 40-80 ug. Increase in abso rbancy was monitored

for 3 minutes at 240 nm.

Fumarate reductase (Succinate: ( accep to r ) oxido r e d u c t a s e , EC 1.3.99.1);

The a s say based on tha t d e s c r i b e d by P r i c h a r d (1973) was performed

with mitochondria l p repa ra t ions . - Reaction mix ture contained Tr i s

buffer (pH 7 . 2 ) , 100 mM; MgCl2, 10 mM; NADH, 0.3 mM; K" fumarate,

22

10 umol; CaCl^, 1.5 umol and enzyme protein 0.5-1.5 mg.

Succinate dehydrogenase (Succinate: (acceptor) oxido r e d u c t a s e ,

EC 1.3.99.1): It was assayed according to Metzger and Duwel (1973)

and was performed with mitochondria l p r e p a r a t i o n . The reactipn

mixture contained sodium potassium phospha te buffer (pH 7 . 4 ) ,

EDTA 1 mg/ml; 2,6 d ich lo ro phenol indophenol (Na DCIP 15 mg/100

ml ) ; sodium succinate (4.05 g/?5 ml) and enzyme prote in 0.5-1.5

mg. The decrease in ext inct ion was followed at 600 nm.

Defination of enzyme unit and speci f ic act iv i ty

One unit of enzyme a c t i v i t y i s the amount of enzyme r equ i r ed

to ca ta lyse the transformation of one umol subs t r a t e or the forma­

tion of one umol product pe r minute under speci f ied exper imenta l

condi t ions . Specific a c t i v i t y i s unit/mg of enzyme p r o t e i n . The

extinction for oxidat ion or reduct ion of p y r i d i n e nucleot ides was

measured at 340 nm whi le increase in op t ica l dens i ty in fumarase

assay was r ecorded at 240 nm.

6 ? An ext inct ion coefficient of 6.22 xlO cm /mole (Horecker

and Kornberg, 1948) at 340 nm was used for NAD or NADP.

For fumarase the molar ext inct ion coefficient used was

3 -1 -1 2.44 X 10 x m M at 240 nm (Mahler et_ al_., 1958). For succinate

6 ? dehydrogenase the molar ext inct ion coefficient was 2.06x10 cm /molar

at 600 nm.

The following general equation was a p p l i e d for calculat ing

enzyme a c t i v i t y :

23

lU = OD/min X TRMV X DF Extinction coefficient

lU = Internat ional Unit

OD/min = Change in op t i ca l dens i ty /min

TRMV = Total react ion mixture volume (ml)

DF = Dilution factor of enzyme used

Protein estimation: Protein content in different subce l lu la r f rac t ions ,

column eluates (obta ined during pur i f ica t ion) was es t imated s p e c t r o -

photomet r ica l ly according to Layne (1957) or ca lo r imet r i ca l ly by

the method of Lowry et_ a l . (1951) using bovine serum albumin as

s t anda rd .

II. Purification of PEPCK

Adult females of S . ce rv i were homogenised in ice cold Tr i s

HCl buffer (50 mM pH 6 . 5 , 1:10 w / v ) containing DTT (1.5 mM) and

PMSF (0.1 mM) using a t igh t f i t t ing Po t te r Elvehjam homogeniser

for 2-j min. Centrifugation was done at 800xg for 10 min and at

12,000xg for 30 min. The post mi tochondr ia l supernatant thus o b t ­

ained was used for the pur i f ica t ion of PEPCK.

Step I: Ammonium sulphate fractionation

The supernatant from t h e p rev ious s t ep was brought to

301 saturat ion with r e spec t to ammonium su lpha te and the mixture

was gently s t i r r e d for 1 hou r . After centrifugation at l l ,000xg

for 30 min the p r e c i p i t a t e was d i s c a r d e d . To t h e supernatant

24

ammonium sulphate was added at constant stirring to 70% saturation.

The pH was adjusted to 7.0 with dilute NH_ after each addition

of (NH.)_SO. . The mixture was allowed to stand for 2 hours and

then centrifuged. The supernatant was discarded and the p rec ip i ­

tate was dissolved in a small volume of 50 mM Tris HCl containing'

1.5 mM DTT and 0.1. mM PMSF. The solution was further passed

through the Sephadex G-25 column.

Step II: Desalting on sephadex G-25 column

2 gm of Sephadex G-25 was soaked in about 500 ml of

double dis t i l led water and kept at boiling temperature for 2 hr

with intermittent gentle s t i r r ing. The upper layer of water contain­

ing the fine part icles of Sephadex was decanted. Fresh volume

of dis t i l led water was added and the contents were left overnight

for complete soaking. The sephadex beads were equilibrated with

the suspension buffer and packed in a glass tube for making column

size of 11x1.4 cm. The protein fraction obtained after 70% ammonium

sulphate saturation was layered over this column and washed with

equilibration buffer, 3 ml eluates were collected at a flow rate

of 3 ml/5 min. The protein content of different eluates was moni­

tored at 280 nm and the PEPCK rich fractions were pooled and

used for affinity chromatography.

Step III: Blue sepharose CL-6B affinity chromatography

1 g of Blue Sepharose CL-6B was soaked in dis t i l led water

for swelling of the beads . For the regeneration of Blue Sepharose

25

the gel was washed with regenerat ing buffers I and II followed

by washing with the equ i l ib ra t ing buffer of following composi t ion.

The regenera t ing buffers v i z . , Buffer I contained Tr i s HCl, pH

8.5 (100 mM)and NaCl (0 .5 M); whi le Buffer IT contained sodium

a c e t a t e , pH 4.5 (100 mM) and NaCl (0 .5 M). The equ i l i b r a t i ng

buffer , Buffer III contained Tr i s HCl, pH 6.5 (50 mM) , PMSF (0 .1

mM) and DTT (1.5 mM). These buffers were f r e sh ly p r e p a r e d . The

beads of Blue sepharose were washed a l t e rna t e ly with Buffer I ,

Buffer II and then again with Buffer I and f inal ly with Buffer I I I .

The washed beads were uniformly packed in a small g lass column

(5x0.5 cm) and desa l t ed fraction from Step I I , r i c h in PEPCK a c t i ­

v i t y , was s lowly l aye red over the column. Now equ i l ib ra t ion buffer

(Buffer III) was passed through the column for removing unadsorbed

p r o t e i n s . The dehydrogenases bound to Blue Sepharose were removed

by pass ing Buffer III containing NADH (1 mM) (equiva lent to 4 v o l ­

umes of loaded f r ac t ion ) . Then the adso rbed enzyme was e lu ted

from the column by passing Buffer III containing 0.5 M, 0.75 M,

1.0 M and 2.0 M NaCl in a s tepwise manner. The fraction conta in­

ing maximum PEPCK ac t i v i t y was e luted with IM NaCL.

CHAPTER III

RESULTS AND DISCUSSION

26

The distribution pattern of the enzymes of PEP-succinate

pathway was analysed in different subcellular fractions v i z . , cel l-

free, post mitochondrial and post microsomal fractions of the bovine

filarial worm S.servi and is presented in Table I. Pyruvate kinase,

phosphoenolpyruvate carboxykinase, lactate dehydrogenase, malate

dehydrogenase, malic enzyme and fumarase were localized in 105,000xg

cytosolic fraction of the worm homogenate, while succinate dehydro­

genase and fumarate reductase were found to be particulate bound.

Among the soluble enzymes, malate dehydrogenase was most active

exhibiting 313 units act ivi ty/g wet worms with specific activity

of 17.08. Significant levels of LDH (27 units/g wet worms) fumarase

(14.9 units/g wet worms) and PEPCK (15.0 units/g wet worms) were

observed while the levels of pyruvate kinase and malic enzyme

were very low. Decrease in the level of pyruvate kinase from

4.72 units (12,000xg) to 2.41 units (105,000xg) suggest the removal

of some factor from the cytosol required for enzyme act iv i ty .

The mitochondrial pellet (12,000xg sediment) showed the presence

of succinate dehydrogenase (4.07 units/g wet worms, specific act i ­

vity 0.067) and fumarate reductase (1.35 units/g wet worms; specific

activity 0.02).

Effect of antifilarials on different enzymes of PEP-succinate pathway

The action of a few antifi larial agents v i z . , DEC.centperazine

compound 72/70, levamisole and suramin on the different enzymes

of PEP-succinate pathway was studied by preincubating the enzyme

t/5

X o o o

o

60 X

o

C3

a 3

CO

X o o o

& 3

en

X o o 00

w

E N n

W

u >

to u —^ <Ti

be

< 3

> s

O >

> be •H - ^

o "

^^ c 3

a t

^ •

be

<

o >

at;

> w E o p O

< g S

00 CD

Q

i n

C

tt)

> D

O IS

^ o 1—1

' ^ IT)

O ^ CO O fS]

m oo

> 3 U >- O p . CO

' ^ rt 2 c <u ^ 2 >-a o 2 ^

(1< o

Q 2

sO 00

t n

o

OJ

00

o

00 t — '

o o

vD in t>

o O Z

Q 2

o

CO I—1

CO

O

o

a 2

o

CD (X)

O

OO

CD

I o u

Xi > <u

Ti

0)

rt in

rt (1) 1-^ bC

CD CD • i n i n

I O U

<D

0) OJ

2 bC

" 1 r J

0) E > N

c

o • H r—I

E 3

r--o

CO 1—1

o o

vO vO in

CD (-J 2

o 2

i n i n •^ CD O^ 1—' CD (NJ O

CM ro

I O

0)

c •H

(D .. w

o c 3 0)

to be

Q 2

Q 2

Q 2

Q Z

Q 2

Q 2

Q 2

O

O

(—I (NJ CD

CD

i n CO

Q 2

Q 2

i n o o

fO (NJ

eg r-j

3 Q)

O -t- "+-1 +J C O

bC g C c E o

• H nJ - H

27

(fl

(U

x:

^ 1

o (U

T3

« .5 ai 4)

(U +-' (fl 5H w "J -{^

!* C <" +^ 3 X) iJ

m 3 Ti

og (U X ! -(-> • <D

c X! 2 •

5 2 I S >2 (u -

Ti -2 c z i

r-H (ti O

!i£| ,—I C («

•H -H C

E ^ i J o S o 0)

^ c S >< c ^

(U 0)

— be + j u 6 0)

en (u P-o\o nJ W W CD >-^

& t; '^ >- . (13 J-

13 N U 3 <D C .2 ^ O C C ^ ^ -^ (U -C be u o (S E <i o

• ^ • ^

S E OJ OJ

w en

E

Tl (I) ^1 •H

3 CT (U V4

• 1 - 1

u (U U-

-t-i

o 3

TJ 0

^ 1

o

o

00 (fl • -t->

i-< U)

o

E o >- E N 3 C <U (U

C +H O

o o

28

prepara t ion with the t e s t compound for 15 min at room tempera ture

(28±2 C) in the d e s i r e d buffer and then determining the r e s idua l

ac t i v i t y in the usual way .

Pyruvate kinase: Among the f ive f i l a r i c ida l compounds screened

the enzyme was most affected by suramin exhib i t ing 52% inhib i t ion

at 1 yuM concentration (F ig . 2 ) . Higher concentration of the drug

(100 /iM) caused 76% i n h i b i t i o n . DEC, levamisole and centperazine

could reduce the a c t i v i t y of the enzyme to 50% at 10 jaM concentrat ion.

Phosphoenol pyruvate carboxykinase : CDRI compound 72/70 and s u r a ­

min were found to be more effect ive in reducing the ac t i v i t y of

PEPCK than t h e remaining t h r e e f i l a r i c i d a l compounds. The i n h i ­

bition by former two drugs was 45-50% at 100 /uM concentrat ion.

Compound 72/70 showed 41% inh ib i t ion o f the enzyme at 1 yuM con­

centrat ion whi le l evamiso le , DEC and centperazine d id not show

much effect at 1/iM concentrat ion (F ig . 3 ) .

Lactate dehydrogenase: Suramin e x h i b i t e d maximum inh ib i t o ry action

lowering the LDH a c t i v i t y by 40% at 10 ^ M concentration while com­

pound 72/70, l evamiso le , cen tperaz ine and DEC showed 33, 31, 29

and 27 percen t inh ib i t ion at 100 /aM concentration (F ig . 4 ) .

Malate dehydrogenase: Maximum inh ib i t o ry effect was obse rved

with suramin inh ib i t ing MDH a c t i v i t y by 33% at 10/uM concentrat ion,

while centperaz ine and levamiso le showed only 19% and 7% i n h i b i -

29

o e

0.3 -

0.2 -

0.1

1x10 -6 1x10 " 1x10

Drug cone. (M)

1x10 -3

Fig . 2: Effect of an t i f i l a r i a l s on the ac t i v i t y of py ruva t e k inase of S . ce rv i E DEC, ® cen tperaz ine , E ! 72/70, nri levamisole and [oj suramin.

30

o E

(J

<

0.5 -

0.4

0.3 -

0.2

0.1

1x10 -5 X

1x10 " 1x10

Drug cone (M)

-4 1x10

-3

Fig. 3: Effect of antifilarials on the activity of phosphoenol

pyruvate carboxykinase (PEPCK) of S.cervi . E ] DEC,

fflcentperaTiine, [ 3 72/70, S levamisole and E ) suramin.

31

1.0 -1

0.8

0.6 -

o g

u 0.4

0.2 -

1x10 1x10 1x10

Drug cone (M)

1x10

F i g . 4 : Effect of an t i f i l a r i a l s on the a c t i v i t y of l ac ta te d e h y d r o ­

genase (LDH) of S . c e r v i . K l DEC, '--' cen tperaz ine ,

\Y} 72/70, [ f i 1 evamisole and El suramin.

32

tion at 100 AIM concentration and compound 72/70 was ineffective

(F ig . 5 ) .

Fumaj-ase: The enzyme was i n h i b i t e d by very low concentration

of suramin recording 72% decl ine at 1 AIM concentration while cent -

pe r az ine , DEC and 72/70 showed 32%, 32% and 20% inhibi t ion r e s ­

pec t ive ly at 10 yuM concentration (F ig . 6 ) .

Malic enzyme: Centperazine and suramin were found to be effect ive

to some extent at h i g h e r concent ra t ions . Suramin caused 60% i n h i b i ­

tion at 0.1 mM concentration whi le 1 mM centperazine was r equ i r ed

to get 51% inhib i t ion of the enzyme. Levamisole , DEC and 72/70

were ineffective even at h ighe r concentration (F ig . 7 ) .

Fumarate reductase: The enzyme was inh ib i t ed by 50% and 31%

r e s p e c t i v e l y in presence of 1 mM centperaz ine and DEC. Levamisole

and compound 72/70 were ineffective (Table 2 ) .

Succinate dehydrogenase: About 75% inhibi t ion of SDH ac t i v i t y was

obse rved at 100 /JM concentration of suramin whi le centperazine

levamisole and compound 72/70 showed 50-57% inhib i t ion at t h i s

concentration (Table 3 ) .

Since suramin was found to be a potent i nh ib i t o r of most

of t h e enzymes of PEP-succinate p a t h w a y , t h e va lues of the i n h i ­

bi t ion constant (K.) was determined from the Dixon p lo t s and r e ­

corded in Table 4 . The K. va lues for PK, LDH and fumarase were

33

10.0 -

o E

>

<

IxlO -6 1x10 ^ 1x10

Drug cone (M)

1x10

F ig . 5: Effect of a n t i f i l a r i a l s on the a c t i v i t y of malate d e h y d r o ­

genase (MDH) of S . c e r v i . (AI DEC, E cen tperaz ine ,

t S 72 /70 , I S levamisole and \9} suramin.

34

^ >-

<

1.6

1.4

1.2

0.8

0.4

1x10 -4 1x10

-3 xlO -' 1x10

Drug cone (M)

F ig . 6: Effect of a n t i f i l a r i a l s on the a c t i v i t y of fumarase- of S . c e r v i .

LSj DEC, |A] cen tperaz ine , t S 72/70, CS le vamisole and

suramin .

35

0 . 0 4 - 1

5

<

0.03 -

0.02 -

0.01

1x10 -6 1x10

F i g . 7:

1x10 - 1x10

Drug Cone (M)

Effect of an t i f i l a r i a l s on the a c t i v i t y of malic enzyme

of S . c e r v i . {^ DEC, I S cenp te raz ine , CE] 72/70

f S levamisole and tSJ suramin.

36

Table 2: Effect of an t i f i l a r i a l d rugs on the ac t i v i t y of fumarate reductase of Setaria c e r v i .

Addition Concentration % Act ivi ty (M) Remaining

None - 100

-3 Diethylcarbamazine 1x10 69

IxlO"'^ 83

-3 Centperazine 1x10 50

IxlO"^ 100

72/70 1x10 ^ 100

Ixio"'^ 100

-3 Levamisole 1x10 100

IxlO"'^ 100

37

Table 3: Effect of a n t i f i l a r i a l drugs on the a c t i v i t y succinate dehydrogenase of Setar ia c e r v i .

Addition Concentration (M)

% Act ivi ty remaining

None

Centperazine 1x10 -3

1x10 -4

100

25

43

72/70 1x10

1x10

40

50

Levamisole 1x10

1x10 -4

25

43

Suramin 1x0 25

/ 'A -:a.kO Lixx :-4^^

I ^ f

[ D3/3/7 .-3

38

Table 4: K. value of suramin for the enzymes of PEP-succinate 1

pathway of Setar ia cervi_.

Enzymes K. (M)

-7 Pyruva te kinase 6.5x10

-5 Phosphoenol p y r u v a t e - 2.5x10 carboxy kinase

-7 Lactate dehydrogenase 8.75x10

Malate dehydrogenase 1.00x10

-7 Fumarase 7.00x10

Malic enzyme 1.4x10

39

-7 -7 -7 recorded as 6.5x10 , 8.75x10 and 7.0x10 r e s p e c t i v e l y . There

were lower than the values for PEPCK, MDH and malic enzyme.

Effect of 2J1 v i tro treatment of S. cervi adults on the enzymes of

PEP succinate pathway

Since suramin, centperazine and levamisole showed i n h i b i t o r y

effect on the enzymes of PEP-succinate pa thway , a t t empts were

a lso made for s tudying the effects of these drugs on l i ve worms

during Jji v i t r o incubation for 3 h r in R inger ' s so lut ion. Significant

inh ib i t ion was obse rved when the concentration of t h e s e drugs was

5 mM. At t h i s concentration suramin inh ib i t ed the mi tochondr ia l

enzymes (SDH and FRD) by 75% and 79% and the cy tosol ic enzymes

(PEPCK, PK and LDH) by 44%, 35% and 41% r e s p e c t i v e l y (Table

5 ) . Centperazine (5 mM) caused 62%, 63% inhib i t ion of SDH and

FRD and 51% and 34% inhib i t ion of MDH and malic enzyme (Table

6 ) . Levamisole at 5 mM concentration inh ib i t ed SDH and FRD by

65% and 33% whi le the a c t i v i t y of PK and MDH were lowered by

49% and 30% r e s p e c t i v e l y (Table 7 ) .

Isolation and propert ies of phosphoenol pyruvate carboxykinase

Phosphoenol py ruva te carboxykinase (PEPCK) from S. c e rv i

adul t females was ve ry unstable in nat ive s ta te and the enzyme

was inac t iva ted within 24 hours when kept at 4-5 C. A s tudy

of the effect of a few th io l compounds (y^-mercaptoe thanol , ME

and d i t h i o t h r i t o l , DTT) on the ac t i v i t y of PEPCK ind ica te t h a t

40

Table 5: Effect of ui v i t r o t reatment of l ive Setaria ce rv i with suramin on the a c t i v i t y of the enzymes of PEP-succinate pa thway .

Enzyme Control (/umol/ml)

Exper imenta l (/umol/ml)

Inhibi t ion ( I )

Pyruvate kinase

Phosphoenol py ruva te ca rboxykinase

Lactate dehydrogenase

Malate dehydrogenase

Malic enzyme

0.279±0.010

0.939±0.070

2.450±0.001

31.800±0.001

0.032±0.000

0.182±0.013

0.626+0.040

1.460±0.110

23.30+1.690

0.023±0.001

35

44

41

27

29

Fumarase

Fumarate reduc tase

Succinate dehydrogenase

0.614±0.000

0.062+0.000

0.174±0.001

1,780±0.084

0.016±0.003

0.036+0.009

189(act i -vation)

75

79

Adult motile S .ce rv i were incubated with suramin (5 mM) in R inge r ' s solution at 37 C for 3 h r . A 10% ( w / v ) homogenate of the worms was made in 150 mM KCl. The a c t i v i t i e s of the cytosol ic enzymes were efjtimated in post mi tochondr ia l f rac t ion , whereas the a c t i ­v i t i e s of fumarate reduc tase and succinate dehydrogenase were determined in the mi tochondr ia l f rac t ion , A control batch was also incubated along with e x p e r i m e n t a l .

41

Table 6: Effect of in v i t r o t rea tment of l ive Setaria cerv i with centperazine on the a c t i v i t y of the enzymes of PEP-succinate pathway enzymes .

Enzyme

Pyruvate kinase

Phosphoenol pyruva te carboxykinase

Lactate dehydrogenase

Malate dehydrogenase

Malic enzyme

Fumarase

Fumarate reductase

Succinate dehydrogenase

Adult motile S.

Control (>umol/ml)

0.616±0.05

1.123±0.10

2.670±0.24

31.50±0.35

• 0.027±0.01

0 .76U0.07

0.122+0.02

0.269±0.03

ce rv i were n

Exper imenta l Oumol/ml)

0.481±0.09

0.984+0.09

2.21010.41

15.91±2.05

O.OlSiO.OO

0.49210.04

0.045+0.01

0.10110.06

incubated wi th centperaz ine

Inhibit ion (%)

22

13

17

51

34

35

63

62

(5 mM) in Ringer ' s solution at 37 C for 3 h r . A 10% (w/v ) homogenate of the worms who made in 150 mM KCl. The a c t i v i t i e s of the c y t o -solic enzymes were estimated in pos t mi tochondr ia l f rac t ion , whereas the a c t i v i t i e s of fumarate r educ ta se and succinate dehydrogenase were determined in the mi tochondr ia l f rac t ion . A control pa tch was also incubated along with the e x p e r i m e n t a l .

42

Table 7: Effect of iii v i t r o t reatment of l ive Setar ia ce rv i with levamisole on the a c t i v i t y of the enzymes of PEP-succinate pathway enzymes

Enzyme

Pyruvate k inase

Phosphoenol py ruva te carboxy kinase

Malate dehydrogenase

Lactate dehydrogenase

Malic enzyme

Fumarase

Fumarate reduc tase

Succinate dehydrogenase

Adult motile S,

Control Oumol/ml)

0.56±0.06

1.24±0.25

24.50±0.10

1.80±0.10

0.03±0.01

1.63±0.12

0.03±0.00

0.15±0.04

,cerv i were O

Experimental Inhibi t ion (/umol/ml) (%)

0.29±0.03

1.21+0.22

17.21±0.18

1.61±0.02

0.03+0.01

1.28±0.51

0.0210.00

0.05±0.00

incubated with levamisole (5

49

4

30

11

17

22

33

65

mM) in R inge r ' s solution at 37 C for 3 h r . A 10% (w/v ) homogenate of the worms was made in 150 mM KCl. The a c t i v i t i e s of the c y t o -sol ic enzymes were es t imated in pos t mi tochondr ia l f rac t ion , whereas the a c t i v i t i e s of fumarate reduc tase and succinate dehydrogenase were determined in t h e mi tochondr ia l f rac t ion. A control batch was also incubated along with expe r imen ta l .

43

both DTT and /3-UE a c t i va t ed the enzyme suggesting t h e r e b y the

involvement of -SH grcSups in the ca ta ly t i c reaction (Table 8 ) . Add i ­

tion of p ro tease i n h i b i t o r (phenyl methyl sulphonyl f luor ide , PMSF)

showed s t ab i l i z ing effect on the enzyme during s to rage .

PEPCK from adul t female S .cerv i was p a r t i a l l y pur i f i ed

using a combined p rocedure of ammonium sulphate f rac t ionat ion,

desal t ing through Sephadex G-25 column and affinity chromatography

on Blue Sepharose CL-6B, d e t a i l s of which have been d e s c r i b e d

under Mater ia ls and Methods sec t ion . The post mi tochondr ia l s u p e r ­

natant (12,000xg) p r e p a r e d by homogenising 7 g motile worms in

suspension buffer containing Tr i s HCl (50 mM), DTT (1.5 mM) and

PMSF(0.1 mM) was subjec ted to (NH.)_SO. f ract ionat ion. The p ro t e in s

p rec ip i t a t i ng between 30-70% saturat ion of (NH.)_SO. contained most

of the or ig ina l PEPCK a c t i v i t y . The p r e c i p i t a t e was so lub i l i s ed

in small volume of suspension buffer and passed through a f r e sh ly

p r e p a r e d Sephadex G-25 column (14x1.4 cm) for d e s a l t i n g . The

fract ions r i c h in PEPCK were pooled and app l i ed over a f r e sh ly

p r e p a r e d Blue Sepharose column. The p ro te ins adso rbed on t h e

affinity column were e lu ted with increasing concentrat ions of NaCl

(0 .5 -2 .0 M) in a s t epwise manner. The summary of the pur i f ica t ion

protocol i s shown in Table 9. Over 20 fold pur i f ica t ion of the

enzyme was a c h i e v e d .

Properties of the enzyme: F ig . 8 shows the pH a c t i v i t y curve

of PEPCK using 2 buffer sys tems v i z . , sodium potassium phospha t e

and T r i s HCl. The enzyme showed maximal a c t i v i t y at pH 6 . 5 .

44

Table 8: Effect of phenylmethyl sulphonyl f luo r ide , d i t h i o t h r i t o l and beta-mercaptoethanol on phosphoenol py ruva t e c a r b o x y -kinase ac t i v i t y of Setaria c e r v i .

-

Additions

Control

Phenylmethyl sulphonyl f luor ide

Control

Di th io th r i to l

Control

Beta-mercaptoetha

Concentration (mM)

-

0.1

0.5

1.0

2.0

-

0.5

1.0

1.5

2.0

3.0

5.0

-

nol 0.5

10.0

Act ivi ty

0.279

0.258

0.219

0.206

- 0.183

0.279

0.361

0.578

0.762

0.385

0.376

0.370

0.472

0.665

0.559

% Act ivi ty remaining

100

92

78

72

66

100

129

207

273

137

134

132

100

141

118

The enzyme was incubated with different concentra t ions of p h e n y l ­methyl sulphonyl f luo r ide , d i t h i o t h r i t o l and be ta -mercap toe thanol for 5 min each and then the enzyme a c t i v i t y was a s sayed by s t an ­da rd assay sys t em.

- fH

" >-^ +^

0) Tj

at) tn nj

> U

> ^^ O ° ° o - (U Di

C

o +->

O

<o <o

• .—1

1—1

o^ o

• o

o o 1—1

i n (Nl

• (NJ

un o tNl

• o

00 1—1 r—(

CO

'—' " i n

i n vO '^f

• o

i n

I — (

o o •

(NJ

a r-• 1—i

oo

•>-' 2 o T3

>^: • f H

> • H + J O

<

> U <U > o o (D oi

_E (n

-i-> • H C D '

o\o

^"^

c •H

-(-> O O U

H a

c •r^

-i->

o u P-.

1—1

<i + J

o H

1—1

G " • " ^

bO E

o E ^ 3 ^ -5 E > "^

c o

• H

a •H !H O W <U

Q

CO

(N!

1—1

o i n

0 0

0 0

(NJ

CM CO

OO

ro

^ o •vD

0 0 r-•

CO

o ^ * 1-H

CO CD

o

00

o

rt Vi

+ j

X <u

<u <u ^ m

o 0) o u 00 ^

be

o o o

*•

(M 1—1

1 o -t-" r-t

V <« E -H

^

o o a j :

v ^ O

z * ^

c o

•H

o rt ?H

m

c o

o U

o ^

• ^ I

Z CO

tn O u

r-H , _ | CQ 0)

CO

E

H H D

he c

•H C

•H rt c o u .

c ^ o i n -H

vO u -H

P- u 3

. P-

E o

i n Ti w 0)

tn

m rt

•- 5 ^ ^

C " In .,-1 (U

a D

•5 <u +^ 0) 3

> I E

IS D

u 5-1

OJ P-

tn

tsi • iH

0) bC O E o

J2

u <L)

tn E

o •'^ •H X

< T3 2 1—1

O MH O O

u o •H E , D 5-1

0)

o-B >

o S E

E ^

c 3

<U

E N

c

45

46

0.4

0.3 A / '

• 0.2 J

0.1

-^

(pH)

Fig. 8: Effect of pH on the activi ty of phosphoenol

pyruvate carboxykinase of S.cervi .

47

Since GDP is necessary for the enzyme, i t s effect on PEPCK has

been s tud i ed . Maximum a c t i v i t y was obse rved when 0.5 mM GDP

was added to the react ion mixture (F ig . 9 ) . K value determined m

by studying the r a t e s of the PEPCK ca ta lysed reaction at different

concentration of subs t r a t e (PEP) was found to be 2.0 mM (F ig .

10). The enzyme e x h i b i t e d h y p e r b o l i c sa tura t ion curve with r e spec t

to s u b s t r a t e .

Since PEPCK from seve ra l sources has been shown to r e q u i r e

Mn and HCO, for i t s a c t i v i t y , exper iments were conducted using

different concentrations of anions and ca t ions . The enzyme from

S .cerv i r equ i r e s 10 mM MnCl-, and 50 mM NaHCO, for showing optimal

a c t i v i t y (F ig . 11a,b) MgCl^ showed i n h i b i t o r y effect as 28% ac t i v i t y

was lost when 2 mM MgCl_ was added to t h e react ion mixture (Fig .12)

The effect of FDP and ATP on PEPCK of S . ce rv i was also s t u d i e d .

ATP showed i n h i b i t o r y effect lowering the a c t i v i t y by 69% at 4

mM concentration (F ig . 13a), FDP, howeve r , showed ac t iva t ing effect

when the enzyme was incubated for 15 min in presence of the buffer

(F ig .13b) .

48

0.4 H

o e

•H >

• H +-> O

<

0.2

F i g . 9:

0.5 1.0 1.5

GDP (mM)

Effect of GDP on the ac t i v i t y of p h o s p h o -

enol py ruva te ca rboxykinase of S . c e r v i .

49

L. vO

w 0

h •*

o c (L) O

X a VI o x; a

MH O

• ( - ' • H

> • H 4-> U

I )

c o

(U 4 - ' n) Js

4-1 U)

X I 3 [ f i

4- (

o

c o

• H +->

(5) o c o u

M

c • r - (

<t) 0)

u c

•H H-l

o

u

H H

W

.—1

X I 3 o

TJ

X u 3

0)

> (D ^ <U c

x; >->

o x: tn

+ j (D m n

1—1

> ! H

<D CJ

c/5

O

(D V)

a c

•rH 4«i > X O

X ^ 1

u

0)

> 3 U

>-a

• 0) 3

1—1

n]

> E

fc-i

0) -C 4 J

• H

<ti • — (

3 O

1—1

(D O

U O

"+H

o 1—1

r—1 (TJ o o a

•H

o 0) ^ 1

•H

-[Ui/xouirv A;TA :: ^V

0.8 1 50

o E ? >

+-> • i - t

> •r-i +->

u <

0.6

0.4 -

0.2

10 20

MnCl^ (mM)

F i g . 11a: Effect of Mn on the ac t i v i t y

of phosphoenol py ruva te c a r b o x y -

kinase of S . ce rv i -

o e

NaHCO^(mM)

F i g . llb^: Effect of HCO: on the . , .^i. uix^ enzyme

phosphoenol py ruva t e carboxykinase

of S . c e r v i .

51

0.3 -

o E

0.2 -

OA -

T 4

2 +

10

Mg (mM)

F i g . 12: Effect of Mg''' on the ac t i v i t y of Tshospho-

enol p y r u v a t e ca rboxykinase of S . c e r v i .

0.2 T 52

o E ?

>^ +->

> •H +->

u

0.1 -

"T 4 1 2 3

ATP (mM)

Fig. 13a: Effect of ATP on part ial ly purified

phosphoenol pyruvate carboxykinase

of S.cervi .

< 0.4 -

0.2 _

— 1 ~ 0.2

— r 0.4

T — r 0.8

Fig. 13b:

0.6

FDP (mM)

Effect of FDP on part ial ly purified

phosphoenol pyruvate carboxykinase

of S.cervi .

53

DI SOBS ion

The in te rmedia ry metabolism of p a r a s i t i c nematodes has

r ece ived wide attention during the las t two decades and significant

cont r ibut ions have been made in dec ipher ing different metabolic

pa thways opera t ing in f i l a r i a l and o the r in tes t ina l he lmin ths as

well a s s tudying the p r o p e r t i e s of the regula tory and terminal

enzymes of c a rbohyd ra t e metabolism. These s tud ies enable us

in improving our understanding of the biology of t he se organ isms .

S . ce rv i a p p e a r s to be equipped with a l l t he enzymes of

PEP-succinate p a t h w a y . The a c t i v i t y of some of the key enzymes

in S . ce rv i a p p e a r s to be h ighe r than the corresponding values

r e p o r t e d for o the r f i l a r i i d s ( L . c a r i n i i , S r ivas tava et_ al^., 1970;

C .hawking i , S r ivas tava and Ghatak, 1971) t rematodes (Fasciola

h e p a t i c a , P r i c h a r d and Schofield, 1968) and nematodes (Haemonchus

contortus l a r v a e , Ward and Schofield, 1967; Ascar i id ia g a l l i , S r i v a s t a v a

et a l . , 1970). Invest igat ion on g lycoly t ic and carbon d iox ide fixing

enzymes have shown tha t p a r a s i t e s producing succinic and vo la t i l e

fa t ty ac ids in preference to lac t ic acid as end produc t of c a r b o ­

h y d r a t e u t i l iza t ion have a modified scheme of g lyco lys i s d iverging

at t h e PEP l e v e l . These p a r a s i t e s usual ly pos ses s high a c t i v i t e s

of PEPCK and MDH and low l eve l s of PK and LDH, and the g lyco ­

l y t i c pa thway p roceeds only up to the formation of PEP which

i s then ca rboxy la t ed to oxaloacetate by an ac t ive PEP-carboxykinase

(von Brand, 1973). The worms coming under t h i s group a re Ascaris

lumbr ico ides (Bueding and Saz, 1968). H. contortus l a r v a e (Ward

et a l . , 1968a ,b) , Hymenolepis diminuta (Bueding and Saz, 1968),

54

F.hepatica (Prichard and Schofield, 1968b) and T.spiral is larvae

(Agosin and Aravena, 1959) which convert negligible amount of sugars

to lactic acid. On the other hand, typical lactic acid producers

like S.mansoni {Buoding and Saz, 1968) and C.hawkingi (Srivastava

et a l . , 1968; Srivastava and Ghatak, 1971) converting 80-90% glucose

into lactic acid resemble vertebrate tissue in possessing high levels

of PK and LDH and low levels of PEPCK and MDH. The intermediate

case may also occur as in Dictyocaulus viviparous, a worm depending

essentially on aerobic life where the activit ies of PK and PEPCK

are more or less of the same order (von Brand, 1973).

S.cervi aerobically converts about 28% glucose into lactic

acid (Anwar ei_ aL., 1975) and possess a PK/PEPCK ratio of around

0.4. Hence it appears that both glycolytic and PEP-succinate path­

ways are operating in the bovine filarial parasite and thus it differs

metabolically from other filarial worms (L.carinii and C.hawkingi)

and resemble more closely the intestinal parasite specially with

regard to the PEP metabolising enzymes. The difference in the

metabolic activity can be explained on the basis of the location

of the parasite in the host . S.cervi resides on the peritoneal

folds of the intestine having relat ively high oxygen tension while

C.hawkingi and L.carinii thr ive in heart and pleural cavity of

crow and cotton rat respectively under a rich supply of oxygen.

Studies on the subcellular localization of the enzymes of

PEP-succinate pathway in S.cervi showed that PK, PEPCK, MDH,

55

LDH, fumarase and malic enzyme were present in the soluble fract­

ion of the homogenate. This is similar to the cytosolic localization

of these enzymes observed in vertebrate tissues (Lehninger, 1984).

As in vertebrate tissues and other intestinal parasites SDH and

FRD were localized in mitochondrial pellet of S.cervi .

^" S.cervi the level of PEPCK is more than that of PK,

which is in contrast to the observation made in S.mansoni (Brazier,

1973) and a few other nematode paras i tes . This is possibly due

to the different habitat of the parasi tes as S.cervi is found in

a high oxygen tension. According to Prichard (1976) in F.hepatica,

the observed NADH stimulation of PEPCK under conditions of lower

oxygen avai labi l i ty , higher concentration of NADH and PEP due

to a stimulation of PFK and overall rate of glycolysis may facilitate

PEPCK activity to increase the supply of fumarate for regeneration

of NAD and to maintain pyruvate production via MDH and MDH (decar-

boxylating ) should the PK activity be insufficient due to a lower

concentration of FDP. Thus, the net effect of metabolic control

on the act ivi t ies of PK and PEPCK would be to allow the enzymes

to complement each other for maintaining a steady supply of pyru­

vate in accordance with the demands for NADH to generate energy

while allowing the parasite to adjust the flow of carbon to fumarate

in accordance with supply of NADH and oxygen.

Although the detailed mechanism for controlling fermentation

in different helminths has not been resolved, it is clear from

56

the s tudies made tha t PK and PEPCK play a key role in de te rmin­

ing the fermentation p roduc t s produced by different helminths and

pe rhaps in determining whe the r the products can change in response

to a l tera t ion in environmental demands on the organism.

In most of the helminth p a r a s i t e s PEPCK se rves in the

carboxylat ion of PEP leading to t h e syn thes i s of OAA and thus

regulates g l y c o l y s i s . The enzyme in mammalian t i s sues b r ings

about the syn thes i s of PEP from OAA thus playing an important

role in the regulation of gluconeogenesis .

PEPCK pur i f i ed from S . ce rv i has a r ecovery g rea te r than

100% in post ' mi tochondria l and ammonium sulphate (30-70% sa tura t ion)

fraction (Table 9 ) . Presence of an inh ib i to r has e a r l i e r been

repor ted by Tar tora et_ a l . (1985) in DEAE sephace l fraction from

yeast PEPCK, which was ident i f ied as adenyla te k i n a s e . The enzyme

s tudied in S .ce rv i i s cy toso l ic resembling the analogous enzyme

isola ted from A. lumbr ico ides muscle (van den Bossche , 1969). In

general the p r o p e r t i e s of PEPCK from bovine f i l a r i a l worm d id

not differ s ignif icant ly from t h e analogous enzyme obtained from

some helminths and v e r t e b r a t e t i s s u e s . The enzyme from S .ce rv i

showed maximal a c t i v i t y around pH 6.5 while PEPCK from F .hepa t i ca

(Behm and Bryant , 1982) and H.diminuta (Prescot t and Campbel l ,

1965) were opt imal ly ac t i ve between 6 . 0 - 6 . 5 . PEPCK from S .ce rv i

has speci f ic requirement for Mn which could not be r ep laced

t>y Mg showing s i m i l a r i t y with the enzyme from chicken l i v e r

57

(Kurahashi et_ al_., 1957), E.granulosus (Agosin and Repet to , 1965)

and F . h e p a t i c a (Behm and Bryant , 1982). Among the anions , HCO_

st imulated the carboxyla t ion of PEP in S .ce rv i while PEPCK from

pig l i v e r was i nh ib i t ed by HCO- (Chang et_ a l . , 1966). Among

the s eve ra l nucleot ides inves t iga ted GDP was most effect ive cofactor

for S . ce rv i PEPCK, followed by IDP while negl igible a c t i v i t y was

obse rved with ADP. Similar pa t t e rn was obse rved in M.expansa

(Behm and Bryant , 1982). However, IDP was more ac t ive for the

enzyme from M.s imi l i s (McManus and James , 1975), A.suum muscle

(van den Bossche , 1969), F . h e p a t i c a ( P r i c h a r d and Schof ie ld , 1968)

and H.diminuta (Presco t t and Campbel l , 1965).

PEPCK from S .cerv i d i s p l a y e d t y p i c a l h y p e r b o l i c k ine t i c s

with r ega rd to PEP and nucleot ides (GDP) resembling the enzyme

from many o the r sou rce s . ATP e x e r t s i nh ib i t o ry effect on PEPCK

of S . ce rv i s imi la r to the obse rva t ions made in M.expansa (Behm

and Bryant , 1982). However, t he enzyme from A.suum muscle (van

den Bossche , 1969) and o the r sources were unaffected by ATP.

Since ATP also i n h i b i t s PK in S .ce rv i (Anwar, N. P h . D . t h e s i s ,

1976) i t i s un l ike ly tha t t h i s nucleot ide i s a major regula tor of

PEP/OAA bronchpoint in the forward d i rec t ion to succ ina te . Stimu­

lation of PEPCK a c t i v i t y in S . ce rv i by th io l compounds suggest

the p resence of s u l p h y d r y l (-SH) groups at t h e ac t ive s i t e of the

enzyme molecule . Wilkes et_ a l . (1981) also r epo r t ed t h e presence

of --SH groups in the enzyme of A.suum.

58

The jji vitro effect of a few filaricides on the enzymes

of PEP-succinate pathway was studied for identifying sensitive

molecular targets to provide a more rational approach for the chemo­

therapy of f i lar ias is . Among the different filaricides tested,suramin

was most effective, inhibiting PK and fumarase activity of S.cervi

at 1 /uM concentration, while centperazine and DEC exerted inhibitory

effect on these enzymes at ten times higher concentration. Maximum

inhibition of PEPCK was achieved with CDRI compound 72/70 which

lowered the enzyme activity by 41% at 1 ^M concentration, while

suramin was effective at higher concentration (100 pm). Levamisole,

DEC and centperazine, however, could inhibit the enzyme at 1 mM

concentration. LDH was inhibited by suramin (10 jaM), centperazine,

levamisole, compound 72/70 and DEC at 100 /uM concentration; while

MDH was inhibited by suramin (10 /uM), and levamisole and cent­

perazine (100 /uM). Malic enzyme was inhibited by 100 uM suramin

and 1 mM centperazine respect ively. Among the mitochondrial enzymes,

SDH was inhibited by suramin, centperazine, 72/70 and levamisole

at 100 /uM concentration, while 1 mM centperazine was needed for

exhibiting inhibitory effect on FRD .

A study of the effect of these drugs on live worms during

in vitro incubation indicated that suramin, centperazine and leva­

misole could exhibit inhibitory effect on most of the enzymes of

PEP-succinate pathway only when motile worms were incubated at

37 C for 3 hours at 5 mM concentration. It is quite possible that

the cuticle of the worm is serving as a bar r ie r for the transport

59

of these drugs and hence high concentrations are needed for showing

inhibitory effect on the enzymes of living worms.

The biochemical mode of action of suramin (hexasodium

3,3 ' ureylene-bis-8(3 benzamido-p-toluido)-l ,3 ,5-naphthalene sulpho-

nate) against the filarial worms, hookworms and ascaris has been

the subject of research in this laboratory as well as elsewhere.

Studies conducted so far have indicated that suramin inhibits LDH

from D.immitis at 6 /UM (Walter, 1979); LDH and MDH from 0.volvulus

at 5 yuM (Walter and Shulzkey, 1980), protein kinase I from

0.volvulus at I jaU (Walter and Shulzkey, 1980), phosvitin phosphory-

lating protein kinase from B.malayi, A.viteae and S.cervi at 30

uM (Saxena et_ a3 . , 1984), folate dehydrogenase from D.immitis

and B.pahangi at 5 ^M (Jaffe, 1980); malic enzyme from 0.volvulus

at 1"0 nM (Walter, 1982); phosphofructokinase from S.cervi at 5

nM (Sharma et a l . , 1987); N-acetyl-^-D-glucosamindase from S.cervi

at 30 /UM (Singh et_ al_., 1986) and phosphatidylglycerolphosphate

synthetase, the key enzyme in the biosynthesis of phosphatidyl

glycerol, from 0.volvulus (Srivastava et a l . , 1987). However,suramin

had no inhibitory effect on AChE from S.cervi even at 1 mM con­

centration (Sharma, 1988 Ph.D. t h e s i s ) . All these studies were

conducted with adult stage of these paras i tes .

For onchocerciasis suramin remains the only acceptable

microfilaricidal drug. It also acts on adult worms of L.carinii

(Lammler e^ a l . , 1971) and W.bancrofti (Hawking, 1978). After

60

intravenous injection it combines with serum proteins (histones,

serum globulins , albumin and casein) and persists for long periods

in the blood stream.

Diethylcarbamazine, the currently used filaricide in t r ea t ­

ment of fi lariasis does not exhibit significant effect on the enzymes

of PEP-succinate pathway from adult S.cervi . It is a microfilari-

cidal agent active against W.bancrofti and B.malayi (Peters, 1979)

as well as 0.volvulus (Hawking, 1978). Sufficient evidence exists

to show that piperazine induces revers ible paralysis by acting

on neuromuscular system (van den Bossche, 1976). Electron micros­

copic studies on the mf of A.viteae and L.carinii isolated from

infected hosts indicted cellular damage on treatment with DEC which

could be the direct effect of the drug or secondary manifestation

of its action (Melhorn et_ a^. , 1981).

DEC along with other drugs (centperazine, levamisole and

CDRI compound 72/70) significantly inhibit glucose utilization, syn­

thesis of glycogen, protein synthesizing capacity and release of

mf from adult S.cervi females (Anwar et a l . , 1978; Srivastava,

1980).

Comley et_ al_. (1981) observed significant inhibition of

SDH and FRD activi t ies from A.tetraptera and A.suum during in

vitro incubation with 5 mM levamisole, which is in agreement with

our findings showing that 5 mM levamisole was needed for exhibi t ­

ing inhibitory effect on SDH and FRD of S.cervi .

61

Thus success has been achieved in purifying and character is­

ing PEPCK from S.cervi, and studying the effect of a few filaricides

on the enzymes of PEP-succinat^ pathway. Strong inhibition _of

LDH and MDH by low concentration of suramin would interfere with

the reoxidation of NADH generated by glyceraldehyde 3-phosphate

dehydrogenase and lead to the eventual arrest of glycolysis of

the parasi te . Further, attempts would now be made for studying

the kinetic properties of the nea^ homogenous preparation of PEPCK

(free of dehydrogenases) in relation to the effect to act ivators ,

inhibitors and anthelmintic agents.

CHAPTER IV

SUMMARY AND CONCLUSION

62

Filariasis is a major public health problem affecting millions

of people in the tropical and subtropical regions of the world.

Though the disease is not fatal. Taut it is responsible for consider­

able disabi l i ty and social stigma due to elephantiasis caused by

immunopathological reactions of parasite with the host . Effective

chemical or immunological remedies against filariasis are s t i l l lacking

and this is mainly because of poor understanding about the parasite

biochemistry. It i s , therefore, necessary to have a sound knowledge

of the morphology, physiology, sepcially of the biochemistry of

these parasi tes for developing drugs specifically inhibiting the

metabolic processes of the parasi te .

The filarial parasites derive energy for their survival*

mainly through carbohydrate utilization, however, the processes

involved in carbohydrate degradation are not identical with those

established in vertebrate tissues as none of these parasites possesses

the abil i ty of oxidising the carbohydrate completely to COp and

H_0. PEP-succinate pathway has been shown to have a crucial

role in the channeling of carbon as well as in the regeneration

of energy in many helminth paras i tes . Therefore, a study of PEP-

succinate pathway enzymes with special reference to the action

of antifilarials may provide useful leads for identifying paras i te-

specific enzyme targets for chemotherapy. In view of the non­

availabil i ty of human filarial parasites (W. bancrof t i / B. malayi) in

sufficient amounts, Setaria cervi , a bovine filarial paras i te , resembl-

63

ing the human filarial worm in microfilarial periodicity and antigenic

pattern has been used in this investigation.

The present stu^y shows the subcellular localization of

the enzymes of PEP-succinate pathway. The enzymes-pyruvate

kinase, phosphoenol pyruvate carboxykinase, lactate dehydrogenase,

malate dehydrogenase, malic enzyme and fumarase were localized

in the cytosol whereas succinate dehydrogenase and fumarate reduct­

ase were found to be particulate bound. Malate dehydrogenase

was found to be most active among the soluble enzymes.

Action of antifilarials on the enzymes of PEP-succinate

pathway wa"! also studied by incubating the enzyme extract or

live S.cervi with different concentrations of ant i f i lar ia ls . Among

the various compounds tested suramin at low concentration could

cause a marked inhibition of the activit ies of most of the enzymes.

Due to the large size of suramin (hexasodiumS ,3 ' -ureylene-bis -8(3-

benzamido-p-toluido)-l ,3,5-naphthalene sulphonate), it is likely

that an extensive biochemical interaction occurs between different

enzymes and the drug. Centperazine also showed significant inhi­

bitory action on pyruvate kinase, lactate dehydrogenase fumarase

and succinate dehydrogenase. Incubation of live worms with th is

drug caused 62%, 63% and 51% inhibition of succinate dehydrogenase,

fumarate reductase and malate dehydrogenase respect ive ly . Diethyl-

carbamazine and levamisole were found to be more or less ineffective

at lower concentration. However, they could inhibit some of these

64

enzymes when used at h i g h e r concentra t ion. Compound 72/70 e x h i ­

b i t ed significant i nh ib i to ry effect only on phosphoenol pyruva te

ca rboxyk inase .

The most important regula tory branchpoint enzymes of PEP-

succinate pathway in S .ce rv i i s phosphoenol py ruva t e carboxykinase

which has been p a r t i a l l y pur i f ied and i t s p r o p e r t i e s were s tud i ed .

The p a r t i a l l y pur i f ied enzyme was v e r y unstable in nature but

i t could be s t ab i l i zed by the addi t ion of 0.1 mM phenylmethyl

sulphonyl f luoride (a p ro tease i n h i b i t o r ) and 1.5 mM d i t h i o t h r i t o l

( th io l compound) when added in the ex t rac t ion buffer during homo-

genisa t ion. These also prevented the inact ivat ion of the enzyme

during purif icat ion p r o c e d u r e .

Purif icat ion was done by a t h r e e s t ep p rocedure employing

(NH.)-SO^ fract ionat ion, gel f i l t ra t ion on Sephadex G-25 and Blue

Sepharose affinity chromatography which r e su l t ed in 20 fold p u r i f i ­

cat ion. The enzyme was found to be opt imal ly ac t ive at pH 6.5

and had a K value at 2.0 niM for s u b s t r a t e ( P E P ) . MnCl^ (10 m L

mM)and NaHCO-(50 mM) a re r e q u i r e d for maximal enzyme a c t i v i t y ,

2+ whi le Mg ions (2 mM) exe r t ed i n h i b i t o r y effect on the enzyme.

FDP was found to ac t iva te while ATP i n h i b i t e d the enzyme.

These s tud ies indicate the s e n s i t i v i t y of some enzymes

of PEP-succinate pathway in f i l a r i a l p a r a s i t e s to known drugs and

some candidate drugs being deve loped at t he i n s t i t u t e . They may.

65

therefore, serve as a potential metabolic targets for antifilarial

drug design. Further attempts would be made for purifying the

enzyme (PEP-carboxykinase) to homogeneity for studying the kinetic

properties in relation to act ivators, inhibitors and anthelmintic

agents. Oxidative activit ies in mitochondria like particles from

S.cervi would also be examined. The sensitive molecular targets

identified during this study are expected to provide a more rational

approach to filarial chemotherapy.

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