ENERGY METABOLISM OF SCHISTOSOMES by Clement Antonio … · 2016-07-29 · ENERGY METABOLISM OF SCHISTOSOMES by Clement Antonio Earle B.Sc (Hons). A thesis submitted for the degree

ENERGY METABOLISM OF SCHISTOSOMES

by

Clement Antonio Earle B.Sc (Hons).

A thesis submitted for

the degree of Doctor of Philosophy

in'the University of London

and for the Diploma of Imperial College

Department of Pure and Applied Biology,

Imperial College of Science and Technology,London Slii7 2BB September, 1983

ABSTRACT

Studies on carbohydrate catabolism in several species of

adult schistosomes, in particular Schistosoma mansoni and

S.margrebowiei, have been carried out. The basic body constituents i.e. protein, glycogen and lipid, together with wet weight, were measured in these two species. The specific

activities of certain key glycolytic, tricarboxylic (TCA) cycle

and hexosemonophosphate (HMP) shunt enzymes have been measured in S.bovis, S.leiperi, S,haematobium and S,.japonicum to provide an indication of the relative significance of these pathways in

these parasites. A more detailed examination of these pathways

using enzyme activity measurements and determination of steady

state levels of intermediary metabolites has been performed for

5.mansoni and S.marqrebowiei.

The release of end products and changes in endogenous levels

of carbohydrate were measured during incubation experiments involving 5.mansoni, S.marqrebowiei, S.haematobium and

Schistosomatium douthitti to establish which catabolic pathways the parasites utilise. Additional studies included an assessment

of the regulatory role of pyruvate kinase in 5.mansoni, .

S.marqrebowiei, S.japonicum and S.bovis. A study of the nin vivo”

activity of the enzyme from 5.mansoni has been performed using

"physiological”Llevels of reactants, determined from the previously measured metabolite levels. Oxygen uptake measurements and

determination of cytochrome spectra have been carried out in

S,mansoni to establish the role of oxidative phosphorylation inthis species

nc.

The results indicate that in general, schistosomes rely

heavily on glycolysis for energy production and that the TCA and

HMP pathways appear to contribute little in terms of ATP synthesis.

The incubation experiments showed that female schistosomes channel

approximately half of their glucose consumption into lactate produce

tion. The fate of the remaining glucose is unknown but a signifi- ) cant proportion could be accounted for in S.mansoni by oxidative

phosphorylation coupled via a glycerol shuttle mechanism.

Enzyme kinetic studies indicate that pyruvate kinase is

playing a regulatory role in glycolysis in S.mansoni. The male

and female enzymes showed a differential response to fructose-1,

6- bisphosphate (FBP) but were inhibited to the same degree by ATP. Evidence for pyruvate kinase regulation in other schistosome

species is not conclusive.In general, schistosomes seem to have wider metabolic

capabilities for ATP production than previously assumed. The

female parasites in particular are not homolactic fermenters as

they channel half their glucose consumption into non-glycolytic

energy synthesis and possibly anabolic metabolism.

ACKNOWLEDGEMENTS

I would like to express my thanks to Dr. D.P. McManus for

his help and discussion throughout this study. I would also

like to thank Mr. R.J. Knowles of the British Museum of Natural

History for his kind provision of some of the schistosome material.

In addition, I am grateful to Dr. D. Hayes of St Bartholomew^

Biochemistry Department and Miss Sheila Lanham of the London

School of Hygiene and Tropical Medicine for their assistance andoadvice regarding the cytochrome and electrophretic analyses.

I am indebted to Mr. D. Featherston, Mr. R.J. Cripps and

Mrs. J Crombie for their constant friendship and enthusiastic support. Also, I would like to thank the Science and Engineering

Research Council, Miss S.E. Revington and my mum and dad for their

financial assistance and encouragement throughout this project. Finally, I am extremely grateful to Mrs. J. True for her patience,

skill and generosity in typing this manuscript.

CONTENTS

CHAPTER PAGE NO.

Abstract 1

Acknowledgements 3

1. Introduction 1 o

1.1 General Introduction 10

1.2 Energy metabolism in adult schistosomes 12

1.2.1. Basic considerations 12

1.2.2. Aims 18

2. Materials and methods 20

2.1. Maintenance 20

2.1.1. Snails 20

2.1.2. Mice 202.1.3 Infections 202.1.3.1. Egg collection, hatching and collection

of miracidia 20

2.1.3.2. Snail infections 21

2.1.3.3. Collection of cercariae 21

2.1.3.4. Mouse infections 222.1.4. Recovery of adult parasites 23

2.2. Chemicals 24

2.3. Carbohydrate and wet weight determinations 242.4. Protein determinations 252.5. Lipids 26

PAGE NO

2.6* Enzyme assays 272.7. Metabolites 382.7.1. Spectrophotometry 382.7.1.1. Preparation of material 382.7.1.2. Assays 392.7.2. Fluorimetry 402.7.2.1. Preparation of material 402.7.2.2. Assays 412.8. Incubations 432.8.1. End product analysis 452.8.1.1. Medium 452.8.1.2. Endogenous metabolite levels 492.8.1.3. ^ C 02 measurement 502.8.2. Glucose incorporation 512.9. Pyruvate kinase kinetics 522.9.1. Physiological enzyme activity 532.9.1.1. Male enzyme 532.9.1.2. Female enzyme 532.9.2. Effects of M(f+/Mrt'+ on enzyme activity

and enzyme stability 542.9.3. Host enzyme activity in parasite gut

contents 542.9.4. Mouse erythrocyte and serum pyruvate

kinase 55

PAGE NO

*

*

*

*

♦

*

2.10. Electrophoresis 56

2.10.1. PAGE 562.10.2. Starch gel 57

2.10.3. Cellulose-acetate 57

2.10. A. Isoelectric focusing 56

2.11. Cytochromes 61

2.12. Oxygen uptake 63

3. Results 64

3.1. Maintenance and collection of parasites 64

3.1.1. Molluscan hosts 643.1.2. Infections 64

3.1.2.1. Recovery of adult parasites 653.2. Statistical analyses 653.3. Carbohydrate determination 57

3.3.1. Glucose levels 673.3.2. Glycogen levels 68

3.4. liJet weight determinations 71

3.5. Protein determinations 71

3.6. Protein : Wet weight ratios 72

3.7. Total lipids 78

3.8. Enzyme analysis 783.8.1. S.mansoni and S.marqrebowiei 783.8.1.1. Glycolysis 78

PAGE NO

3.8.1.2. TCA cycle 79

3.8.1.3. HMP shunt 79

3.8.2. Enzyme activities in other species 79

3.9. Metabolites 87

3.9.1. Analysis of metabolites of mouse liver 87

3.9.2. Analysis of standard metabolite solutions 873.9.3. Analysis of parasite-..metabolite levels 90

3.9.3.1. S.mansoni 90

3.9.3.2. S.marqrebouiei 903.9.4. Mass action ratios 94

3.10. Incubations 94

3.10.1. S.mansoni 943.10.1.1. Glucose uptake and lactate production 94

3.10.1.2. Metabolite levels 95143.10.1.3. CO2 production 95

3.10.1.4. Glucose incorporation 99

3.10.2. S.marqrebouiiei 99

3.10.2.1. Glucose uptake and lactate production 99

3.10.2.2. Metabolite levels 102143.10.2.3. CO2 production 102

3.10.3. 5.haematobium 102

3.10.3.1. Glucose uptake and lactate production 1023.10.3.2. Metabolite levels 103

3.10.4. S.douthitti 103

103

3.10.4.2. Metabolite levels 103

3.10.4.3. ^ C 02 production 1063.10.5. Interspecific comparisons 106

3.11. Kinetic study of pyruvate kinase 1073.11.1. S.mansoni 107

3.11.1.1. ATP inhibition 111

3.11.1.2. FBP activation 111

3.11.2. S.marqrebouiiei 112

3.11.2.1. ATP inhibition 112

3.11.2.2. FBP activation 1123.11.3. S..japonicum 115

3.11.4. S.bovis 115

3.11.5. Interspecific comparisons 123

3.11.6. Pyruvate kinase activity under physiological

substrate and modulator concentrations 1233.11.6.1. Enzyme activity in parasite gut contents 1243.11.6.2. Kinetics of PK from male S.mansoni 124

3.11.6.3. Kinetics of PK from female S.mansoni 126

3.11.6.4. Stability and effect of cations on theactivity of PK 150

3.11.6.5. Pyruvate kinase from mouse erythrocyte 1533.12. Electrophoresis 1533.12.1. Isoelectric focusing 157

3 ,1 0 .4 .1 G lucose uptake and la c ta te prod uction

PAGE NO.

3.13. Cytochromes 1373.13.1. Controls 1373.13.1.1. Whole mouse blood and rat liver 1373.13.1.2. Digested mouse blood 1383.13.1.3. Haematin 1383.13.2. S.mansoni 1383.13.2.1. Males 1423.13.2.2. Females 1423.14 Oxygen uptake 1424. DISCUSSION 1484.1. Glucose, glycogen, protein, uiet weight and

lipid levels 1484.2 Enzyme analysis 1514.3. Metabolites 1544.4. Incubations 1564.5. Pyruvate kinase 1654.6. Oxygen uptake and cytochromes 1734.7. Summary 180

References 183»

CHAPTER 1

INTRODUCTION

1.1. GENERAL INTRODUCTION

Schistosomiasis is one of the most prevalent parasitic

diseases in man. Currently, it is second only to malaria in

terms of public health importance in tropical and sub-tropical

regions. The disease is now endemic in seventy-three countries

and recent estimates indicate that nearly 500 million people world wide are at risk of infection (Iarotski & Davis, 1981). In

contrast to malaria, little advance has been made with regard

to the chemotherapy of schistosomiasis since the introduction of antimonials in 1918 (Huang, 1981). Further studies of

schistosome biochemistry are suitable, as comparatively little

detail is known concerning the basic metabolism of these parasites

Moreover, conflicts of opinion exist regarding certain aspects of

the available data. A clearer knowledge of basic schistosome

metabolism may eventually result in wider, more rational methods of drug control.

At present, Schistosoma mansoni is the most widely studied

species, primarily because of its pathogenicity and the relative

ease with which it can be maintained in the laboratory. The

general schistosome life cycle is shown in Fig.T. There are two parasitic and free living stages in the cycle and it has been

*

Figure 1.A

The general life cycle cf schistosomes.*

%

*

<*

ft

Adult host

Adult parasitesEggs retained in tissues

A

Common environment in water

Free swimming Cercariae

Free swimming Miracidium

Intermediate host

demonstrated that the energy metabolism of the different stages

alters in relation to the helminth's environment.

The miracidial and cercarial stages are capable of operating

a fully oxidative metabolism (Bruce et al, 1971; Coles, 1972 b,

1973a). As these forms are free living, oxidative metabolic

processes are a more efficient means of coupling the greater

oxygen content of the external environment to energy synthesis.

Examination of sporocyst (intramolluscan stage) metabolism is

complicated due to the difficulties of separating the parasite from host tissue. Consequently details of sporocyst metabolism

are lacking.On invasion of the definitive (adult) host the cercaria moves

into an environment with a lower oxygen tension. After penetration,

the cercaria becomes a schistosomulum and there is a change from

aerobic metabolism to anaerobic lactate production (Coles, 1972a, 1973b, 1973c; Bruce et al, 1969; Lawson & Wilson, 1980). The

schistosomulum migrates via the tissues and blood system to the

final adult sites which are usually the portal or mesenteric

vessels.

1.2. ENERGY METABOLISM IN ADULT SCHISTOSOMES

1.2.1• Basic considerationsAdult helminths are totally reliant on carbohydrate as a

source of metabolic fuel. They are also generally unable to

catabolise glucose completely to carbon dioxide (C0£) and water, hence they produce a variety of reduced organic end-products

*

♦

♦

♦

*

♦

*

(Barrett, 1981). Schistosomes take up glucose (Fripp, 1967;Uglem & Read, 1975; Cornford & Oldendorf, 1979) and also contain

large quantities of glycogen (Bueding & Koletsky, 1950; Lennox

& Schiller, 1972; Magzoub, 1974) which^depleted during incubation

(Bueding, 1950).In contrast with most other parasitic helminths, adult

schistosomes have been shown to produce only lactate under aerobic and anaerobic conditions (Bueding, 1950; Schiller et al, 1975).

Certain nematodes e.g. Chandlerella hawkinqi and Setaria cervi have a similar metabolism (Barrett, 1981) but no other trematode

appears to exhibit this feature.

The main area of debate regarding energy synthesis in the

adult parasite concerns the extent to which oxidative processes participate in adenosine -5*-triphosphate (ATP) generation.

Bueding and co-workers (1950, 1982) have shown that L-lactate is the major end-product of glucose catabolism in S.mansoni. They also failed to demonstrate a Pasteur effect and so have concluded

that anaerobic glycolysis (Fig.2) is the sole mechanism for

energy production in the parasite. Therefore, schistosomes are

frequently referred to as "homolactic fermenters".

In view of the oxygen tension of blood (49-66 mm Hg, Smyth,

1976), some oxygen would presumably be available to the parasites.

It would be physiologically advantageous for the worms to be able

to harness any available oxygen for more efficient means of energy production. All helminths studied so far consume oxygen

when available (Barrett, 1976a) and S.mansoni is no exception

(Bueding, 1950; Schiller et al, 1975), The exact physiological

role of the oxygen remains to be elucidated, Schillex et al (1975)

oogenesis. Large numbers of mitochondria have been found in

association with the reproductive tissues of female S.mansoni and

5,haematobium (Erasmus, 1973; Burden & Ubelaker, 1981), There

fore, oxidative phosphorylation may be of great significance in the reproductive biology of the parasite. Coles (1972 b, 1973a)

suggested that oxygen is used to generate ATP via an electron transport chain. From incubation experiments, Bueding & Fisher

(1982) maintain that no oxidative processes contribute to energy

formation in schistosomes because of the exact balance obtained

between glucose depletion and lactate production. Additionally,

Bueding & Charms (1952) failed to find sufficient quantities of

cytochromes to account for more than one tenth of the oxygen taken

up by the worms.

More evidence does exist regarding oxidative phosphorylation

in S.japonicum in that Huang (1980) has confirmed the presence of

a succinate - cytochrome C reductase, cytochrome C oxidase,

riboflavin and an ubiquinone - like substance. There have also

been reports of tricarboxylic acid (TCA) cycle (Fig.3) activity in S.mansoni and S.japonicum (Coles, 1973b; Smith & Brown, 1977a;

Huang, 1980). liiaitz (1964) also suggests that schistosomes are capable of operating a hexosemonophosphate (HMP) shunt (Fig.4) and

the first two enzymes of the pathway have been found in S.mansoni

(Smith & Brown, 1970, 1977; Coles, 1973b).

have oxygen is used in the tanning processes during

Figure 2

The glycolytic pathway.

Glucose

ATP AOP

Glycogen

Pho&phoryfaSQ

Glucose-1-P

Phosphogiucomufosa

Glucose-6-P

Glucota-phosphata isomarasa

Fructose-6-P ATP----J

PhotphofructoklnasaAOP

Fructose-1.6-bisP

AidoiaM

Dihydroxyacetone-P Tfiow-*'*orr»rs»«Glyceraldehyde-3-PMAO*--vL„ J G

H* + NAOH 4. — A Glycaraidaftyda-3-P danvdroganaaa

2x1.3-OiphosphoglycerateAOP----S

1 Phoapfioglycarata kmasa ATP A — -A,

2 x 3-Phosphoglycerate

Phosphoglycaromutasa

2 x 2-Phosphoglycerate

FhoiaaaHjO4

Pyruvafa k/naaa

2xPhosphoenolpyruvateaop----JA T P + — A

2 x Pyruvate H* • NAOH--- \J

Lactata dahydroganaiaNAO*

2 x Lactate

*

*

Figure 3.

The tricarboxylic acid cycle.

•f

%

0

*

Pyruvate

Oxaloacetate-----------

NADH + H *Maiatedehydrogenase

r^ — N A D *

Malate

Fumarase

H20

Fumarate

Succinatedehydrogenase

i

r

fpH2

fp

Succinate

i ;Citrate synthase

Citrate

CoAAconitase

Isocitrate

Isocitratedehydrogenase

Oxalosuccinate

c o 2

2-Oxoglutarate

Succinyl-CoA

Figure 4.

The hexosemonophosphate shunt.

GlucoseATP

MAOP NAOPH+H' ADP

Glucose-6-P « 6-Phosphogluconate

Schistosomes ingest large quantities of red blood cells

(Chappell, 1980), From the quantities of haematin visible in the

parasites it is evident that the females ingest more erythrocytesthan the males. Adult erythrocytes are primarily lactate

producers but younger cells are capable of oxidative metabolism

(Harris & Kellermeyer, 1967). Therefore, if erythrocyte enzymes

are active within the parasite gut then assays will include host

enzyme activity. The extent of contamination will depend on the<*l

proportion and development^status of the erythrocyte material in

the schistosome gut. Timms & Bueding (1959) and Coles (1970b)

suggest that erythrocyte enzymes will be denatured in the gut but

Smith & Brown (1970) reported that host enzymes are still func

tional after ingestion. However, this aspect has been largely

neglected by other researchers.

1.2.2. Aims

The present study attempts to evaluate the relative con

tributions made by glycolysis, TCA and HMP pathways to overall

energy metabolism in schistosomes, particularly S.mansoni. This

evaluation is based on the results of in vitro incubation

experiments, intermediary metabolite and specific enzyme analyses.

The study also examines the role of pyruvate kinase in glycolytic

regulation as Brazier & Jaffe (1973) suggested that the enzyme

is not allosteric. Additionally, S.mansoni!s potential foroxidative phosphorylation is investigated

Due to problems of supply and life cycle maintenance, many previous studies have been restricted to S.mansoni, S.haematobium

and S.japonicum. Consequently there is a paucity of comparative

interspecific data relating to these and other species. During

the course of this study, small quantities of schistosomes other than S.mansoni, became available intermittently. Hence it was

possible to extend the range of certain areas of study to include

the following species, S.marqrebowiei, S.japonicum, 5.haematobium,

S.bovis, S.leiperi, S.intercalatum and Schistosomatium douthitti.

Further information relating to schistosomes of primarily veterinary

significance may prove valuable as there have been reports of

S.bovis (Blair, 1966) and 5.marqrebowiei (Walkiers, 1928; Lapierre

& Hein, 1973) infections in man.

Whilst much of the previously published work on schistosome

biochemistry has been concerned with paired worms, the present study is concerned with the separate examination of male and

female metabolism. Also this study attempts to quantify the effect

of schistosome gut contents on specific enzyme analyses.

CHAPTER 2

MATERIALS AND METHODS

2.1. MAINTENANCE

2.1.1. Snails

Uninfected, albino Biomphalaria qlabrata were reared in

dechlorinated tap water, in covered plastic trays (50 x 20 x 4 cm)

at 25°C. Snails were fed with washed lettuce and small quantities

qf commercial fish food (e.g. Tetramin) and the trays were cleaned

once every two weeks. Infected snails were maintained in a similar

fashion except that the snails were isolated in a special bio

hazard area and fed on boiled lettuce only. Bulinus tropicus were maintained in the same manner except that the snails were fed with

pre-soaked and dried sycamore leaves.

2.1.2. Mice

Outbred LACA and To mice of both sexes were used to harbour

S.mansoni and S.marqrebowiei infections respectively. The animals

were maintained under standard animal house conditions.

2.1.3. INFECTIONS

2.1.3.1.Egg collection, hatching and collection of miracidia

Livers and small intestines of mice of greater than six weeks

past-infection were homogenised in approximately 30 ml of 0«9j

(w/v) saline in an MSE blender set at maximum speed for 7 min.The slurry was washed through 500, 300 and 200 mesh sieves with

several volumes of saline. The filtrate was left to stand for

5 min*in a large crystallising dish, after which it was decanted

slowly, leaving the eggs in a small volume of saline. The eggs

were then washed into a smaller glass dish with saline and left to

stand for a further 5 min. Most of the saline was then withdrawn

using a Pasteur pipette and several ml of aquarium water were

added. The dish was then placed near a 15 w bench lamp and

examined under a dissecting microscope after 5 min. Miracidia

were collected using a finely drawn Pasteur pipette and used to infect the appropriate molluscan host.

2.1.3.2. Snail infections

Miracidia of S.mansoni and S.marqrebowiei were used to infect

S.qlabrata and B.tropicus respectively. Individual snails were placed in separate wells containing approximately 5 ml of de-

chlorinated tap water in plastic trays. B.qlabrata were infected

with 4 - 6 miracidia each and B.tropicus with 2 miracidia each.

The trays were then covered and left at room temperature for

between 12 - 24 h. The snails were then transferred to an isolated

biohazard area and maintained as described previously.

2.1.3.3. Collection of cercariae

Snails were examined at 35 days post infection (DPI) for

cercarial production (shedding). Small glass jars containing

about 50 ml of dechlorinated tap water were used to hold 1 0 - 1 5

infected snails each. The jars were placed as close as possible

to a 15 w bench lamp and examined at 15 min intervals. Cercariae were collected using a finely drawn Pasteur pipette. All

procedures involving collection and handling of cercariae were

carried out in an isolated biohazard area whilst observing the

utmost safety precautions.

On occasions, cercariae of S.bovis, S.leiperi, 5.haematobium

S.marqebowiei, S.douthitti and 5..japonicum were obtained from the

British Museum of Natural History and Winches Farm field station,

cercariae were transported to the department in sealed 20 ml

plastic vials enclosed in four layers of polythene. On arrival,

the cercariae were taken to the biohazard room and treated as above

2.1.3.4. Mouse infections

Infection procedures were based on the method of Smithers

& Terry (1965). Mice older than 5 weeks were used to harbour

adult parasites. The animals were anaesthetised by intraperitoneal injection of sodium pentabarbitone (Sagatal, May & Baker, Ltd)

diluted 1:9 with sterile distilled water at a dosage of 0*1 ml/g body weight. This level generally maintained anaesthesia for

between i - 1 h. 16 mm, 25 G hypodermic syringes were used to administer the drug.

As much hair as possible was removed from the ventral abdominal region of the mice using sharp scissors. The animals

23

were then placed in rows, on their backs, between close fitting

slats of wood. Depilated areas were moistened with dechlorinated

water and metal rings (1 cm internal dia x 0*5 cm high) were applied. Cercariae were placed in the lumen of each ring in as small a volume as practicable. The routine infection level was approximately

200 cercariae per mouse. The animals were left for at least 30 min

after which the rings were removed and the mice returned to their

cages. All working surfaces were then doused with copious amounts

of absolute alcohol and left overnight.

2.1.4. RECOVERY OF ADULT PARASITES

Techniques for the recovery of adult parasites were based on

the method of Yolles et al (1947) and Duvall & De bJitt (1967).

Animals were killed by placing them in a chloroform jar. A circumferential dermal incision was made at approximately the stomach region and the fore and hind areas of skin were pulled away to

expose as much of the abdomen as possible. The ventral body wall

was cut away to expose the viscera and part of the rib cage was

removed to allow access to the dorsal aorta. The hepatic portal vessel was severed as close to the liver as possible and the

visceral organs displaced over the lip of a glass funnel. A 25 mm

19 G hypodermic syringe was inserted into the dorsal aorta and used to inject 15 - 20 ml of saline (0»9% w/v Na Cl) or citrated

saline (5 mg sodium citrate/ml saline). The perfusate was

allowed to run into a collecting dish through a nylon fine meshtea strainer

The funnel was washed through with several ml of saline

and perfused worms were removed from the strainer with fine forceps

and placed in a petri dish containing 0*9? (w/v) saline. The

visceral surfaces and mesenteric vessels were examined and any remaining parasites were removed using fine forceps and transferred

to the saline pending further analysis.

2.2. CHEMICALS

All enzymes and other reagents were obtained from Sigma or

Boehringer Mannheim unless otherwise stated.

2.3. CARBOHYDRATE AND WET WEIGHT DETERMINATIONS

Glucose and glycogen levels in male and Pemale S.mansoni and

S.marqrebowiei were measured at 42, 56, 70 and 84 days post infection.

Worms were removed as described in section 2.1.4, separated with

fine forceps and rinsed three times in ice-cold 0-9$ (w/v) saline.

Batches of 5 male and 8 female parasites were blotted dry on tissue2paper and placed onca pre-weighed 1 cm stainless steel grid. A

hot air drier was held for 10 sec at a distance of about 15 cm to

remove additional surface water. The worms were then weighed on a Mettler analytical balance accurate to 0*1 mg. The parasites

were then removed and placed in 100 |jl of ice-cold 0«6N perchloric

acid in pre-cooled, glass, 0*1 ml mini-homogenisers (Jencons)

and disrupted with approximately 60 passes of the pestle.

Homogenates were then transferred to 1*5 ml Eppendorf tubes

and left at 4°C for 45 min. The tubes were then centrifuged at

14000 g for 10 min in a Jobling mini-centrifuge and the supernatants

used for glucose and glycogen determinations according to the

method of Keppler & Decker (1974) (section 2.8.1,2.). The centri

fuged pellets were assayed for total protein using a modified

method of Loiury et al (1951) (section 2.4.).

2.4. PROTEIN DETERMINATIONS

Protein measurements tuere performed on perchlorate pre

cipitated pellets using a modified method of Lowry et al (1951). Constituent reagents were reduced proportionately so that the final

assay v/olume in each tube was 3 ml. Protein pellets were re

suspended in 0*5 ml of 0*5 M NaOH and heated at approximately 80°C in a water bath until they dissolved. Bovine serum albumen

(BSA) was made up to 1 mg/ml with 0«5 M NaOH and heated with the

samples. Standards (20, 50, 100 & 200 pg protein) and samples

were made up to 0«8 ml with 0*5 M NaOH in-separate ts3t -tubes.

2 ml of Lowry reagent was added-to each sample and standard and

the-tubes were left for 10 min at room temperature. 0-2 ml of Folin - Ciocalteau reagent were added to each tube and readings were taken 30 min later on a Cecil 202 Spectrophotometer at

750 nm. A comparison was made between this method and the Bradford dye binding technique (1976) using BSA standards.

2.5. LIPIDS

Total lipid levels in male and female S.mansoni were measured

at 42, 50, 70 and 84 days post infection. Adult parasites were

obtained and washed as described in section 2.1.4. 5 - 10 malesand 1 0 - 2 0 females were homogenised in 100 |jl of a chloroform :

methanol (2:1) mixture in glass, 0*1 ml, mini-homogenisers (Jencons)

with about 60 passes of the pestle. The homogenates were transferred to 1*5 ml Eppendorf tubes and left overnight at 4°C. The tubes

were then centrifuged in a Jobling mini-centrifuge for 5 min (14000 g) and the supernatants were removed and stored at 4°C. The pellets

were re-suspended in 100 jjl of chloroform/methanol and left at 4°C

for 60 min. The tubes were centrifuged for 5 min and the supernatants

combined with the respective previous fractions in separate tubes.

The pellets were re-suspended in 100 il each of ice-cold 0-6 N

perchloric acid and left at 4°C for 45 min. The pellet material

was then centrifuged for 10 min as above, the supernatants discarded

and the pellets assayed for protein as previously described. The

lipid samples were evaporated to dryness under a stream of nitrogen.

2 ml of concentrated sulphuric acid was added to each test tube and the tubes heated in a boiling water bath for 10 min. The total lipid levels were determined using the method of Zollner & Kirsch

27

*

%

%

A

%

2.6. ENZYME ASSAYS

Enzyme analyses were performed on extracts of adult males and

females of six schistosome species, namely S.mansoni, S.marqrebowiei,

S.bovis, S.leiperi, S..japonicum and 5.haematobium. Worms were removed as previously described and rinsed with three changes of

ice cold 0*9? (w/v) saline. Male and female parasites were separated

and homogenised in pre-chilled, glass mini-homogenisers (Jencons)

using the extraction medium of Zammitt etal^ (1976); 50 mM

Triethanolamine (TRA)/HCL, 1 mM ethylenediamine tetra-acetic acid

(EDTA), 2 mM MgCl2 and 30 mM 2 - mercaptoethanol, at pH 7.5. The

volume of medium was adjusted to give approximately a 1:5, worm : pi

medium ratio. The extraction of phosphofructokinase was performed

using the medium of Opie & Newsholme (1967) ; 50 mM Tcis/HCL,1 mM EDTA and 5 mM MgSO^ at pH 8*2. The homogenates were centrifuged

at 30,000 g for 10 min at 2°C in an M.S.E. High Speed 25 centrifuge and the supernatants taken for enzyme and protein analysis.

Tricarboxylic acid enzyme activities in S.mansoni were assayed

in sonicated and intact homogenates using up to 70 pg of soluble

protein per assay. Ultrasonic disintegration was performed using

an M.S.E. Soniprep 150 using 3 x 10 sec phases of 20 p with 30 sec

intervals, with the sample packed in ice.

Assays were perfomed at 30°C in a total assay volume of 1 ml using a Cecil 505 recording spectrophotometer. All reactions were

followed at 340 nm except where stated, initiated by the addition

of supernatant (except pyruvate kinase) and measured using the following methods:-

The analysis was based on the method of McManus 4 Smyth

(1982).The final reaction mixture contained: 50 mM Phosphate, pH

7»4; 0 1 mM EDTA; 0*5 mM NADP; 10 mg Glycogen; 5 y M Glucose-1,

6 - bisphosphate; 15 mM Mg C^; 2 mM AMP; 71) Phosphoglucomutase;

5U Glucose -6- phosphate dehydrogenase.

Hexokinase (E.C. 2.7.1.1.)The assay was based on the method described in Biochemica

information (Boehringer Mannheim, 1975). The final reaction

mixture contained: 50 mM TRA/HC1, pH 7*4; 200 mM D-Glucose;

10 mM MgC^J 3 mM ATP; 0*7 mM NADP; 5U Glucose -6- phosphate

dehydrogenase.

Glucosephosphate isomerase (E.C. 5.3.1.9.)The assay was based on the method of McManus & Smyth (1982).The final reaction mixture contained: 100 mM TRA/HC1, pH 7»45

2 mM Fructose -6- phosphate; 0*5 mM NADP; 7 mM MgCl^; 5U Glucose

-6- phosphate dehydrogenase.

Phosphoqlucomutase (E.C. 2,7.5.1.)The assay was based on the method of McManus 4 Smyth (1982).

The final reaction mixture contained: 100 mM TRA/HC1, pH 7*4; 5 mM Glucose -1- phosphate; 0*2 mM Glucose - 1, 6 - bisphosphate; 1 inM

EDTA; 0.5 mM NADP; 7 mM MgC^; 5U Glucose -6- phosphate dehydrogenase.

Phosphorylase a + b (E .C . 2 . 4 . 1 . 1 . )

P h osphofructokinase (E . C . 2 . 7 . 1 . 1 1 . )

The assay was based on the method of McManus & Smyth (1982) The final reaction mixture contained: 100 mM TRA/HC1, pH 8*0;

100 mM Ammonium sulphate; 1 mM EDTA; 5 mM Fructose -6- phosphate;

2 mM ATP; 0»1 mM NADH; 5: mM Mg 12U Glucose -3- phosphate

dehydrogenase; 10U Aldolase; 10U Triosephosphate isomerase.

Aldolase (E.C. 4.1.2.13).

The assay was based on the method of McManus & Smyth (1982)

The final reaction mixture contained: 100 mM TRA/HC1, pH 7*4; 0*4

mM Iodoacetate; 5 mM Fructose - 1 , 6 - bisphosphate; 0.1 mM NADH; 12u Glycerol -3- phosphate dehydrogenase; 10U Triosephosphate

isomerase.

Triosephosphate isomerase (E.C. 5,3.1.1.)


The final reaction mixture contained: 200 mM TRA/HC1, pH 7«4;

5 mM Glyceraldehyde -3- phosphate; 0*1 mM NADH; 12u Glycerol -3-

phosphate dehydrogenase,

Glyceraldehye -3- phosphate dehydrogenase (E.C. 1,2.1.12.)


The final reaction mixture contained: 100 mM TRA/HC1, pH 7*A;

1 mM ATP; 1mM EDTA; 2 mM Mg SO^; 6 mM Glycerol -3- phosphate;

0*1 mM NADH; 15U. Phosphoglycerate kinase.

Phosphoqlycerate kinase (E.C. 2.7,2.3.)


The final reaction mixture contained: 100mM TRA/HC1, pH 7*4;1 mM ATP; 6 mM Glycerol -3- phosphate; 1 mM EDTA; 2 mM Mg S0^;□ •1 mM NADH; 12U Glycerol -3- phosphate dehydrogenase.

Phosphoqlycerate mutase (E.C. 2.7.5.3.)


The final reaction mixture contained: 100 mM TRA/HC1, pH 7*4;

1 mM Mg S0^; 1 mM ADP; 0-1 mM NADH; 5 mM Glycerol -3- phosphate; 0*1 mM 2, 3- Diphosphoglycerate; 10U L-lactate dehydrogenase;

8U Enolase; 10U Pyruvate kinaSe.

Enolase (E.C. 4.2.11.)


The final reaction mixture contained: 100 mM TRA/HC1, pH 7*4;1 mM Mg S0^; 1 mM ADP; 0*1 mM NADH; 1 mM 2- Phosphoglycerate;10U Lrlactate dehydrogenase; 10U Pyruvate kinase.

Pyruvate k in ase (E . C . 2 . 7 , 1 . 4 0 ).

The assay was based on the method of Brazier & Jaffe (1973).The final reaction mixture contained: 100 mM TRA/HC1, pH 7«4;

5 mM Mg 50^; 40 mM KC1; 5 mM ADP; 5 mM Phosphoenolpyruvate;

0*1 mM NADH; 1QU L-lactate dehydrogenase. The reaction was started

by the addition of Phosphoenolpyruvate.

Lactate dehydrogenase (E.C. 1,1,1.27)

The assay was based on the method of Opie & Neusholme (1967).

The final reaction mixture contained: 50 mM Tris/HCl; pH 7*4;

□•1 mM NADH; 1 mM KCN; 5 mM pyruvate.

Alcohol dehydrogenase (E.C. 1.1.1.1.)

The assay was based on the method shown in Biochemica

information (Boehringer Mannheim, 1975). The final reaction

mixture contained: 75 mM Glycine - Sodium pyrophosphate, pH 9*0;70 mM Semicarbazide; 0-1 ml Ethanol; 1 mM NAD; 10 mM Glutathione.

Pyruvate dehydrogenase (E.C. 1.2.4.1.)

The assay was based on the method of Tai at al (1982) and

the final reaction mixture contained: 200 mM Tris/HCl, pH 8; 20 mM

NAD; 20 mM Acetyl Co enzyme A; 50 mM Dithiothreitol; 500 mM MgC12J

200 mM Thiamine pyrophosphate; 0*5 Arylamine acetyl transferase;

0«5U 4- Aminoazobenzene -4*- sulphonic acid; 10 pi Triton x-100;

500 mM Pyruvate, Activity was determined from the decrease in

optical density at 460 nm.

Citrate synthase (E,C. 4.1.3.7.)

The assay was based on the method of McManus & Smyth (1982) and the final reaction mixture contained: 100 mM Tris/HCl, pH 7*4;

6 mM Malate; 0*2 mM NAD; ; 0»2 mM Acetyl Coenzyme A;

10U Malate dehydrogenase; + 10 pi Triton x-100.

Aconitase (E.C. 4,2.1.3.)

The assay was based on the method of McManus & Smyth (1982).

The final reaction mixture contained: 100 mM Tris/HCl, pH 7*4;

100 mM NaCl; 0*1 mM Cis-aconitate. Activity was measured from the change in absorbance at 240 nm.

Isocitrate dehydrogenase (E.C.1.1.1.41.)

The assay was based on the method of McManus & Smyth (1982).

The final reaction mixture contained: 100 mM Tris/HCl, pH 7*4;

2 mM ADP; 2 mM NAD; 8 mM Mg C ^ or Mn C^j 5 mM DL-isocitrate.

I s o c it r a te dehydrogenase (E . C . 1 . 1 . 1 . 4 2 . )

The assay uias based on the method of McManus & Smyth (1982) and the final reaction mixture contained: 100 mM Tris/HCl, pH 7»4;

0*05 mM NADP; 0*8 mM Mg or Mn 5 mM DL-isocitrate.

2 - oxoqlutarate dehydrogenase (E.C, 1.2.4.2.)

The assay was based on the method of McManus & Smyth (1982). The final reaction mixture contained: 100 mM Phosphate, pH 7*4;

5 mM KCN; 1 mM Fe (CN)g; 5 mM 2 - oxoglutarate. The change in absorbance was measured at 420 nm.

Succinate dehydrogenase (E.C. 1.3,99.1.) (FUM-SUCC)


and the final reaction mixture contained: 100 mM Phosphate, pH 7»4; 0«05 mM Ca C 2 mM Mg Cl^J 0*1 mM NADH; 30 mM Fumarate. The reaction was measured at 420 nm.

Succinate dehydrogenase (E.C. 1.3.99.1.) (5UCC.-FUM)


and the final reaction mixture contained: 100 mM Phosphate, pH 7»4; 1 mM i<2 Fe (CN)gj 40 mM Succinate; 1 mM KCN; 1 mM EDTA. The change

in absorbance was followed at 420 nm.

Fumarase (E . C . 4 . 2 . 1 . 2 . )

The assay was based on the method of McManus & Smyth (1982) and the final reaction mixture contained: 100 mM Phosphate, pH 7»4;

50 mM Malate. The change in optical density was followed by 240 nm.

Malate dehydrogenase (E.C. 1.1.1,37.) (HAL- QAA)


and the final reaction mixture contained: 50 mM Tris/HCl, pH 7«4;1, mM NAD; 1 mM KCN; 1 mM Malate.

Malate dehydrogenase (E.C. 1.1.1.37) (QAA-MAL)

The assay was based on the method of McManus & Smyth (1982) and the final reaction mixture contained: 50 mM Tris/HCl, pH 7*4;0»1 mM NADH; 1 mM KCN; 1 mM Oxaloacetate.

Qctopine dehydrogenase (E.C. 1.5.1.11.)

The assay was based on the method of Storey & Dondo (1982)

and the final reaction mixture contained: 100 mM Imidazole/HCl, pH 7*0; 10 mM L-arginine; 0«1 mM NADH; 1 mM Pyruvate.

Phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32.)

The assay was based on the method of Opie & Newsholme (1967)

and the final reaction mixture contained: 70 mM Tris/HCl, pH 7*4;

1 mM Mn C^; 0*1 mM NADH; 2 mM Inosine -51- diphosphate 15 mM NaHCO^J 10U Malate dehydrogenase.

Glucose -6- phosphate dehydrogenase (E.C, 1,1,49.)


and the final reaction mixture contained: 100 mM TRA/HC1, pH 7*4;

2 mM Glucose -6- phosphate; 0«5 mM.NADP; 7 mM MgCI^.

6 - Phosphoqluconate dehydrogenase (E.C. 1,1,44.)



2 mM PHosphogluconate; 0«5 mM NADP; 7 mM Mg

Transaldolase (E.C. 2,2,1,2.)

The assay was based on the method shown in Biochimica Information (Boehringer Mannheim, 1975) and the final reaction

mixture contained: 80 mM TRA/HC3, pH 7*4; 8 mM EDTA; 0*5 mMErythrose -4- phosphate; 7 mM Fructose -6- phosphate; 0«1 mM NADH;

5L) Glycerol -3- phosphate dehydrogenase; 10U Triosephosphateisomerase.

M a lic enzyme ( E . C . 1 . 1 . 1 . 4 0 . ) (MAL-PYR)


and the final reaction mixture contained: 100 mM Tris/HCl, pH 7-4

7 mil Mg C12; 0*5 mM NADP; 20 mM Malate.

Malic enzyme (E.C. 1.1.1.40.) (PYR-MAL)


and the final reaction mixture contained: 100 mM Tris/HCl, pH 7*4

50 mM Pyruvate; 80 mM KHCO^; 1 mM Mn Cl^; 0*1 mM NADPH.

Nucleoside - 5 dipfaosphate kinase (E.C. 2.7.4.6.)

The assay was based on the method shown in Biochimica

Information (Boehringer Mannheim, 1975) and the final reaction mixture contained: 80 mM TRA/HC1, pH 7*4; 2*25 mM ATP; 1 mM

Phosphoenolpyruvate; 17 mM Mg C^; 65 mM KC1; 0*1 mM NADH; 10U

Pyruvate kinase; 10U L-lactate dehydrogenase; 1 mM Deoxythymidine -5T- diphosphate.

Glutamate - oxaloacetate transaminase (E.C. 2.6.1.1.)

The assay was based on the method shown in Biochimica Information (Boehringer Mannheim, 1975) and the final reaction

mixture contained; 95 mM Phosphate, pH 7*4; 180 mM L-aspartate;

19 mffl 2 - oxoglutarate; 0*1 mM NADH; 8U malate dehydrogenase.

Enzyme activities are expressed in terms of n moles of product formed/min/mg soluble protein. Protein determinations were performed on 20 - 50 pi aliquots of supernatant as outlined in section 2.4

after precipitation with 20-50 ul of ice-cold 0*6 N perchloric acid and centrifugation at 14000 g for 10 min.

2.7. METABOLITES

*

♦

*

#

sWteSteady^levels of certain intermediary and end-product meta

bolites were measured in both sexes of S.mansoni and S,marqrebowiei

using spectrophotometric and fluorimetric methods of analysis. Com

parisons between these procedures were made using known concentra

tions of standard solutions (100, 120 & 140 nM) and mouse liv/er

material.

2.7.1. SPECTROPHOTOMETRY

2.7.1.1. Preparation of materialj

Parasites were obtained as described in section 2.1.4. rinsed

rapidly in ice-cold 0»9$ (w/v) saline and plunged into liquid nitrogen.

Parasites between 42 - 84 days post infection (DPI) were used for

determinations. Frozen material was rapidly weighed (if sample

was large enough) and either processed immediately or stored in

liquid nitrogen until required. Parasite tissue was powdered in

liquid nitrogen using a porcelain mortar and pestle. Ice-cold 0«6N

perchloric acid was added to the samples in the ratio of approximately 20$ w/v. Continual mixing with the pestle thawed the perchloric acid and the powdered material and the samples were left

for 45 min at 4°C. After this, extracts were transferred to

Eppendorf tubes and centrifuged in a Jobling mini-centrifuge at 14000g for 10 min. The protein pellets were assayed as described

in section 2.4. The supernatants were neutralised by addition of

20 pi aliquots of 3*75 M KHCO^ and then re-centrifuged as before. The supernatants were removed with a Pasteur pipette and used for

assay. Samples of mouse liver were dissected from unifected chloroformed LACA mice. Pieces of liver were plunged into liguid

nitrogen, rapidly weighed and treated as for the parasite material.

2.7.1.2. Assays

All assays (0*5 or 1 ml final volume) were performed on a

Cecil 505 recording spectrophotometer at room temperature using

the techniques described below. Optical density changes were

followed at 340 nm.

Adenosine -51- triphosphate

The assay was based on the method of Jaworek et al (1974) and the final reaction mixture contained: 100 mM Triethanolamine

(TRA)/HC1, pH 7»4; 5 mM 3 - Phosphoglycerate; 3 mM Mg S0^; 1 mM

Ethylenediaminetetra-acetic acid (EDTA); 0*1 mM NADH; 12U

Phosphoglycerate kinase; 10U Glyceraldehyde -3- phosphate dehy

drogenase.

Adenosine -5! - diphosphate . & Adenosine -5!- monophosphate

The assay was based on the method of Jaworek et al (1974)

and the final reaction mixture contained: 200 mM TRA/HC1, pH 7«4;

100 mM l^CO^; 1 mM Phosphoenolpyruvate; 30 mM Mg S0^; 100 mM KCl;

0*1 mM NADH; 10U L-lactate dehydrogenase; 10U Pyruvate kinase; 15U

Myokinase.

Fructose - 1 , 6 - bisphosphate

The assay was based on the method of Mschal et al (1974) and the final reaction mixture contained; 200 mM TRA/HC1, pH 7*6; 0*1

mM NADH; 10U Glyceraldehyde -3- phosphate dehydrogenase; 10U

Triosephosphate isomerase; 10U Aldolase.

Oxaloacetate

As described in section 2.8.1.1.

Phospoenolpyruvate> Pyruvate & 2-Phosphoqlycerate

The assay was based on the method of Czok & Lamprecht (1974) and the final reaction mixture contained; 300 mM TRA/HC1, pH 7*4; 0-1 mM NADH; 20U L-lactate dehydrogenase; 1 mM ADP; 10 mM Mg S0^;

35 mM KC1; 10U Pyruvate kinase; 20U Enolase.

2.7.2. FLU0RIMETRY

2.7.2.1. Preparation of material

Parasite and mouse liver samples were prepared as for the spectrophotometric analysis except that the final, neutralised

sample was filtered across a Gelmann Acrodisc 0*2 |j filter (FLOlii) in order to exclude as much dust as possible.

2,7.2.2. Assays

Metabolites were determined using standard enzymatic tech

niques and the serial addition of linking enzymes and co-factors

enabled several metabolites to be determined in a single sample.

Oxidation of NADH was measured on a Perkin-Elmer 2000 fluorescence

spectrophotometer (excitation wavelength 340 nm, emission wave

length 464 nm). The initial assay volume was 2*2 ml and contained between 0*1 - 0*5 ml of neutral perchlorate extract. The order of

addition of co-factors and enzymes (Table 1) was based on the work

of Colson & Klapper (1979) and Barrett & Butterworth (1982).

Known amounts of metabolites ( 5 - 1 0 nmoles) were added as internal

standards after each determination. Concentrations of metabolites

in samples were calculated from the relative changes in fluores

cence caused by endogenous metabolites and their respective

standards. Linking enzymes were also checked for inherent

fluorescence by adding a further aliquot after each metabolite

measurement to determine if there were any subsequent changes in

fluorescence. The increase in volume after each individual add

ition to the cuvette has to be taken into account for the calculations.

A control cuvette, using triple distilled water in place of

the sample, was run with each assay. Fluorescence readings were taken at 30 sec intervals for 5 - 1 0 min. The data was subsequently

fitted with regression lines and the straight line portions of the

plots were used to calculate changes in fluorescence.

Table 1

The sequential assay of ten metabolites in S.mansoni and

S.marqreboujiei by fluorimetry.

Metabolite determined Enzyme and/or co-factors added

Oxaloacetate Malate dehydrogenase (30 U/ml)

Dihydroxyacetonephosphate Glycerol-3-phosphate dehydrogenase (9U/ml)

Glyceraldehyde-3-phosphate Triosephosphate isomerase (10 U/ml)

Fructose-1, 6-bisphosphate Aldolase (3 U/ml)

2-0xoglutarate NADH (0*03 mM), Aspartate (0*4 mM) Glutamate - oxaloacetate transaminase (6 U/ml)

Pyruvate L-lactate dehydrogenase (21 U/ml)

Phoshoenolpyruvate ADP (0*2 mM), Pyruvate kinase (3 U/ml)

2-Phosphoglycerate Enolase (2 U/ml)

3-Phosphoglycerate 2, 3-Diphosphoglycerate (1 uM) Phosphoglyceromutase (11 U/ml)

Isocitrate Pyruvate (0*02 mM), MnCI- (0*1 mM), NADP (0*5 mM), Isocitrate dehydro- • genase (1 U/ml)

The reaction mixture initially contained; 100 mM TRA/HC1, pH 7*5;

0*02 mM NADH; 2 mM Mg C12; AO mM KOI; 0*1 - 0*5 ml sample. The figures in parentheses-represent the concentrations of enzyme/co- factor at the particular stage of the assay.

All data from the spectrophotometric and fluorimetric assays

of parasite and mouse liver material are expressed in terms of

nmoles/g wet weight. If a particular batch of material was too small to be weighed accurately, the protein value obtained was

extrapolated from the protein; wet weight ratios in section 3.6.

2.8. INCUBATIONS

6h incubations were performed under sterile conditions with

single males, females and paired S.mansoni and 5.marqrebowiei, single

males and females of S.haematobium (Morrocan strain) and males of

S.douthitti.

The incubation medium consisted of Earle's balanced salt

solution (DIFCO), containing 10$ inactivated newborn calf serum

(GIBC0BI0CULT) with 1 /ml of a penicillin/streptomycin mixture

(1000 /1000 ug per ml respectively)(FLOlii). All incubations were carried out in autoclaved 1*5 ml Eppendorf tubes using a maximum

volume of 1*2 ml of medium in each tube. The adult worms were

obtained as described in section 2.1.4. and all subsequent operations involving the transfer of parasites from their host to

the incubation tubes were carried out in a Microflow lamina flow cabinet. All instruments used throughout the operation were sterilised by boiling for at least 15 min.

Earle's solution was made up with sterile triple distilled

water and the antibiotics added prior to filter sterilisation

across a 0*2 |j filter using a vacuum pump. This solution was made up the day before each experiment and stored at 4°C until

required. The correct volume of serum was added under sterile

conditions before each incubation. An aliquot was decanted for pH measurement and the complete medium was pre-incubated in a water

bath at 37°C. The blood glucose levels in the host mice had

previously been measured as 0*88 ± 0-002 mg/ml (4.9 mM) using the

method of Keppler & Decker (1974). Therefore, the glucose concen

tration in the medium was accordingly adjusted to 5 mM by addition

of an appropriate volume of sterile triple distilled water prior

to filter sterilisation. The same batches of Earle*s salts,

serum and antibiotics were used for all experiments to ensure consistent incubation conditions. The ratio of parasites to

incubation medium used was approximately 1 worm: 200 pi medium.

Aliquots of medium were pipetted into Eppendorf tubes, sealed and pre-incubated at 37°C in a water bath. Several petri dishes

containing 10 ml of sterile medium each were maintained at 37°C

on a thermostatically controlled heating dish within the flow

hood and used for washing the worms. The parasites were removed

from mice, placed in 0*9% (w/v) saline, the majority separated

under a dissecting microscope and transferred to the petri dishes.

The worms were washed with four changes of medium and then divided

into two batches. One batch of worms we s- transferred to Eppendorf tubes and placed in a shaking water bath (at minimal setting) at 37°C. Control tubes containing no worms were run concurrently.

The tubes were inverted once every hour. The remaining worms were

transferred to clean Eppendorf tubes and stored in liquid nitrogen and were subsequently used as unincubated controls.

After incubation, the worms were removed and each batch was

washed in three changes of sterile, ice-cold 0 9 $ (w/v) saline with vortexing on a rotamix (Tucker Ltd) for 10 sec. This was

done to remove incubation medium from the parasites. The worms

were then transferred to clean Eppendorf tubes prior to storage in liquid nitrogen, 50 pi aliquots were removed from each incubate

for pH measurement. Protein in the media samples was precipitated

by adding M % (v/v) of ice-cold 0*6N perchloric acid and maintaining

the samples at 4°C for 1 hour. Each tube was then spun at 14000 g

in a Jobling mini centrifuge for 10 min, the supernatants decanted,

neutralised with solid KHCO^ and then re-centrifuged. The super

natants were pipetted into clean Eppendorf tubes and stored in liquid nitrogen.

2.8.1. END PRODUCT ANALYSIS

2.8.1.1. Medium

Media samples (10 - 50 pi) were assayed at room temperature

for the presence of alanine, acetate, citrate, ethanol, glucose,

glycerol, D & L-lactate, oxaloacetate, pyruvate, propionate and

succinate using a Cecil 505 recording spectrophotometer in a total cuvette volume of either 0*5 ml or 1 ml at 340 nm unless otherwise stated. The assay methods used were as follows:-

AlanineThe assay was based on the method of Grassl (1974) and the

final reaction mixture contained: 92 mH Tris/HCl, pH 7*4; 7 mH

2-oxoglutarate; 0«1 mH NADH; 6 U L-lactate dehydrogenase; 10 U

Glutamate-pyruvate transaminase.

Acetate

The assay was based on the method of Bergmeyer & Hollering(1974) and the final reaction mixture contained: 140 mH Triethano

lamine (TRA/HC1, pH 8-4; 30 mH Halate; 10 mH HgC12; 1 mH NAD;0*2 mH Coenzyme A; 3 mH ATP; 20 U Halate dehydrogenase; 5 U Citrate

synthase.

Citrate

The assay was based on the method of Hollering & Gruber (1966)

and the final reaction mixture contained: 200 mH Glycyglycine, pH

7*8; 0«1 mH ZnCl; 0*1 mH NADH; 10 U Halate dehydrogenase; 10 U

L-lactate dehydrogenase; 2 U Citrate lyase.

Ethanol

The assay was based on the method of Bernt & Gutmann (1974) and the final reaction mixture contained: 60 mH Phosphate, pH 8*5;

60 mH Semicarbazide; 15 mH Glycine; 0*5 mH NAD; 20 U Alcohol dehydro

genase.

Glucose

4 mM MgSO^; 1 mM ATP; 1 mM NADP; 5U Glucose -6- phosphate dehydrogenase; 15U Hexokinase.

Glycerol

The assay was based on the method of Eggstein & Kuhlmann(1974) and the final reaction mixture contained: 0*25.mM Glycyl-

glycine, pH 7*5; 3 mM MgSO^ 0*1 mM NADH; 1 mM Phosphoenolpyruvate;

10U Pyruvate kinase; 10U L-lactate dehydrogenase? 5U Glycerokinase.

D or L-Lactate

The assay uias based on the method o f K epp ler & Decker (1974)

and the f i n a l re a c t io n m ixture con ta in ed : 200 mM TRA/HC1, pH 7*4;

The assay was based on the method of Noll (1974) and the final

reaction mixture contained: 0*22 mM Glycylglycine, pH 10; 35 mM

L-glutamic acid; 3 mM NAD; 12U Glutamate-pyruvate transaminase;20U D or L-lactate dehydrogenase.

Oxaloacetate

The assay was based on the method of liJahlefeld (1974) and the

final reaction mixture contained: 133 mM TRA/HC1, pH 7*6; 10 mM

Ethylene diaminetetra-acetic acid (EDTA); 0*1 mM NADH; 5ll Malate

48

4

4

4

4

4

dehydrogenase.

Pyruvate

The assay was based on the method of Czok & Lamprecht (1974)


0*1 mM NADH; 7 mM EDTA; 20U L-lactate dehydrogenase.

Propionate

The assay was based on the method of Holz & Bergmeyer (1974)

and the final reaction mixture contained: 50 mM TRA/HC1, pH 7*4:

20 mM MgC^J 15 mM ATP; 0*8 mM Hydroxylamine; 1 mM Acetate; 20U Acetate kinase; 0*1 M Trichloroacetic acid; 10 mM FeCl 3. The

reaction was followed at 492 nm. Acetate kinase catalyses the phosphorylation of acetate and propionate, therefore it is necessary to perform a separate specific assay for acetate (described pre

viously) to determine propionate levels.

Succinate

The assay was based on the procedure described in Methods

of Enzymatic Food Analysis (1977) and the final reaction mixture

contained: 100 mM Glycylglycine, pH 8»5; 15 mM Mg S0^, 0»1 mM NADH;

0.5 mM Coenzyme A; imM Inosine-51- triphosphate; 1mM Phosphoenol pyruvate; 10LI Pyruvate kinase; 10U L-lactate dehydrogenase; 1 U Succiny1-Coenzyme A synthetase (Succinate thiokinase).

2 . 8 . 1 . 2 . Endogenous m etab o lite le v e ls

Incubated and control (i.e. unincubated) parasites were homo

genised by hand in ice-cold 0»6N perchloric acid in pre-cooled glass

mini-homogenisers (Jencons) at a ratio of approximately 1 worm: 20- 30 pi acid. Homogenates were transferred to Eppendorf tubes and

left at 4°C for 1 hour. Samples were then centrifuged in a Jobling

mini-centrifuge1-.(14000 g) for 10 min. The supernatants were decanted

and neutralised with solid KHCO^,

The centrifuged pellets were assayed for protein as described

in section 2.4 and the neutralised supernatants were re-centrifuged at 14ooo g in a Jobling mini-centrifuge. The supernatants were

subjected to similar metabolite analysis as described for the incu

bation media samples. No analyses were carried out for ethanol,

citrate, oxaloacetate, propionate or pyruvate due to lack of material

Glycogen levels were determined using the method of Keppler &

Decker (1974). This technique entails the enzymatic digestion of

glycogen by amyloglucosidase (AGS) and determination of the resul

ting total hexose levels. Approximately 0*05 ml of worm samples were incubated with 0*05 ml of 1M KHCO^ and 0*4 ml AGS (0*4 mg protein) each, in stoppered Eppendorf tubes in a shaking water bath

(minimal setting) at 40°C for 2 h. 0*3 ml of 0*6Aperchloric acid

was then added to each tube and the samples were subsequently

neutralised with solid KHCO^ and centrifuged at 14000 g for 10 min in a Jobling.mini-centrifuge. The supernatants were then removed and used for assay. Glycogen levels were obtained after the sub-

traction of free glucose levels in undigested samples. Frozen

media and parasite samples were thawed prior to metabolite analysis,

which was performed on the day of thawing.

142.8.1.3. CCU Measurement

14The evolution of CC^ by S.mansoni, S.marqrebowiei and14S.douthitti was measured using D - U - C glucose (250 mCi/mmol)

(Amersham Radiochemicals). The radiolabelled glucose was added to

the Earle*s solution prior to filter sterilisation and the final

concentration in the assay medium was adjusted to 0*5 p Ci/p mol

glucose. Modified Eppendorf tubes were used for this series of

incubations. Two Durham tubes with internal measurements 3 mm diax 9 mm long were fixed with Araldite into each Eppendorf tube at

a position to allow the cap to be sealed. Incubation procedures

were carried out as previously described except that 50 pi of

filter sterilised 0 3 M NaOH was placed into each Durham tube to 14collect any CO2 and the maximum volume of incubate per tube was

□•5 ml. Controls for the experiment consisted of intact tubes (with

medium and NaOH) without worms and intact tubes containing 40 and

20 pi aliquots of mouse blood. The latter was used to assess the14contribution, if any, of mouse blood to CO2 evolution. The blood was

removed before perfusion using a sterile hypodermic syringe and the aliquots quickly pipetted into the control tubes.

After incubation, 0*4 ml of absolute alcohol was added to each

tube by injection using a 16 mm 24 G hypodermic syringe. The puncture

holes were rapidly closed with molten sealing wax and the tubes were

left to equilibrate overnight at room temperature. The worms were

then removed, washed several times in 0*9? (w/v) saline and assayed

for protein as described in section 2.4. The NaOH samples were

removed and subjected to scintillation counting.Determination of radioactivity in the NaOH samples were per

formed using a Tracerlab Spotmatic counter. 100 pi samples and

standards (volumes equivalent to 1000, 5000 and 10,000 disinte

grations per minute (d.p.m,)) were mixed with 1 ml each of Beckman

Ready-Solv scintillation fluid (Beckman Instruments) in 1 ml plastic

inserts. The inserts were capped and each placed in a 20 ml glass

scintillation vial. Counting was performed with 1 cycle of 3

phases of 3 min for each tube. Prior to measuring samples and standards, the background count for eqch insert and vial was deter-

^2.^0- ioctO cJp/^ C '* < lvxoos-ft}

mined. Standards/ywere run with every batch of samples to assess

counting efficiency.

2.8.2. Glucose Incorporation

Tissue incorporation of D - u - glucose by S.mansoni

was measured by incubating worms as described for the ^ C 02

evolution experiments using unmodified Eppendorf tubes. The level

of radioactivity used for this investigation was raised to 1 p Ci/ pmol glucose. After 6 h the worms were removed and washed with

three changes of sterile ice-cold 0 9 % (w/v) saline. The worms were then homogenised by hand in 70% ethanol (1 worm/50pl) using

52

glass mini-homogenisers (Jencons). The homogenates were then trans

ferred to Eppendorf tubes and left overnight at 4°C. The tubes

were centrifuged for 10 min in a Jobling min-centrifuge (14000 g)

and the supernatants discarded. The pellets were suspended in 70$

ethanol (50 pl/worm) and left for 1h at 4°C. The tubes were re- centrifuged as before and the supernatants discarded. The pellets

were re-suspended in 0*5 ml of 0-5 M NaOH each and then heated in a water bath at 50°C until they dissolved, 100 pi aliquots of the solutions

were taken for scintillation counting and the remainder used for

protein determination. The controls used for the scintillation

counting consisted of 100 pi aliquots of 0*5M NaOH minus digested

worm material.The results of the incubation experiments are expressed in

14terms of p moles or nmoles per mg total protein# .CO2 production is expressed in terms of glucose consumption attributable to tricarboxylic acid cycle operation. As the mechanism(s) of glucose

incorporation into parasite tissues is not known, the number of counts per minute as measured by the scintillation counter, were

related directly to the initial glucose concentration in the medium.

2.9. PYRUVATE KINASE KINETICS

The activity of pyruvate kinase (PK) from both sexes of S.mansoni, S.marqrebowiei, S.jeponicunyand male S.bovis was

determined at phosphoenol pyruvate concentrations[PEFj between 0*01 - 5 mM, using the assay outlined in section 2.6. The effects of fructose -1, 6- bisphosphate (FBP) and adenosine -5’- triphospate

(ATP) on enzyme activity were examined separately in both sexes

of S.mansoni and S.marqrebowiei using the following variations in

substrate and modulator concentrations:-

- ATP + FBP (0*001 - 0*125 mM); 0*1 mM PEP + ATP - FBP (Inf'll, 1 pM & 1 m); 1*0 mM PEP

2.9.1. "PHYSIOLOGICAL" ENZYME ACTIVITY

Using the data obtained from the metabolite determinations

(section 3.9.2.1.) PK activity in both sexes of S.mansoni was

assayed at "physiological" levels of phosphoenolpyruvate (PEP)

and adenosine -5*- diphosphate (ADP). In addition, the effects of

"physiological" levels of ATP and FBP on enzyme activity under these conditions were examined individually and jointly using the following methods:-

2.9.1.1. Male enzyme

The reaction mixture contained: 100 mM Triethanolamine

(TRA)/HC1, pH 7*4; 5 mM MgSO^; 40 mM KC1; 0*5 mM ADP; 0*1 mM NADH;Vw

10-50 pM PEP; ± 23 pM FBP; ± 2*25 mM ATP; 20IJ L-lactate deyhfdro- genase.

2.9.1.2. Female enzyme

The final reaction mixture contained: 100 mM TRA/HC1, pH

7*4; 5 mM MgS04; 40 mM KC1; 0*4 mM ADP; 0*1 mM NADH; 10 - 50 pM

PEP; ± 50 pM FBP; 0-6 mM ATP; 20U L-lactate dehydrogenase

2.9.2. Effects of Mg /Mn on enzyme activity and enzyme stability

The effect of 5 mM MiJ'and Mi*f on PK activity of both sexes of

S.mansoni was examined using the "physiological” regimes previously

described in the absence of ATP and FBP. The stability of PK from male and female S.mansoni was determined as previously described in the absence of ATP or FBP. Enzyme activity was measured as

soon as possible following centrifugation of the homogenates and

subsequently after 2, 4 and 7 h.

2.9.3. Host enzyme activity in parasite gut contents

Gut contents of female S.mansoni were obtained by cutting the parasites at approximately half-way along their bodies and

allowing the digest to diffuse into the normal enzyme extraction buffer (section 2.6.) (10 pl/worm) in an Eppendorf tube at 4°C for about 1h. The buffer containing gut contents was decanted and

centrifuged as described in section 2.6.

Samples were assayed for PK activity according to the method

presented in section 2.6. in the presence of 5 mM PEP. Samples

were also examined for glucose -6- phosphate dehydrogenase, malate dehydrogenase (malate - oxaloacetate) and citrate synthase activity

using the methods outlined in section 2.6. This was done to

assess the relative contributions, if any, of host gylcolytic,

tricarboxylic acid and hexosemonophosphate enzymes to the deter-

mination of parasite enzyme activity

2.9.4. House erythrocyte and serum pyruvate kinase

For the purposes of comparison, PK from mouse erythrocytes and serum was examined using the assay shown in section 2.6. Nice

were killed as in section 2.1.4 and blood was obtained from the posterior vena cava of the animals using a 16 mm 25 G hypodermic

syringe. The blood was treated as follows, based on the method of Gutmann & Bernt (1974), House blood was added to a solution

of sodium citrate to give a final concentration of approximately

1 mg citrate/ml sample. For a whole blood preparation, 0«5 ml of

citrated blood was added to 5 ml of ice-cold triple distilled

water and left to stand for 15 min at 4°C to complete haemolysis.

Prior to assay, the haemolysate was centrifuged at 3000 g at 4°C

for 10 min and the supernatant taken for determination.

For the separation of erythrocytes and serum, 2 0 0 j j I aliquots of citrated blood were centrifuged as described above and the super

natants (serum fraction) used for assay. The pellets were sus

pended in 1 ml each of ice-cold 0 * 9 % (w/v) saline and then re-

centrifuged. This washing was performed three times and the final

pellets, were suspended in 2 ml each of ice-cold triple distilled

water and left at 4°C for 15 min. The samples were centrifuged as above and the supernatants containing erythrocyte enzymes were assayed. Serum and erythrocyte samples were also examined for

glucose -6- phosphate dehydrogenase, malate dehydrogenase (malate -

oxaloacetate) and citrate synthase activity as outlined in section

2.6 to assess the relative activities of host glycolytic, tri

carboxylic and hexosemonophosphate shunt pathways.♦

*

2.10 ELECTROPHORESIS

Pyruvate kinase (PK) from male and female S.mansoni was

examined using polyacrylamide gel(PAGE), cellulose-acetate, starch

gel and isoelectric focusing techniques. Parasites were obtained

and washed as described in section 2.3 and homogenised in ice-cold 100 md TRA/HC1 buffer at pH 7*4, in a ratio of approximately 2:1

males and 4:1 females, worm/jjl buffer respectively. The samples

were then centrifuged at 20,000 g at 2°C for 10 min in an dSE High

Speed 25 centrifuge and the supernatants taken for examination.

2.10.1. PAGE

Slab gels and buffers were prepared according to the method

of Hames (1981) and the protocols for gel and resevoir buffer pre

paration are shown in Tables 2 and 3 respectively, douse erythrocyte

samples were also examined using this system and the material was obtained as described in section 2.9.4.

The gels were run on an LKB Vertical Electrophoresis system

at 4°C for 8h at 200 V and 14 h at 20V. Gels were then removed and st-ained using the method of Harris & Hopkinson (1976) and the final reaction mixture contained:

0*5d Tris/HCl, pH 7*4; 9 md Phosphoenolpyruvate; 8 md ADP;

0*01 mM NADH; 5 mM Fructose - 1 , 6 - bisphosphate; 20 mM Mg 50^;

67 mM KC1; 15U/fal L-Lactate dehydrogenase.

The reaction mixture was applied to the gels using filter paper overlays and the gels were left at room temperature to

develop. Gels were examined periodically under an ultra violet

(UV) light source after 5 min and the staining patterns were photo

graphed.

2.10.2. STARCH GEL

Parasite and mouse er^hrocyte samples were prepared as

described in section 2.6 and 2.9.4 respectively. Cotton threads

(5 mm long) were dipped into the samples and pressed into pits

in 9«6j, 1 mm thick, starch gels. The gels were run at 4°C on

horizontal apparatus at 200 V, 10-11 mA for 2h using a 0*1 M Tris/

HC1/0«1 M NaH2 P0^ reservoir buffer at pH 7.5.On completion, the gels were stained for PK activity as

previously described.

2.10.3. CELLULOSE-ACETATE

Parasite and mouse erythrocyte samples were prepared as

described above and separated on cellulose-acetate sheets at room temperature on horizontal apparatus at 250 V. The reservoir

buffer was Tris/Barbital (pH 8*6-9*0) at an ionic strength of 0«03

58

*

m

*

ur

♦

The samples were run for 30 min after which, PK patterns were

developed as described above using filter paper overlays and

recorded photographically.

2.10.4. ISOELECTRIC FOCUSING

Homogenates of male and female S.mansoni and whole mouse

blood were prepared as described above in the ratio of 10 worms/50^1 buffer and 15 worms/SOpl buffer respectively. Separation of proteins

was carried out at 4°C using an LKB ampholine polyacrylamide gel

plate, pH range 3*5 - 9*5 on an LKB multiphor with an LKB 2103

power supply. 5, 15 and 20 pi aliquots of each sample were applied cathodally to the gel on small (5 x 10 mm) pieces of filter paper

and 5pl aliquots of haemolysed human blood were also used to act as a marker. The cathode and anode wicks were soaked in 1M sodium

hydroxide and 1 M phosphoric acid respectively. The power pack

was set to deliver 1*4 KV at 30 Id and the pieces of filter paper

were removed after 1h to prevent tailing of proteins.The experiment was stopped after 2i h and the cathode and

anode wicks were removed together with the underlying gel. The pH

gradient was measured on the gel using an Ingold membrane electrode (Pye Unicam) with readings being taken at 1 cm intervals from the

cathodic end of the gel. The gel was then placed on a clean glass plate and stained for PK as described previously. The gel was left to develop at room temperature and the pattern of staining was

visualised and recorded as described above. Subsequently, the out-

Table 2

Protocols for polyacrylamide gel preparation.

A # 4r

Stackinq Gel

Acrylamide/bisacrylamide(37-5:1)

2-5

Stacking gel stock buffer 5-0

Resolving gel stock buffer1*5% Ammonium persulphate 1 -0

Water 11-5

TEWED 0-015

TEWED = , l\l, - tetramethylenediamine

All values are shoun in ml.

Resolving Gel

7-5< 105S

7-5 10

3-75

1*517.25

0-015

3-75

1-5

14-75

0-015

Table 3

Protocols for stacking gel, resolving gel and resevoir buffer

prepration for polyacrylamide gel electrophoresis.

*

Stacking gel buffer : Tris/HCl (pH 6-8); 6 g Tris was dis

solved in AO ml HÔ and titrated to pH 6*8 with

iM HC1. Water was added to make up to 100 ml. Resolving gel buffer : Tris/HCl (pH 8-8); 36*3g Tris and

* 48 ml; HC1 were mixed and made up to 100 ml

final volume with water. The solution was titrated

to pH 8*8 with HC1, if necessary,rResevoir buffer : Tris/glycine (pH 8*3); 15g Tris andK

72g glycine were dissolved in and made up to

51 with water.

*

*

lines of the patterns were drawn in with ink and the distance from

the centre of each band to the origin was measured. A graph plot

of these distances against pH readings was fitted with a regression

line and thus used to derive the isoelectric points for each band.

2.11. CYTOCHROMES

Cytochrome spectra were measured separately in male and female S.mansoni, whole and pepsinised mouse blood and a haematin solution.

Parasite samples were obtained as described in section 2.1.4.

Approximately 200 male and female worms were homogenised separately

in 200|j1 of the normal enzyme extraction buffer (section 2.6) at

4°C. 50 jjI and 100 pi aliquots of male and female samples respect

ively, were used for scanning. Whole mouse blood was obtained from

infected animals as described in section 2.9.4 and 100 pi aliquots

were used for the measurement of cytochromes. Spectral scans were also performed on a sample of rat liver to provide a comparison for the parasite material. 0*5 g of rat liver was homogenised in

5 ml of a 225 mM Mannitol/75 mM Sucrose buffer and 200 pi taken for assay.

Differential cytochrome scans were performed on an Aminco D.lii. —2 UV/VIS spectrophotometer at -196°C. Samples were added to

the reaction mixtures to give a final volume of 750 pi at the

following final concentrations Measurement cuvette (reduced)

225 mM Mannitol; 75 mM Sucrose; 5 mM Sodium phosphate; 10 mM

♦

*

0

0

4*

Morpholinopropane sulphonic acid (MOPS); CM mM Ethylenediamine-

tetra-acetic acid; 1Q |-in Rotenone; a feu/ crystals of sodium

dithionite.

Reference cuvette (oxidised)

As for the measurement cuvette, plus 25 pM Carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP).

In order to visualise cytochrome b in samples more clearly,10 mM Succinate and 10 pg Antimycin A were added to separate

measurement and reference cuvettes prior to scanning. In addition,

enzymatically digested u/hole mouse blood and haematin samples were

scanned to determine if any intermediary or final breakdown products

of the blood interfered with the parasite cytochrome scans. 10 mg of pepsin were dissolved in 5 ml of in HC1 and the pH was adjusted

to 2*3 by dropwise addition of 4M NaOH. 3ml of the pepsin solution

were added to 1 ml of citrated whole mouse blood in a 20 ml plastic

screw-top tube and subsequently incubated for 30 min at 37°C and 1h at room temperature. Concurrent to this, 5 mg of haematin

were dissolved in 2 ml of 2M NaOH. 50 pi aliquots of the enzyme digest and haematin solution were added separately to 95 pi of

distilled water and their spectra determined against distilled water blanks, at room temperature, using a recording Pye Unicam

SP100 spectrophotometer. After the initial scans were recorded, a few crystals of sodium dithionite were added to each cuvette to

produce reduced conditions and the samples were re-scanned.

2.12. OXYGEN UPTAKE

♦

♦

<6

*

Oxygen uptake measurements mere performed using a YSI Model

53 Oxygen Monitor (Yellow Springs Instrument Co) with a Clark type

electrode, on male and female S.mansoni. The worms were recovered as described in section 2.1,4 and approximately 10 males and 15

females were placed into separate chambers containg 3 ml of the Earle's medium used for the incubation experiments (section 2.8).

The medium was prepared as described in section 2.8 and pre

incubated at 37°C for 5 min prior to assay.The sample: chambers and oxygen probe were thoroughly cleaned

before use to minimise contamination by micro-organisms. The

assay medium was maintained at 37°C by a circulating water jacket

connected to a thermostatically controlled water pump. Measurements were made using a chart recorder over 1 5 - 2 0 min periods.

10 pi aliquots of KCN were added to each sample and control chamber

using a 5 pi Hamilton syringe (Jencons) to give a final concentration of 20 mM. Control values were obtained by measuring any

changes in samples of media without parasites in the presence and

absence of KCN. Worms were removed after assay and washed with 3 *changes of 0*9% (w/v) saline using vigorous manual agitation for

30 sec prior to assaying for protein as described in section 2.4.

CHAPTER 3

r* ftu 4

♦

*

RESULTS

3.1. MAINTENANCE AND COLLECTION OF PARASITES

3.1.1. Molluscan hosts

Maintenance of Biomphalaria qlabrata stocks proved to be

relatively trouble free. Infected animals survived for an average of approximately one month after the onset of cercarial

shedding which began between 28 - 37 days post infection. Great

difficulties were encountered in the maintenance of Bulinus

tropicus colonies. It was generally observed that the mortality rate of the snails increased and egg production decreased after

approximately 3 months. Consequently, it was decided to abandon the colonies and obtain S.marqrebowiei cercariae from the British

Museum qf Natural History.

3.1.2. Infections

Adequate numbers of miracidia were obtained to maintain an infected snail stock. Yet it was noted, that livers and small intestines from animals of between 6-:9 weeks post-infection

yielded greater quantities of viable miracidia. This was probably related to the level of calcification of eggs that would subsequently increase with time.

#•

m

&

3.1.2.1. Recovery of adult parasites

There was no noticeable difference between the numbers of

parasites recovered using normal or citrated saline. The quantity

of S.mansoni recovered from each host was routinely approximately

10? - 20? of the infection dose (about 200 cercariae per mouse). Recovery of other infections was usually much lower (approx

imately 2 - 1 0 worms per host) but occasionally up to 40 worms

per host were obtained.

3.2. STATISTICAL ANALYSES

All experiments yielding data on the variability of one

parameter against serial increments of another were first tested

for linear or logarithmic relationship using the algebraic form of analysis of variance for testing regression goodness of fit as

described by Clarke (1980) (Table 4). Logarithmic relationships

i.e. log Y = a + bx and Y = a +b logx were tested as outlined in Table 4 by substituting the Y or X values by their logarithms

and retaining the corresponding X or Y values in their original

units. The resulting F values were read from tables of F distribution. If no relationship was established, the differences

between means were compared using the following form of Student*s

t - test (from Clarke, 1980):-

(l^+.l^ - 2 ) = X - Y+ S

N

72

2

t

Table 4

Algebraic form of analysis of variance for determiningregression goodness of fit.

SOURCE OF VARIATION

DEGREES OF FREEDOM

SUM OF SQUARES

MEANSQUARES

TEST

REGRESSION 1 SR MR = SR 1

MR = F(1,N-2)MD

DEVIATION FROM LINE

N-2 SD=S-SR MD = SD N-2

TOTAL N-1 S

5 = (1/N) (M.Zx2 - £ x . £ y )SR - (1 /M) (N.Sxv -Sx.Syl2

(1/N) (N.Ex2 - (£x)2)SO = S - SR

N = number of observations MD = deviation mean of squares

S = total sum of squares MR = regression mean of squares

S2 = sample variance x/y = sample meanSD =s deviation sum of squares FE = sum of observationSR = regression sum of squares FE2= sum squares of observation

The resulting values were compared with tables of t distri

bution. The explanation of the symbols is given in Table 4 .

For all statistical tests, the 95$ probability level was chosen

as the threshold of significance. In analyses involving a time course of measurement, if no linear or logarithmic relationship

could be established, overall changes in levels were determined by

comparing individual mean values against each other using the t -

test.

3.3. CARBOHYDRATE DETERMINATION

3.3.1. GLUCOSE LEVELS

Glucose levels in male S.mansoni showed no overall changes from 42 - 84 days except for a decline (P<0*02) at 70 days post

infection (DPI)(Fig 5). Levels of glucose in female S.mansoni

were similar to the males* from 42 - 70 DPI after which they began

to fall (Fig 5). Glucose levels in male S.marqrebowiei at 42,56 and 84 DPI were statistically similar but the levels at 70 DPI were significantly higher (P<0*05) (Fig 7). The levels of

glucose in female S.margrebowiei at each period post infection

were not statistically different (Fig 7). Glucose levels in male worms were significantly lower than in females at 56 and 84 DPI (P«= 0»05).

Glucose levels in male S.mansoni were significantly higher (P<0.001) than in male 5.marqrebowiei at each measurement period

by approximately 50%. Differences between the females of both

species were only found at 42 and 70 DPI when levels in S.mansoni were 40% higher.

3.3.2. GLYCOGEN LEVELS

Glycogen levels in male S.mansoni at 42, 70 and 84 DPI

were similar but the level at 56 DPI cPo-s1 statistically higher (P<0*01) (Fig 6). The levels in female S.mansoni were similar at

each period post infection (Fig 6). The glycogen content of male

S.mansoni was approximately 50% higher than in female worms at each period post infection.

The glycogen content of male S.marqrebowiei was stable from

42 - 70 DPI but the level of 84 DPI showed a marked statistical increase (P«= 0*001) (Fig 8). Levels in female parasites (Fig 8)

were similar at 42 and 56 DPI but a significant increase (P«=Q*05)

was noted at 70 and 84 DPI. Glycogen levels in male S.marqrebowiei

were apprqximately 40% higher than in female worms at each period post infection.

Glycogen levels in male S.mansoni were higher than in maleS.marqrebowiei at 56 DPI by approximately 50%. This situation was

threversed by the 84 day when levels in male S.marqrebowiei were

45% higher than in S.mansoni. The glycogen content of female

S.mansoni was approximately 42% higher than in female S.marqrebowiei at 42 DPI. After this point, there were no statistical differences between the two species.

Figure 5

The values represent the means of 10 determinations ±

standard deviation.

Glucose levels in male and female S.mansoni over a period

from 42 -84 days post infection.

Figure 6

Glycogen levels in male and female S.mansoni over a period

from 42 - 84 days post infection.

The values represent the means of 10 determinations ±

standard deviation

(jg g

lyco

gen

/ m

g pr

otei

n ug

glu

cose

/ m

g pr

otei

n

Fig . 5

Fig jj

Figure 7

The values represent the means of 10 determinations + standard

deviation.

Glucose levels in male and female S.marqreboiijiei over a period

from 42 - 84 -.post infection.

Fioure 8

Glycogen levels in male and female S.marqreboiuiei over a period


The values represent the means of 10 determinations + standarddeviation.

Fig -7

* Fig .8

7

3.4. UJET WEIGHT DETERMINATIONS

*

#

©

©

The wet weight of male S.mansoni showed no overall signifi

cant change between 42 - 84 DPI (Fig. 9). A similar situation was

found regarding female S.mansoni (Fig. 9). Male parasites were

approximately 63% heavier than the females at each period post

infection.

The wet weight of male S.margrebowiei showed an overall significant increase from 42 DPI onwards (Fig. 11). Female worms also

increased in wet weight from 42 - 56 DPI after which the level re

mained constant (Fig. 11). The wet weight of male S.marqrebowiei was significantly higher (P«=0*001) than the females’ at each period

post infection by approximately 64%.

The wet weight of male S.margrebowiei was approximately 72% higher than male S.mansoni from 42 - 84 DPI. Female S.marqrebowiei

and S.mansoni showed similar wet weights at 42 DPI, after which,

the former showed a 78% increase over the remainder of the time

course.

3.5. PROTEIN DETERMINATIONS

Protein levels in male S.mansoni showed no statistical change over the 6 week time course (Fig. 10). In contrast, the level in

female parasites showed a significant decline after 56 DPI (Fig. 10).

Up to this point, there was no statistical difference in protein

levels between both sexes, but from 56 DPI onwards, protein in the females fell to about 50% that of the males.

Protein in male S.marqrebowiei showed a significant (P<Q-05) linear decrease with time (Fig. 12). Levels of protein in female

S.marqrebowiei showed no change from 42 - 70 DPI, after which there was a significant decline (P«=0*01) of approximately 40?

(Fig. 12). Protein levels in the male parasites were significantly

(P«= 0*001) higher than in the females by approximately 52?.

Male S.marqrebowiei had higher levels of protein (approximately

63?) than S.mansoni males over 42 - 84 DPI. Female S.mansoni and

S.marqrebowiei showed similar protein levels at 42 DPI but subsequently, the latter were found to have levels approximately 52? higher.

Analysis of the values in Table 5 showed that the Lowry

technique of protein determination was significantly more accurate

(P<0*02) than the Bradford method.

3.6. PROTEIN : WET WEIGHT RATIOS

The ratios in male S.mansoni showed no overall significant

decrease (P< 0*001) between 42 - 84 DPI (Fig. 13). In female

S.mansoni the ratios showed no statistical differences up to 70 DPI (Fig. 13) after which, there was a significant decline (P«=0*001)

of about 45?. The ratios in male S.marqrebowiei decreased significantly (P«=0*001) between 42 - 56 DPI, after which the levels remained constant (Fig. 14). The ratios in female S.marqrebowiei showed an overall decrease with time (Fig. 14).

Figure 9

The values represent the means of 10 determinations ± standard deviation.

liiet weight of male and female S.mansoni over a period from 42 -

84 days post infection.

Figure 10

Protein content of male and female S.mansoni over a period from 42 - 84 days post infection.

The values represent the means of 10 determinations ± standard

deviation.

MS p

rote

in /

wor

m

mg

wet

Wei

ght

/ w

orm

Figure 11


deviation.

Wet weight of male and female S.marqrebowiei over a period from

42 - 84 days post infection.

Figure 12

Protein content of male and female S.marqrebouiei over a period



deviation

pg p

rote

in /

wor

m

mg

wet

wei

ght

/ w

orm

Fig. 1 1

Fig. 12

Table 5

Comparison of Lowry and Bradford protein assay techniques.

♦

4

m

m-

STANDARD LOWRY BRADFORD

20 1 9 + 2 (5) 13 + 0*3 (5)30 2 6 + 2 (5) 21 + 0.4 (5)50 44 + 1 (5) 39 + 1 (5)70 ' 67 + 2 (5) 58 ± 5 (5)

100 96 + C M (5) 90 ± 1 (5)

Values = pg protein + standard error, with the number of

determinations in parentheses.

Fiaure 13

Protein : liiet weight ratios in male and female S.mansoni over a period from 42 - 84 days post infection.


deviation.

Figure 14

Protein : Wet weight ratios in male and female S.margrebowiei

over a period from 42 - 84 days post infection.

The values represent the means of 10 determinations + standarddeviation.

Fig.13

Fig. 14

Figure 15

Total lipid content of male and female S.mansoni over a period


The values represent the means of 10 determinations + standarddeviation

Fig. 15

1600 " i

Days post infection

TOTAL LIPIDS3,7.

Levels of total lipids showed no statistical changes between 42 - 84 DPI in male S.mansoni (Fig. 15), whereas the levels in

female S.mansoni showed an overall decline over the same period

(Fig. 15). Total lipid content in both sexes showed no statistical

difference until 84 DPI when the level in males was 82$ higher than in females.

3.8. ENZYME ANALYSIS

3.8.1. S.MANSONI AND S.MARGREBOWIEI

3.8.1.1. Glycolysis

The activities of glycolytic enzymes of S.mansoni and S.marqre-

bowiei were higher than enzymes of the tricarboxylic acid (TCA)

cycle and hexosemonophosphate (HMP) shunt (Tables 6, 7, 8, 9 and 10).

In addition the glycolytic enzyme activities of male schistosomes

were higher than those of the females (Fig. 6). The phosphoenol-

pyruvate carboxykinase (PEPCK): pyruvate kinase (PK) activity ratios of male and female S.mansoni and S.margrebowiei were 0*1,0»3, 0.01 and 0»002, respectively. The activities of malic enzyme,

fumarase and nucleoside diphosphokinase of both species were either

low or not detectable and lactate dehydrogenase activities were relatively high (Tables 6 and 7). This indicates that lactate

formation is a more important route of energy synthesis than CO2

fixation and the TCA cycle in these parasites. Malate dehydrogenase (OAA - MAL) in males and females of bothnspecies was very active

in comparison with other enzymes (Tables 6 and 7). The alternative terminal glycolytic enzymes i.e. octopine dehydrogenase and alcohol

dehydrogenase were not detectable in either species.

3.8.1.2. TCA cycle

TCA enzymes were more easily detectable in S.marqrebowiei than

in S.mansoni, although isocitrate dehydrogenase was not measurable

in either species (Tables .8 and 9). Citrate synthase-activity in female S.mansoni increased significantly (P<0*05) in the pre

sence of Triton x - 100 (10 pl/ml)(Table 8 ). Pyruvate dehydrogenase activity was not detectable in homogenates of S.mansoni even in the presence of Triton x - 100 (10 pl/ml) and 5 mM MgC^ at protein levels of up to 50 pg/cuvette.

3.8.1.3. HMP shunt

Glucose -6- phosphate dehydrogenase and 6- phosphogluconate

dehydrogenase activities were detectable in both species of

schistosome (Table 10). In addition, transaldolase activity was measurable in S.mansoni homogenates.

3.8.2. ENZYME ACTIVITIES IN OTHER SPECIES

Glycolytic, TCA and HMP enzyme activities in males and females of S.bovis, S.haematobium (S.African strain), S.intercalatum,

Table 6

Glycolytic and associated enzyme activities in male and femaleS.mansoni

ENZYME MALES FEMALES

Phosphorylase a + b 64 ± 0 (3) 92 + 13 (3)Hexokinase 130 + 19 (3) 89 + 11 (3)Phosphoglucomutase 1113 + 59 (3) 388 ± 13 (4)Glucosephosphate isomerase 49 + 3 (4) 12 + 2 (4)Phosphofructokinase 10 + 1 (4) 40 ± 10 (4)Aldolase 466 + 23 (4) 139 + 14 (4)Glycerol-3-phosphatedehydrogenase 442 ± 48 (3) 361- + 39 (3)Triosephosphate isomerase 5811 ± 114 (4) • 2063 + 458 (4)Glyceraldehyde-3-phosphatedehydrogenase 183 ± 15 (4) 56 ± 2 (4)Phosphoglycerate kinase 459 + 42 (4) 108 ± 6 (4)Phosphoglycerate mutase 3645 + 216 (4) 2475 ± 50 (4)Enolase 2162 + 291 (3) 1775 + 268 (3)Pyruvate kinase 370 ± 4 (7) 182 + 34 (3)L-lactate dehydrogenase 9314 + 1291 (6) 610 ± 100 (5)

Phosphoenolpyruvatecarboxykinase 37 + 6 (10) 58 ± 4 (10)Alcohol dehydrogenase n.d. n.d.Octopine dehydrogenase n.d. n.d.Malate dehydrogenase (OAA-MAL) inCDCD<r + 238 (5) 957 ± 101 (5)Malic enzyme (MAL- PYR) n.d. n.d.Malic enzyme (PYR-MAL) n.d. n.d.Nucleoside -5'- diphosphokinase 34 ± 2 (4) 32 ± 1 (4)Glutamate-oxaloacetatetransaminase 17 ± 3 (3) 32 ± 5 (3)n.d. = activity not detectable

Results are expressed as nmoles product formed/min/mg protein + standard error. Figures in parenthesis represent the number ofdeterminations.

Table 7

Glycolytic and associated enzyme activities in male and female

5.margreboudei.

4ENZYME MALES FEMALES

*

*

Phoshorylase a + bHexokinasePhosphoglucomutaseGlucosephosphatsPhosphofructokinaseAldolaseGlycerol-3-phosphate dehydrogenase Triosephosphate isomerase Glyceraldehyde-3-phosphate dehydrogenase Phosphoglycerate kinase Phosphoglycerate mutase EnolasePyruvate kinase L-lactate dehydrogenasePhosphoenolpyruvate carboxykinase Alcohol dehydrogenase Octopine dehydrogenase Malate dehydrogenase (OAA-MAL) Malic enzyme (MAL- PYR)Malic enzyme (PYR-MAL)

11146

±±

0-01 3) 15 (3)

2414

++

0-01 (3) 67 (3)

1086 ± 100 (3) 776 ± 97 (3)4546 ± 366 (3) 2319 + 118 (3)1464 ± 35 (3) 561 + 44 (3)

888 ± 79 (3) 448 + 18 (3)325 + 10 (3) 178 + 9 (3)

3698 ± 876 (3) 2789 + 641 (3)1292 + 56 (3) 959 + A0 (3)213 + 1 (3) 22 + 1 (3)

1449 ± 333 (3) 1264 + 269 (3)256 ± 30 286 ± 24 (3)155 + 10 (4) 577 + 151 (3)2056 + 318 (it) 2004 ± 401 (4)

2 + 1 (4) 1 + 0*2 (4)n,.d. n.,d.n,,d. n.,d.

712 ± 165 (5) 628 + 106 (5)n,,d. n,.d.n,,d. n..d.

n.d. = activity not detectable

Results are expressed as nmoles of product formed/min/mg protein + standard error. Figures in parentheses represent the number ofdeterminations

#

Table 8

♦

Tricarboxylic acid cycle enzyme activities in male and female

S.mansoni.

#

ENZYME MALES FEMALES

Pyruvate dehydrogenase n.d. n.d.(6)Citrate synthase 2 + 1 (6) 2 ± 1

Citrate synthase + Triton x - 100 - 5 + 0*1 (3)Aconitase ^ 2 ± 1 (4) 11 ± 2 (4)Isocitrate dehydrogenase (Mi?,NAD)

" " (Mq ,NADP)n.d. n.d.n.d. n.d.

,f " (Mn,NAD) n.d. n.d." " (Mn,NADP) n.d. n.d.

2-0xoglutarate dehydrogenase n.d. n.d.Succinate dehydrogenase (SUCC - FUM) n.d. n.d.Succinate dehydrogenase (FUM -SUCC) n.d. n.d.Fumarase n.d. n.d.Malate dehydrogenase (MAL - OAA) 31 + 4 (4) 29 + 9 (4)


= assay not performed

Results are expressed as nmoles of product formed/min/mg protein

+ standard error. Figures in parentheses represent the number of

determinations

Table 9

Tricarboxylic acid cycle enzyme activities in male and female

S.marqrebouiei.

*

m

♦

ENZYME

Citrate synthase AconitaseIsocitrate dehydrogenase (Mcf,NAD)

" n (Mq,NADP)" " (MiT,NAD)

2-Qxoglutarate dehydrogenase Succinate dehydrogenase (SUCC - FUN) Succinate dehydrogenase (FUM-SUCC) FumaraseMalate dehydrogenase (MAL- OAA)

MALES FEMALES

10 ± 1 (5 ) 25 + 5 (4 )0.2 + 0 *03 (3) 0-2 ± 0-1 (3 )

n,,d. n,,d.n,,d. n,,d.n,.d. n,,d.

12 + 11 (3) 17 + 12 (3 )a + 1 (3 ) 10 + 1 (3 )

0*2 ± o.1 (3 ) 0-3 0-1 (3 )1 + 0 «2 (3) 2 + 0*2 (3 )

13 ± 3 (5 ) 17 + 5 (4 )


Results expressed as nmoles product formed/min/mg protein +

standard error. Figures in parentheses represent the number of

determinations.

*

m

Table 10

Hexosemonophosphate pathway enzyme activities in male and female

S.mansoni and S.margrebowiei.

S.MANSQNI S .MARGREBOUilEI

ENZYF1E MALES FEMALES MALES FEMALES

Glucose-6-phosphate dehydrogenase 82 + 15 (A) 3 5 + 2 (4) 166 + 7 (3) 126 + 1 (3)6-Phosphogluconate dehydrogenase 83 ± 14 (4) 9 + 2 (3) 3 7 + 2 (3) 37 + 1 (3)

Transaldolase 2 2 + 3 (4) 22 + 1 (A) - -

- = assay not performed.

Results are expressed as nmoles product formed/min/mg protein + standard error. Figures in

parentheses represent the number of determinations.

Table 11

Enzyme activities in male S.bovis, 5.haematobium, S.intercalatum,

S.japonicum and S.leiperi.

Results are expressed as nmoles product formed/min/mg protein +

standard error. Figures in parentheses represent the number of

determinations

ENZYME S.BOVIS

Glucosephosphate isomerase Triosephosphate isomerase Glycerol-3-phosphate dehydrogenase Pyruvate kinase L-lactate dehydrogenase

5 + 1 (7) 785 + 110 (7) 335 + 10 (7)

Phosphoenolpyru\/ate carboxykinase Malate dehydrogenase (OAA-MAL)

" n (ma l-o a a)Citrate synthaseIsocitrate dehydrogenase (Mg,NAD)

" " (Mg,NADP) " " (Mn,NAD)" " (Mn,NADP)

2-0xoglutarate dehydrogenase Fumarase

3 3 + 4 (7) 4 7 + 5 (6) 16 + 1 (5)

n.d. n.d. n.d. n.d.

Glucose-6-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase

147 + 18 (7) 3 7 + 6 (5)

n.d. = activity not detectablei

= assay not performed.

u Species originated from Durban, South Africa.

f 0-ip *

S. HAEMATOBIUM# S. INTERCALATUM S. J APONICUM

- - 24 + 1 (3)- - 60 + 1 (3)- 82 + 20 (6) -

• 11 (6) 5419 + 447 (7) 486 + B7 (3); 87 (0) 4925 + 566 (7) 797 + 25 (3)

- - 20 + 3 (3)- 2727 + 194 (7) 818 + 41 (3)- 44 + 5 (5) 26 + 5 (3)- 64 + 6 (5) n,.d.

n.d. 3 + 1 (3) -

n.d. n,,d. -

n.d. n,,d. -

n.d. n,.d. -

- - 5 + 0*3 (3)- - n,,d.- 62 + 2 (3) 59 + 4 (3)- 39 + 1 (3) 0 + 2 (3)

S.LEIPERI

n.d.1355 ± 480 (3)2524 + 879 (3)2284 + 977 (3)

1 8 + 4 (3) 1 7 + 3 (4)

n.d. n.d. n.d. n.d.

44 + 16 (4) 11 + 1 (4)

Table 12

Enzyme activities in female S.bouis, 5.haematobium, S.intercalatum,

5,.japonicum and S.leiperi.

Results are expressed as nmoles product formed/min/mg protein +

standard error. Figures in parentheses represent the number ofdeterminations

ENZYME S.BOVIS

Glucosephosphate isomerase Triosephosphate isomerase Glycerol-3-phosphate dehydrogenase Pyruvate kinase L-lactate dehydrogenase Phosphoenolpyruvate carboxykinase Malate dehydrogenase (OAA - MAL) Malate dehydrogenase (MAL - OAA) Citrate synthaseIsocitrate dehydrogenase (McnNAD)

" " (Mg,NADP)" " ( m , M Q )

" " (Mn,NADP)2-Qxoglutarate dehydrogenase FumaraseGlucose-6-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase

3 + 0-2 (7)403 + 96 (6)258 + 5 (7)23 + 2 (7)65 + 13 (4)35 + 5 (4)

n«.d.n,,d.n,,d.n.,d.

113 + 24 (5)90 ± 15 (5)

n.d. = activity not detectable.

= assay not performed

* Species originated from Durban, S.Africa

* ♦

S.HAEMATOBIUM* S.INTERCALATUM S.JAPONICUM S.LEIPERI

- 191 + 42 (3)416 + 82 (3) 2405 + 435 (3)38 ± 12 (3) 2775 + 255 (3)

_ 1278 + 75 (3)- 47 ± 2 (3)- 31 + 3 (3)- n.d.- n.d.- n.d.— n.d.—

74 + 3 (3)- 33 + 3 (3)

19 + 1 (3) _

52 + 2 (3) -

- n.d.480 + 80 (3) 1308 + 460 (3)180 + 17 (3) 1796 + 597 (3)25 + 3 (3) -

739 + 61 (3) 1737 + 562 (3)26 + 5 (3) 29 + 3 (3)

n,,d. 24 + 17 (3)- n,,d.- n,.d.- n,.d.- n.,d.

7 + 1 (3) -

n..d. -

54 hh 3 (3) 46 + 3 (3)12■ it 1 (3) 11 + 2 (3)

5.japonicum and S.leiperi showed similar relationships to those

observed for S.mansoni and S.mararebowiei (Tables 11 and 12).

The malate dehydrogenase activities (OAA - HAL) in all species except S.bovis were high in comparison u/ith other enzymes (Tables

11 and 12). The lactate dehydrogenase activities of male and

female S.bovis and 5.haematobium were lower than the activities

of the same enzyme from other species. Isocitrate dehydrogenase

activity was only detected in male S.intercalatum (Table 11) and

glucose -6- phosphate dehydrogenase measurable in all species

investigated.

3.9. METABOLITES

3.9.1. ANALYSIS OF METABOLITES OF MOUSE LIVER

The comparative levels of selected metabolites assayed by spectrophotometry or fluorimetry showed no significant differences

(Table 13). The levels of fructose -1, 6- bisphosphate (FBP) and

pyruvate were significantly lower (P<0*02) than those published

by Williamson and Brosnan (1974) but the levels of phosphoenol-

pyruvate (PEP) and 2- phosphoglycerate (2 - PG) were found to be significantly higher (P«=0*01j.

3.9.2. ANALYSIS OF STANDARD METABOLITE SOLUTIONS

Table 14 shows the results of spectrophotometric and fluorimetric assay methods for measuring known concentrations of selected

Table 13

Comparison of metabolite levels in mouse liver measured by

spectrophotometry and fluorimetry.

METABOLITE SPECTROPHOTOMETRY FLUORIMETRYWILLIAMSON # & BROSNAN

Fructose - 1, 6-bisphosphate 7 + 3 (3) 5 ± 1 (3) 24

Pyruvate 152 + 5 (3) 145 + 4 (3) 170

Phosphoenolpyruvate 273 + 18 (3) 224 + 12 (3) 110

3-Phosphoglycerate 263 t 8 (3) 270 + 3 (3) 220

Values = nmoles/g wet weight + standard error, with the number of determinations in parentheses.

* = Data taken from Williamson & Brosnan (1974).

Table 14

Comparison of standard solutions of metabolites measured by

spectrophotometry and fluorimetry.

METABOLITE STANDARD SPECTROPHOTOMETRY FLUORIMETRY

Oxaloacetate 140 145 + 15 (3) 137 ± 4 (3)Pyruvate 120 123 ± 1 (3) 119 + 4 (3)2-Phosphoglycerate 100 97 + 3 (3) 103 + 6 (3)

Values = nM + standard error, uith the number of determinations in parentheses.

metabolites. No significant differences were found between the

data obtained using both techniques.

3.9.3. ANALYSIS OF PARASITE METABOLITE LEVELS

3.9.3.1. S.mansoni

Table 15 shows the metabolite levels in male and female

S.mansoni using a combination of fluorimetric and spectrophoto-

metric assays. All the glycolytic intermediates investigated

were detected and L-lactate was by far the most abundant metabolite

detectable in both sexes of schistosome. Of the TCA cycle intermediates only oxaloacetate (OAA) was detectable. OAA levels in

male worms were significantly higher (P«=0*02) than in females. Similarly, ATP levels were far higher in males than in females

(P«0»01) but AMP levels were higher in the females (P«=0*Q2).The adenylate charge and ATP:AMP ratios were both higher in male

parasites than in females (Table 17).

3.9.3.2. S.marqrebowiei

All the glycolytic intermediates investigated in S.marqrebowiei were detected except dihydroxyacetone phosphate (DHAP) in the

females (Table 16). L-lactate levels were relatively high and

similar in both sexes. OAA was the only TCA cycle metabolite

measurable and levels were significantly higher (P«=0#01) in the males. Similarly DHAP and 3- phosphoglycerate (3-PG) levels were also higher in male parasites. ATP, ADP and AMP levels were not

Table 15

Metabolite levels in male and female S.mansoni.

METABOLITE MALES

Fructose -1, 6-bisphosphate 23 ± 7 (11)Dihydroxyacetone phosphate 9 + 1 0 )Glyceraldehyde-3-phosphate 5 + 1 (8)3-Phosphoglycerate 33 + 9 (5)2-Phosphoglycerate 54 ± 12 (5)Phosphoenolpyruvate 26 + 7 (10)Pyruvate 31 + 9 (12)Lactate# 5553 + 680 (6)

DL-Isocitrate n.d.

2-0xoglutarate n.d.

Oxaloacetate 1894 + 385 (5)

ATP# 2184 + 119 (3)

ADP# 529 + 420 (4)AMP# 14 ± 1 (3)

Values = nmoles/g wet weight + standard error, with determinations in parentheses.

# = metabolites assayed by spectrophotometry.

50 + 18 (4)

88 + 33 (4)

3 + 3 (5)

2 0 + 3 (3)32 + 10 (4)

1 8 + 2 (4)

33 + 12 (4) 4247 + 1544 (6)

n.d.

n.d.

4 5 + 7 (3)

631 + 23 (3)

426 ± 16 (3) 179 + 17 (3)

the number of

FEMALES

n.d = not detectable.

Table 16

Metabolite levels in male and female 5.margrebowiei.

METABOLITE MALES FEMALES

Fructose -1, 6-bisphosphate 14 + 4 (5) 28 + 11 (3)Dihydroxyacetone phosphate 72 + 26 (3) n,>d.Glyceraldehyde-3-phosphate 7 ± 3 (3) 2 + 0*2 (3)3-Phosphoglycerate 59 + 9 (3) 4 + 1 (4)2-Phosphoenolpyruvate 32 + 12 (6) 8 + 3 (3)

Phosphoenolpyruvate 11 + 3 (4) 6 ± 1 (3)Pyruvate 10 + 6 (5) 12 + 5 (3)Lactate # 480 + 280 (6) 876 + 472 (6)DL-Isocitrate n.-d. n,.d.2-0xoglutarate n,.d. n.>d.Oxaloacetate 677 + 185 (5) 6 + 2 (4)

Values = nmoles/g wet weight + standard error, with the number of

determinations in parentheses,

# = metabolite assayed by spectrophotometry.n.d = not detectable

Table 17

Adenylate charge and ATP : AMP ratios in S.mansoni

MALES FEMALES

Adenylate charge 0*89 0*70

ATP : AMP 156 3*53

Adenylate charge = [ATP] + 2 [AD§[AMP + [ADP] + [A TPJ

measured in S.marqrebowiei due to lack of material.

A number of interspecific differences were revealed in this

study. L-lactate and QAA levels were significantly higher ((P**0*001) and (P<0*02) respectively) in male S.mansoni than in male

5.marqrebowiei. However, the latter contained higher levels of DHAP (P<0*05). Female S.mansoni contained higher levels of OAA

(P-s=0«01), DHAP (P«=cO*OT) and PEP (P<CU01) than female S.marqreboujiei.

3.9.4. Hass action ratios

The available data was used to calculate the mass action ratios

(MAR), of a few glycolytic enzymes in S.mansoni and S.marqrebowiei.

The MAR is the ratio of concentrations of products:reactants in

a given reaction (Price & Stevens, 1982). The MAR: equilibrium

constant ratios for both male and female S.mansoni PKs was 1 x 10"^ and so according to Rolleston (1972) the enzymes are catalysing

non-equilibrium reactions in vivo. The other enzymes i.e. aldolase, triosephosphate isomerase, 2-phosphoglycerate mutase and enolase

were, as expected, regulating equilibrium reactions.

3.1Q. INCUBATIONS

3.10.1. S.MANSONI

3.10.1.1. Glucose uptake and lactate productionof L-lactate produced by female and paired S.mansoni

was not significantly different from the amount.of glucose con

sumed from the medium and endogenous sources (Fig. 16). The

glucose consumed:lactate^produced (G:L) ratios were 1:1 *0 and

1:0.9 respectively. In single males, lactate production was

significantly greater than glucose consumption (P«= 0*001) and

the G:L ratio was 1:2*2 (Fig. 16). The comparative levels of

glucose consumption over the 6h incubation period were in the order; pairs, single females (P<0*05) single males (P< 0*002)

and the comparative levels of lactate production were in the order pairs, single females (P«=0*02) single males (P<0*001) (Fig.16). The initial incubation pH level of 7*5 fell to an average of 7*2

+ 0*1(9 determinations) after the experiment.

3.10.1.2. Metabolite levelsAlanine, acetate, citrate, ethanol, glycerol, D-lactate,

oxaloacetate, pyruvate, propionate and succinate were not detectable in incubation samples. The only endogenous metabolites

detectable were glucose, glycogen, glycerol and L-lactate (Figs.

17 and 18). Endogenous glucose and glycogen levels in female S.mansoni showed no significant change during the incubation but

levels in males showed a marked reduction of 56$ (P<0*05) and 55$ (P<0*001) for glucose and glycogen respectively (Fig. 17).

Glycerol and L-lactate levels in female S.mansoni did not change

during the incubation (Fig. 18). A similar situation was found

with glycerol levels in male S.mansoni but the quantities of

endogenous L-lactate declined by 82$ (P-=0*001).

143.10.1.3. COq productionPaired and single S.mansoni liberated ^ C 02 from D - U -

Figure 16

Glucose consumption from the medium and L-lactate production

by S.mansoni during 6h incubation. The values represent the

means of 9 determinations + standard error. The data represents

the values obtained after correcting for changes in endogenous

metabolite levels and controls.

Figure 17

Endogenous glucose and glycogen levels in S.mansoni before and

after 6h incubation. The values represent the means of 9

determinations + standard error.

Fig. 16

Glucose consumption | | L-lactate production urn

FigOZ

Before incubation! 1 After incubation\//A

Glucose ^ Glycogen

Figure 18

Endogenous L-lactate and glycerol levels in S.mansoni before

and after 6h incubation.The values represent the means of 9 determinations + standarderror

nmol

es /

mg

prot

ein

Fig. 18

♦

%

Before incubationj 1 After incubation (ZZZ

1 2 0 -

80-

40-

$

L-lactate Glycerol

♦

Figure 19

CO2 production by S.mansoni and mouse blood during 6h incubation.

The values represent the means of 6 determinations, accounting for

controls, + standard error. The data is expressed in terms of the

amount of glucose that would be consumed if the TCA cycle alone

is operating.

14

Figure 20

14D - U - C glucose incorporation into the tissues of S.mansoni during 6h incubation.

The values represent the means of 6 determinations, accounting

for controls, + standard error.

<#* 4ft-

nmoles glucose incorporated / mg protein

Fig.20

Mouse blood

w 0 04Ji>

6r§!d

glucose (Fig. 19). Single male parasites produced significantly / > 1Amore (P<001) CC^ than single females or paired worms. Paired

14worms and single females showed no difference in levels of CC^

production (Fig. 19). House whole blood controls also produced14 / \low levels of CC^ during incubation (Fig. 19).

3.10.1.4. Glucose incorporation14Single male and female S.mansoni incorporated D - U - C

glucose into body tissues with the males showing higher levels of incorporation (P<0*002) than the females (Fig. 20).

3.10.2. S.MARGREBOUJIEI

3.10.2.1. Glucose uptake and lactate production

The quantity of L-lactate produced by single and paired worms

was significantly different (P<0«02) from the amount of glucose

consumed. Lactate production in single males was greater than

glucose consumption with the G:L ratio being 1:1*7 (Fig. 21).

In contrast, single females and paired worms consumed more glucose

than they produced lactate and the G:L ratio was 1:0*7 for both

groups (Fig. 21). The comparative order of glucose consumption during incubation was as follows :- single females and paired

worms, single males (P<0*01) and the order for lactate production was:- single males, paired worms (P<0*05). There was no statistical difference in lactate production between single males and females or single females and paired worms. The initial pH level

Figure 21

Glucose consumption from the medium and L-lactate production

by S.marqrebouiiei during 6h incubation.The values represent the means of 3 determinations + standard

error. The data represents the values obtained after correcting

for changes in endogenous metabolite levels and controls.

Figure 22

Endogenous glucose and glycogen levels in S.marqrebouiiei before

and after 6h incubation.The values represent the means of 3 determinations + standard

error.Vfr GnS'yc&cyZ-'A, vJc\Wv\\c„

pmol

es /

mg

prot

ein

Hm

oles

/ m

g pr

otei

nFig.21Glucoseconsumption

L*lactateproduction EZ2

Fig.22

Before incubationl | After incubationT 7 7 7 \,

<? ? Cf $

Glucose * Glycogen

Figure 23

Endogenous L-lactate levels in S.margrebowiei before and after

6h incubation.


error.

Figure 24

14 .CO2 production by S.marqrebowiai during 6h incubation.The values represent the' means of 3 determinations minus controls,

+ standard error. The data is expressed in terms of the amountof glucose that would be consumed if the TCA cycle alone is

operating.

(+

nmoles glucose consumed / mg protein

Fig. 24

♦

nmoles / mg protein

CD<Do-ICD=3OcCT 0)o'3

Fig.23

102

*

*

w

of the medium (7*45) fell to an average of 7*1 + 0-4 (3 determin

ations) after incubation.

3.10.2.2. Metabolite levelsAnalysis of the incubation medium failed to detect any

metabolites except glucose and L-lactate. Moreover, glucose,

glycogen and L-lactate were the only endogenous metabolites detectable (Figs. 22 and 23). Glucose levels in single males and

females showed no significant changes during incubation. A

similar situation occurred with glycogen levels in single male

worms but the females showed a significant increase (Fig. 22).

Endogenous lactate levels in male worms remained constant but the

females showed a significant decrease (P<0*01) during incubation

(Fig. 23).

143.10.2.3. Co-production14 14Each experimental group produced CO^ from the D -U - C

glucose in the medium and the comparative levels of production were single females, single males and paired worms (P<0*01) (Fig.24).

3.10.3. S.HAEMATOBIUM


In single male and female worms, glucose consumption was

significantly higher ((P<-0*02) and (P<0*001) respectively) than L-lactate production and the respective G:L ratios-were 1:0*4 and

1:0*5 (Fig. 25). Glucose depletion by female worms was higher

than for males (P«=0*01) and the same occurred with lactate production (P<0*05) (Fig. 25). The initial pH of the medium (7*6)

fell to an average of 7 + 0*2 (2 determinations) after incubation.

3.10.3.2. Metabolite levels

The end product metabolites assayed in the medium in addition

to L-lactate were succinate, pyruvate and acetate. The latter

was detectable in low but similar levels in male and female worms

(Fig. 25). Glucose and glycogen were the only endogenous meta

bolites assayed. Glucose levels in male and female parasites

increased significantly during incubation (P<0*05) (Fig. 26), whereas no changes were observed in the glycogen levels of either

sex (Fig. 26).

3.10.4. S.douthitti


Female parasites were not available for study as the material

was obtained from a unisexual infection. L-lactate production in single male S.douthitti was significantly higher (P<0*02) than glucose consumption and the G:L ratio was 1:2*4 (Fig. 27).

3.10.4.2. Metabolite levelsEndogenous glucose levels remained constant during incubation

but glycogen was completely depleted (Fig. 28). Tissue levels

Figure 25

Glucose consumption from the medium, L- lactate and acetate

production by S.haematobium (Morroccan strain) during 6h

incubation.


error. The data represents the values obtained after correcting for changes in endogenous metabolite levels and controls.

Figure 2G

Endogenous glucose and glycogen levels in S.haematobium (Morroccan)

before and after 6h incubation.


error.

£iSi25

(f ? cf $

Fig.26

Before incubationL___ l After incubation E Z 2

d 1 £ d 1 2

Glucose * Glycogen

Figure 27

1AGlucose consumption from the medium,L-lactate and CO^

production by male S.douthitti during 6h incubation.


error. The data represents the values obtained after correcting14for changes in endogenous metabolite levels and controls. CC^

production is expressed in terms of the amount of glucose that

would be consumed if the TCA cycle alone operating.

Figure 28

Endogenous glucose, glycogen and L-lactate levels in male

S.douthitti before and after 6h incubation.


error.

pmol

es /

mg

prot

ein

Fig.27

production

Fig.28

Before incubation f I After incubation V / A

Glucose Glycogen L-1 act ate

of L-lactate did not change significantly during incubation (Fig, 28)

D-lactate, alanine, acetate, glycerol, pyruvate, propionate and

succinate were undetectable in tissue or media samples.

143.10.4.3. COq productionMale S.douthitti produced low levels of ^CO^ from D- U -

glucose during incubation (Fig. 27).

3.10.5. Interspecific comparisons5.haematobium males and females consumed the most glucose

during 6h incubation followed by S.marqrebowieir S.mansoni and

S.douthitti. Exogenous glucose appeared to be used in preference

to endogenous sources as there were no large depletions in glycogen

except with S.douthitti. In each case, female schistosomes pro-14duced at least as much lactate as their male counterparts. CO2

was produced in each species investigated hence suggesting thatsome oxidative pathway is functioning but the levels indicate

that the pathway is operating at a low rate. The mouse blood14controls also produced CC^ but not enough to seriously interfere

with the experiment. The levels of mouse blood contamination

will be even lower in the actual experimental tubes as the

number of worms used would contain smaller volumes of gut contents.

Radiolabelled glucose was incorporated into worm tissue during incubation thus suggesting that glucose is not solely

used for catabolic processes. The pH levels of the media also

remained within those described as being optimal by Bueding (1950).

Overall it was found that male parasites were using almost all

their available glucose for glycolysis but the females were channelling a large proportion (approximately 50%) of their

glucose into alternative pathways.

3.11. KINETIC STUDY OF PYRUVATE KINASEIt was decided to draw in lines of best fit by 11 eye" for

all pyruvate kinase (PK) saturation curves and to rely on logarithmic or inverse transformations to derive Hill Co-efficients

(HCo) and apparent Km (K1) and Umax (^m) values.

3.11.1. S.MANSQNIThe saturation curves for male and female enzymes, were

sigmoidal and rectangular hyperbolic respectively (Fig. 29); the data transformed to Hill plots is shown in Eig. 30. The K! and

HCo values were determined as set out in Fig. 30 and are shown

in Table 18. The respective HCo values for male and female PK were 1*20 and 0»96 thus according to Price & Stevens (1982) and

Lehninger (1977) the enzymes exhibited positive and negative

co-operativity respectively. Yet, as the HCo of the female enzyme was nearly 1 (i.e. denoting normal Michaelis/Menten

kinetics) the data has been transformed to a Woolf (Hanes) plot

(Price & Stevens, 1982) (Fig. 31). The K! value for male PK was lower than that of the females (Table 18), indicating that the

male enzyme has a higher affinity for phosphoenolpyruvate (PEP). The Vmax for the male enzyme was also higher than that for the

Figure 29

The effect of PEP concentration on the activity of pyruvate kinases from male and female S.mansoni.The values represent the means of 6 determinations + standard

error

*

♦

*

♦

T1

14

Figure 3D

Hill plot of the effect of PEP concentration on the activity

of pyruvate kinases from male and female S.mansoni.

Slope=n=Hill coefficient

Intercept Y =-log. K* = -nlog,o [PEd‘° 50

[PEf = concentration at Vmax/2 50

K* = Apparent Km

110

#

Figure 31

Woolf (Hanes) plot of the effect of PEP concentration on the

activity of pyruvate kinase from female S.mansoni.I

*

#

*

0

[PEP] mM

Intercept X = —Km

Intercept Y = Km/Vmax

%

female (Table 18).

3.11.1.1. ATP inhibition

In male S.mansoni 1nM ATP had no effect on PK activity but

between 1nM - 1|jffl there was a 23? inhibition and between 1pM - 1mM

there was a further 23? inhibition (P<0»001) (Table 19). Simi

larly, female PK is not inhibited by 1nM ATP but there was a

significant decline (P<0*002) of 19? in activity at 1pM. No

further inhibition occurred at 1mM ATP (Table 19), therefore, the

female enzyme is less susceptible to inhibition than male PK over

the entire ATP concentration range but equally as sensitive

between 1nM - 1pM ATP.

3.11.1.2. FBP activation

Male and female PKs both showed increased activity in the

presence of FBP (Table 19). Male PK showed a significant overall

increase of 59? from 1 pM — 25|jM FBP (P<0«001). There was a 10?

decrease in activity from 25jjM - 125pM FBP, suggesting that 25jjM

FBP is the concentration at which maximal activation occurs.

Female PK showed a 54? increase in activity from 0 — 1 jjM FBP but

from 1pM - 25jjM there was no further significant change in activity.

From 25jjM - 50|jM FBP there was a further increase of 35? but there

was no significant change in activity from 50|jM - 125|jM (Table 19).

Thus it appears that an FBP concentration of 50|jM is necessary

for maximal activation of the female enzyme. In comparison with

the male enzyme, female PK required twice the concentration of FBP

to achieve maximal and similar levels of activity. The respec

tive levels of FBP which cause maximal activation are similar to the endogenous levels in the parasites (section 2.9.1.).

3.11.2. S.MARGREBQWIEIThe saturation curves of PKs from males and females were

both sigmoidal (Fig. 32) and the Hill plots are shown in Fig.33.

The male enzyme was approximately twice as active as the female at PEP concentrations from 2mM - 5mM. The K* for male PK was

lower than for female PK and the male enzyme also had a higher

HCo (Table 18). Hence the male enzyme appears to be more positively co-operative and has a higher affinity for PEP than

the female enzyme.

3.11.2.1. ATP inhibition

Male PK showed no significant alteration in activity at

1nM ATP (Table 20). Between 1nM - 1pM there was a significant (P<0*02) 27% decrease in activity and from 1pM - 1mM ATP there

was no further change. Female PK showed no significant change in activity from 0 - 1pM ATP but from 1pM - 1mM there was a

significant (P<0-01) decrease of 41% (Table 20). Therefore,

male PK appears to be inhibited by lower levels of ATP.

3.11.2.2. FBP activation

PK activity of males and females increased in the presence

of FBP (Table 20). Male PK activity was increased by a factor

Figure 32

The effect of PEP concentration on the activity of pyruvate

kinases from male and female S. margrebouiei.

The values represent the means of 4 determinations + standarderror

5000

•5 4000-

bo

^ 3000-cE

(/>0)oEc

2000-

< 1000 -

*

?T

— j------------------------ 1-------------------------- 1

3 4 5

[PEFj mM

•qj|> a #

Figure 33


of pyruvate kinases from male and female S.marqrebouiei.

of 28 from 1nM - 1mM FBP and the activity of the female enzyme

showed a 23 fold enhancement over the same range. The male enzyme

was more significantly activated (P<0*001) over the range InM -

1|#1 than the female enzyme, but in the range - ImM female

PK was more significantly activated (P<=001) than the male enzyme.

3.11.3. 5.JAPDNICUMThe saturation curves of PKs from males and females were

both sigmoidal (Fig. 34) and the Hill plots are shown in Fig. 35.

The HCo and K ! for the male enzyme were higher than those for the

female, thus indicating that the former has a comparatively lower

affinity for PEP and appears to: be more positively co-operative

(Table 18).

3.11.4. S.BQVI5

It was not possible to determine whether the saturation

curve for the male enzyme was a sigmoid or a rectangular hyper

bola (Fig. 36). This was because the lowest recorded activities did not allow the determination of the shape of the lower portion

of the curve. Therefore the available data was transformed to

Hill and Woolf (Hanes) plots (Figs. 37 & 38 respectively) and the derived K* and U’m values shown in Table 18. The K ’ from the Hill plot appeared to be rather high and so the alternative

data was regarded as being more appropriate.

Figure 34


kinases from male and female 5.japonicum.


error.

c0)4—*ov_CLboEc

CO0)oEc

o<

20 0 0 -

1600

1200-

800-

400

4

$ ,

--------------------- ,--------------------------1--------------------------1

3 4 5

[pEP]mM

4 ♦ *

Figure 35


of pyruvate kinases from male and female 5. japonicum.

log [PEI mM

71

1 1 8

Figure 36

The effect of PEP concentration on the activity of pyruvate*kinase from male S.bovis.


error.*

m

*

[PEP] m M

F ig u re 374


of pyruvate kinase from male S.bovis.

4

Figure 38

UJoolf (Hanes) plot of the effect of PEP concentration on the activity of pyruvate kinase from male S.bovis.

•#

4

F ig .3 7

Fig. 38

[P E P ] mM

Table 18

Apparent Km (K!) and Vmax (\y*m) values for the pyruvate kinases

from S.mansoni, 5.marqreboiuiei, S./japonicum, S.bovis and mouse

blood.

S.MANSONI S.MARGREBOUIEI S.JAPONICUM S.BOVIS

Males Females Males Females males Females males mOUSE BLOOD

K* □ •6 1-1 (1.6) 1-1 1-2 2-7 1-5 44 (0*1) 4*5

U‘m (1427) (1796)

\l max 1550 120b 4554 2147 208B 1212 1702 2740

HCo 1-20 0.96 1-73 1-46 1-5 1.4 0-75 0.8

K* and W'm denote the values from Hill or Woolf (Hanes) plots (the latter are shown in parentheses).

HCo = Hill Co-efficient.

K' is expressed in [#1 and V'm and V/max are expressed as nmoles product formed/min/mg protein respect

ively.

Table 19

ATP inhibition and FBP activation of the pyruvate kinases from

male and female S.mansoni

ACTIVITY

EFFECTOR CONCENTRATION (uM) HALES FEMALES

ATP

FBP

0 1027

1 x 10"3 1045

1 810

1 x 103 625

0 255

1 239

12.5 465

25 580

50 520

125 510

+ 22 (6) 460 + 25 (6)

± 48 (6) 450 + 0 (6)

± 14 (6) 365 + 17 (6)

± 10 (6) 386 ± 14 (6)

+ 16 (6) 130 + 11 (6)

+ 26 (6) 280 + 63 (6)

± 8 (6) 275 ± 55 (6)

+ 14 (6) 383 + 23 (6)

± 9 (6) 586 + 71 (6)

+ 5 (6) 572 + 32 (6)

Enzyme activity is expressed as nmoles product formed/min/mg

protein + standard error. Figures in parentheses represent the

number of determinations.The PEP concentrations used in the presence of ATP and FBP were 1mM and 0*1 mM respectively.

Table 20

ATP inhibition and FBP activation of the pyruvate kinases from

male and female S.marqreboudei«

ACTIVITY

EFFECTOR CONCENTRATION (uM) HALES FEMALES

ATP

FBP

0 772 ± 13 (A) 297 + 17 (A)1 x 10-3 749 + 2 (4) 309 + 14 (4)

1 546 ± 59 (4) 274 + 26 (4)

1 x 1Q3 3 9 9 + 1 2 (A) 161 + 7 (A)

0 5 7 + 4 (4) 52 CD+1

0*001 ■7A ± 10 (A) 57 1 + CD -O

1 719 ± 23 (A) 85 1 +

1000 2044 ± 25 (4) 1357 + 86 (4)

Enzyme activity is expressed as nmoles product formed/min/mg protein

+ standard error. Figures in parentheses represent the number of

determinations.

The PEP concentrations used in the presence of ATP and FBP were

1mM and 0*1mM respectively.

123

*

♦

4

3.11.5. Interspecific comparisons

All the enzymes studied exhibited positively co-operative

characteristics with the exception of those from female S.mansoni and male S.bovis (Table 18). The PK from male S.marqrebowiei was the most co-operative and active enzyme, although the PK from

male S.mansoni showed the greatest affinity for PEP. The maximum

specific activities of the female enzymes were lower than the males for each species examined.

3.11.6. PYRUVATE KINASE ACTIVITY UNDER "PHYSIOLOGICAL" SUBSTRATE

AND MODULATOR CONCENTRATIONS.

The approximate "physiological” levels of FBP, PEP, ATP and

ADP in both series of S.mansoni were calculated using the metabolite

data given in section 3.9.2. One gram of parasite material was

considered to be equivalent to 1ml of water, thus allowing the in vivo molarity of the metabolites to be calculated.

The K' and V' values for male and female PK were calculated

from Lineweaver/Burk transformations (Conn & Stumpf, 1976) as follows:-

1. Absence of activator/inhibitor : Intercept X axis = - 1Km

2. Competitive inhibition

Intercept Y axis = 1V max

Intercept X axis = ~VKm

Gradient

i-----II ^'hh*'—’ -H

E+

IIITT__

1

KiVmax

[i] = inhibitor concentration Ki = inhibitor constant

3. Uncompetitive inhibition : Intercept X axis = /Km

Intercept Y axis = 1Vmax

The increase in Umax due to FBP was measured using the protocol for competitive inhibition.

3.11.6.1. Enzyme activity in parasite gut contents

It was not possible to quantify the volume of gut contents

present in each sample using the method described in section

2.9.3. The results showed that activity due to gut contents was

low (Table 21). Therefore, only minor contamination of parasite

enzymes by host enzymes will occur.

3.11.6.2. Kinetics of PK from male S.mansoni

Pk activity from male S.mansoni was linear with respect to PEP concentration only under -FBP, -ATP or +FBP, - ATP assay

conditions (Fig. 39). No linear or logarithmic relationship could be established under -FBP, +ATP or +FBP, +ATP conditions.

Lineweaver/Burk transformations were carried out to elucidate

the effects of FBP and ATP on unmodulated enzyme activity (Fig. 41).

; j*Ki

1 +JSLKi

Table 21

Enzyme activity in gut contents of female S.mansoni.

ENZYME ACTIVITY

Pyruvate kinaseGlucose-6-phosphate dehydrogenase

Malate dehydrogenase (MAL-OXO)

Citrate synthase

2 + 0.01 (5)

□•1 + 0-01 (4)

0*02 + 0*001 (4)

n.d.

n.d. = activity not detectable.

Results are expressed in nmoles product formed/min/mg protein

+ standard error. Figures in parentheses represent the number

of determinations

Regression plots, K! and Vfm values produced by -FBP, -ATP

and -FBP, +ATP conditions, did not fit any of the standard inhi

bitor classes i.e. non, un or competitive inhibition (Fig. 41,

Table 22) (Lehninger, 1977). However, statistical comparison of mean activities at each PEP concentration showed that the

addition of ATP produced an overall significant (Pc:0*02) 37% reduction in enzyme activity. +FBP, -ATP and +FBP, +ATP

conditions resulted in regression plots which indicated that ATP

inhibited the erlzyme uncompetitively. In support of this, the calculated values of K* and V'm showed a large decline (Table

21) and subsequent statistical analysis of the untransformed data (Fig. 39) showed a significant (P<0*02) overall 43% re

duction in enzyme activity. The presence of FBP raised the V*m

of the enzyme (Fig. 41, Table 22). This was confirmed by

analysis of the untransformed data (Fig. 39) which showed a significant (P<0*05) 69% increase in activity.

3.11.6.3. Kinetics of PK from female S.mansoni

PK activity in female S.mansoni was linear with respect to PEP concentration under all assay conditions (Fig. 40). The

data was transformed to Lineweaver/Burk plots which were subse

quently fitted with regression lines (Fig. 42). Comparison of PK activity under -FBP, -ATP and -FBP, +ATP, indicated that ATP

exerted an uncompetitive inhibitory effect. However, statistical

comparison of the untransformed data (Fig. 40) showed that ATP did not significantly inhibit PK activity. Comparison of PK

Table 22

Apparent Km (K')» Vmax (V!m) and inhibitor constant (Kl) for the pyruvate kinases from male and female 5«mansoni.

♦ # * *

MALES FEMALES

MODULATOR K' (pM) \ltm KI (pM) k * (urn) V'm KI (pKl)

- FBP - ATP 61 201 28 153

- FBP + ATP 99 475 0.1 22 88 769

+ FBP - ATP 704 1504 20 195

+ FBP + ATP 39 11 90 72

K' = apparent Km.

W'm = apparent maximum velocity (nmoles product formed/min/mg protein).

KI = inhibitor constant.

Figure 41

Lineiueaver/Burk plot of the effect of "physiological" levels of PEP (10 - 50pM), ADP (0-5r#l), FBP (23pl*l) and ATP (2*25mM)

on the activity of pyruvate kinase from male S.mansoni.

Figure 42

Lineueaver/Burk plot of the effect of "physiological" levels of PEP (10 - 50|j|Yl), ADP (0-4r#l), FBP (50pl>l) and ATP (0-6r#l)

on the activity of pyruvate kinase from female S.mansoni.

Fig. 41

■ -F B P -A TP o — FBP+ATP a +FBP—ATP • +FBP+ATP

Fig. 42

b _FBP—ATP o —FBP+ATP a +FBP-ATP *+FBP+ATP

[PEP] pM

Figure 39

The effect of "physiological" levels of PEP (10 - 50^),ADP (O'SmM), FBP (23^) and ATP (2-25mM) on the activity of

pyruvate kinase from male S.mansoni.The values represent the means of 5 determinations +

standard error.

Figure 40

The effect of "physiological" levels of PEP (10 - SOjjM),

ADP (0*4mfl), FBP (50^) and ATP (O'BmM) on the activity of

pyruvate kinase from female S.mansoni.

The values represent the means of 5 determinations +

standard error.

Activ

ity(n

mol

es/m

in/m

g pr

otei

n)

Activ

ity(n

mol

es/m

in/m

g pr

otei

n■ —FBP—A TP o—FBP+ATP a +FBP-ATP • +FBP+ATP

■ — FBP—ATP o -FBP+ATP a +FBP-ATP • +FBP+ATP -

behaviour under +FBP, -ATP and +FBP, +ATP conditions indicated

that competitive inhibition was occurring (Fig. 42). Although

the K! increased, the V'm decreased (Table 22) indicating that

this effect does not strictly fit the standard inhibitor classes

(Lehninger, 1977). Analysis of the untransformed data (Fig. 40)

showed that PK activity was significantly reduced (P<=0*02)

overall by 33?.The addition of FBP alone (+FBP, -ATP) showed that the K ’

was lowered and the M ' m was increased (Fig. 42, Table 22). This

indicates that enzyme activity was increased and statistical analysis of the untransformed data (Fig. 40) showed that activity

was significantly enhanced (P<G»05) overall by 22?.

PK activity in male S.mansoni was significantly (P<0«02)

higher than in females by an average of 65? at all PEP concen

trations except lOpM. At this concentration there was no sig

nificant difference in activity. In the presence of FBP, male

PK was eight times more active than the female enzyme. Male PK

in the absence of FBP, was more significantly (P<0*001) inhi

bited by ATP than the female enzyme. In the presence of FBP the

enzymes from both sexes were inhibited by ATP, to the same degree.

3.11.6.4. Stability and the effect of cations on the activity of PK

The activity of male and female PK in the absence of FBP

and ATP showed no significant changes over 7h- (Fig. 43). Male and female PKs showed no significant alterations in activity when assayed in the presence of Mg’ or Mn" (Figs, 44 & 45).

Figure A3

Stability of pyruvate kinase from mal-e and female S.mansoni

over a 7h period. [PEP] = 50jjm

The values represent the mean of 5 determinations + standarderror

Fig.43

Figure 44

The effect of Mg and Mn on the activity of pyruvate kinase

from male S.mansoni.

The values represent the means of 5 determinations ± standard

error. The concentration of cation used was 5mM.

Figure 45

The effect of Mg and Mn on the activity of pyruvate kinase from female S.mansoni.


error. The concentration of cation used was 5mM.

*

Activity(nmoles/min/mg protein)r—*

CJ1 O? ________£

vx‘-fc*cn

*-0S

I

lAfri

§13d

]

* *

Activity(nmoles/min/mg protein)i-j. |\i CJo o o

Fig. 44

3.11.6.5. Pyruvate kinase from mouse erythrocytes

Using the technique outlined in section 2.9.4. it provedfeimpossibleôbtain a serum sample that was free of haemoglobin.

As other intracellular components may have leaked into the serum

fraction, only the data for mouse erythrocyte enzymes were recorded.

In the case of erythrocyte PK, insufficient data was available to

determine the shape of the graph at lower levels of PEP concentration. Therefore it was not possible to determine whether the

enzyme oo«sexhibiting Michaelis-Menten or sigmoidal kinetics (Fig.46).

The K ’ and HCo for the enzyme are shown in Table 18.

Glucose-6-phosphate dehydrogenase (1955 + 64 nmol/min/ml)

and malate dehydrogenase ( 8 8 + 3 nmol/min/ml) activity was found

in erythrocyte samples but citrate synthase was not detectable.

3.12. ELECTROPHORESIS

Experiments with polyacrylamide gels were not successful as

many samples failed to migrate into the resolving gel. The PK from mouse erythrocytes produced a single band (Plate 1). Starch gels were more productive as two PK isoenzymes consisting of one major and one minor staining band were detected in male and fe

male S.mansoni (Plate 2). In the females, the minor staining

isoenzyme took longer to develop than that from the males. In

the case of cellulose-acetate gels, parasite samples produced

a single major staining band for PK as did the enzyme from mouse

blood (Plate 3).

Figure 46


kinase from mouse erythrocytes.

The values represent the means of 4 determinations + standarderror

♦

Plate 1

Isoenzyme pattern of mouse erythrocyte pyruvate kinase using

polyacrylamide gel electrophoresis.

Plate 2

Isoenzyme pattern of pyruvate kinase from

male and female S.mansoni using starch gel electrophoresis.

Plate 3

Isoenzyme pattern of pyruvate kinase from mouse erythrocytes and

male and female S.mansoni using cellulose-acetate electrophoresis.

Plate.1

Plate.2

Plate.3

Haemoglobin

Erythrocyte PK

Plate 4

Isofocusing patterns of pyruvate kinase from male and female

S.mansoni.

Plate.4

3*8-i

4-3- 4*G“

p H 4-9-

6-2-

7 0 J

cf $ Haemoglobicontrol

3.12.1 Isoelectric focusingIsoelectric focusing rev/ealed that 6 similar isoenzyme bands

were produced by both male and female S.mansoni (Plate 4). 5pl aliquots produced the clearest patterns and these corresponded

to 50pg and 30|jg of protein for male and female samples respect

ively. One major band of activity, with an isoelectric point of

6*2 was evident in both samples and these bands developed at the

same rate.

3.13. CYTOCHROMES

Quantitative calculations of cytochrome levels were perfor

med using the wavelength pair data of Wilson & Epel (1968)

according to the following equation:-

0.D.1 - 0.D.2 x \I = mmoles/ml sample (E) ( U U.F.)(u)

O.D.1/2 = optical density at specific wavelengths (nmetres).

\J = Cuvette volume

E = Extinction co-efficient.

L = Light path (cm).I.F.= Intensification factor (7) (Wilson, 1967).

v = Sample volume.

3.13.1. CONTROLS

3.13.1.1. Whole mouse blood and rat liver

Three minor peaks were found in the citrated blood samples

at 454, 530 and 575 nm (Fig, 47). The scans obtained from rat

liver showed peaks at the expected wavelengths for cytochromes a, a^» c, c and b i.e. 602, 445, 550, 556 and 564 respectively

(Hayes, 1983) (Fig. 48).

3.13.1.2. Digested mouse bloodThe scans of pepsinised mouse blood are shown in Fig. 49.

The areas of scans that overlap, represent the wavelengths that

would be cancelled out in the spectrophotometer during assay i.e. those wavelengths that would not be recorded as an increase

or decrease in absorbance. Therefore it is evident that the

presence of blood digestion products will interfere significantly with any scans between 484 - 680 nm.

3.13.1.3. HaematinThe scans of the haematin solution are shown in Fig. 50.

These results show that there are very slight differences in absorbance between the reduced and oxidised states from 400 -

497 nm and 647 - 700 nm. However, between 497 - 647 nm large differences in absorbance occurred. Therefore, haematin

represents a comparatively larger source of contamination than the digested blood preparation, particularly in the range cover

ing cytochromes c» Oj and b i.e. 550, 556 and 564 nm respectively

3.13.2. S.MANS0NI

Figure 47

Cytochrome scan of whole mouse blood.

Figure 48

Cytochrome scan of rat liver

Abso

rban

ce

Fig. 47

1-1

*

*

454I .....i “ "

530 575

Wavelength(nm)

4-

*•

Fig. 48

556 5 6 4

Wavelength(nm)

Figure 49

Absorbance scan of pepsinised mouse blood.

Abso

rban

ce

* * lyif -(jifc. 9*

Figure 50 4

Absorbance scan of a solution of haematin.

*

4

4

m

Abso

rban

ce

Wavelength(nm)

+ ¥ i I* &-

3.13.2.1. MalesCytochrome peaks were detected at 445, 548, 556 and 564

nm and comparison with Fig. 48 shows these are cytochromes a^»

c, c and b respectively (Figs, 51 & 53). Haematin, although

present, did not prevent the peaks from being recorded. No

peak was recorded at 602 nm suggesting that cytochrome a was not

present but a large increase in absorbance was found at 640 nm

in one sample. Subsequent scans of several different samples

from 600 - 700 nm failed to show similar peaks, so it was

assumed that the first sample included an unknown artefact. Quantitative data for the cytochromes are shown in Table 23 and cytochrome c is the most abundant.

3.13.2.2. FemalesPeaks were detected at 445, 548 and 566 nm thus indicating

the presence of cytochromes a^, c and c^, (Fig. 52). No peaks

were found at 564 nm even in the presence of antimycin A and

succinate, indicating that cytochrome b was not present or levels

were too low to be detected. As with the males, a large peak

was found at 640 nm in one sample but not in others, hence, as

for the male sample, this was presumed to be an artefact. Cytochrome a^ levels were the most abundant, being three times higher

than in the males (Table 23) but levels of cytochromes c and c

were approximately the same in both sexes.

3.14. OXYGEN UPTAKEMale and female S.mansoni both took up oxygen (O2) from the

Figure 51

Cytochrome scan of male S.mansoni

Figure 52

Cytochrome scan of female S.mansoni

Full

scal

e ab

sorb

ance

Fu

ll sc

ale

abso

rban

ce

Fig.51

Fig. 52

Wavelength(nm)

Figure 53

Absorbance scan of cytochrome b from male S.mansoni.

00 p # 0

Full scale absorbance

Table 23

Cytochrome levels in male and female S.mansoni.

CYTOCHROME MALES FEMALES

a 30*06 + 0*001 (6) 0-2 + 0*004 (5)

c 0*14 ± 0*004 (5) 0.1 ± 0*01 (5)

C1 0*07 + 0*001 (5) 0*1 + 0*002 (4)b 0*02 + 0*001 w n.d.

n.d. = not detectable.

Results are expressed as nmoles/mg protein + standard error.

Figures in parentheses represent the number of determinations.

medium but the females took up 8 times as much 02 as the males

(Table 24). The agitation necessary to maintain hyperbaric

conditions did not appear to visibly damage the worms. Males

and females were found to be motile after 30 min, prior to the

addition of KCN. After this point, there was a large decline in the motility of both sexes. O2 uptake by male S.mansoni was inhibited completely by KCN but uptake by the females was only

inhibited by 64%

Table 24

Oxygen uptake by male and female S.mansoni.

MALES FEMALES

- KCN 2 + 0*1 (5) 16 ± 1 (5)

+ KCN 0.0 (5) 5.8 + 0«3 (5)

Results are expressed in jjl 0 ^ consumed/h/mg protein

standard error. Figures in parentheses represent the

number of determinations

CHAPTER 4

#

0

0

DISCUSSION

4.1. GLUCOSE, GLYCOGEN, PROTEIN, WET WEIGHT AND LIPID LEVELS

Both sexes of S.mansoni and S.marqrebouiiei maintained fairly constant levels of glucose between 42 - 84 days post infection.

Glucose was measured relative to protein levels which did change

over the time course of measurement. These alterations were small

and do not affect the overall relationships between glucose levels

in the different species. This highlights the potential problems

of relating metabolite levels to a baseline which changes with time

without assessing the changes. Particular difficulties might be

encountered when comparing values obtained with the data of other workers. Different worm burdens, the species, strain and nutritional status of the host may significantly affect parasite consti

tuents hence making reliable comparisons difficult.The major problem with using wet weight as a baseline is the

difficulty encountered in removing all extracellular water from the parasite (Radke et al, 1957). In addition, the contribution of

parasite gut contents to the measurements would be extremely difficult to account for. Presumably, the gut contents are likely to

cause greater variation with female measurements as these generally

contain more digested blood than the males. Howeveiv the latter

could also retain additional water in the gynaecophoric canal. Dry

weight measurements of samples would serve as a more reliable base-,

line but would preclude the determination of labile components in

the same sample. Expression and comparison of quantities on a

single worm basis are also beset by the same problems. Therefore

the use of protein as a baseline appears to be the most reliable

means of comparing the data of different researchers.

Glycogen levels are higher than those for glucose in both S.mansoni and S.marqrebowiei. These levels remain stable over the

6 week measurement period with the exception of male S.marqrebowiei.

These observations are in keeping with the findings of Bueding & Koletsky (1950). Male levels of glycogen were higher than those of

the females in both species which is also in agreement with the work

of Bueding (1950). As male worms are more mechanically active in vivo

than the females, the higher levels of glycogen are probably essential to meet energy demands. The larger glycogen stores in

the males may also be accessible to the female parasites via a glucose

transfer mechanism (Cornford & Huot, 1981). This may be another aspect of the nutritional dependance of the female on the male

(Senft, 1968) and indicates a division of metabolic labour between the functional schistosome pair.

Smyth (1966) proposed that the best means for assessing tissue

growth is on a weight basis. Therefor^ the present study indicates

that S.mansoni had reached a stable growth level by 42 days post infection, whereas S.marqrebowiei continued to grow after this time.

Female S.marqrebowiei appear to plateau off at about 56 days but

the males increased their weight up to 84 days post infection. This

increase may be linked with the synthesis and storage of glycogen

in this parasite mentioned earlier. Very little is known concerning glycogen synthesis in schistosomes, therefore male S.marqrebowiei may serve as a useful model for future studies in this area.

Cornford et al (1982) found that protein levels in female S.mansoni declined after 98 days post infection. This is thought

to coincide with the chronic phase of infection (Cheever et al, 1980)

and hence the fall in protein levels is thought to be linked with the host^ immune response. In the present study, all but male

S.mansoni showed a decline in protein levels so indicating a possible

response to host immune attack by 56 - 70 days post infection. It

should be noted that higher numbers of cercariae were used for in

fection in the present study than used by Cornford et al (1982).

Therefore, this may have elicited a more rapid host response.

Wilson & Barnes (1977) found a high rate of membrane protein

turnover in S.mansoni and as tegument renewal is thought to be a part of an immune evasion mechanism, more protein may be diverted

to the tegument and subsequently shed. This would account for the decline in parasite protein levels. The quantity of protein per

worm in S.mansoni in the current investigation shows close agree

ment with the values found by Cornford et al (1982) and Watts (1978).

Male S.mansoni contain higher levels of total lipid/mg protein

than female worms. However, if these values are related to the wet weight of the parasites then the levels in males and females

are similar i.e. 11% and 9% respectively. Lipid in the female parasite probably plays a significant role in egg production

(Erasmus, 1973) but its function in males is less clear. Lipid is

also a major component of the schistosome tegument (Smith & Brooks,

1969) and so, relatively high levels would have to be maintained as

the parasites have a double plasmalemma and the rate of tegumental

turnover is high (Wilson & Barnes, 1977).

4.2. ENZYME ANALYSIS

Measurement of the specific activities of enzymes involved in

different metabolic pathways provides a rapid means for a broad comparison between species and affords a general view of which path

ways are predominant. The main drawback with this type of analysis

is the use of standardised assays involving saturating levels of

reactants and co-factors, which do not account for the individual

optimal requirements of each enzyme.

The results of the present study indicate that glycolysis is

a major source of ATP generation in schistosomes and so agree with

the findings of Bueding (1950) and Shapiro and Talalay (1982b). A possible exception to this was S.haematobium (S.African strain) as

the maximal L-lactate dehydrogenase (L-LDH) activities were low in

comparison with other species. The incubation of 5.haematobium (Morroccan strain) also suggested that glucose can also be meta

bolised via a non-glycolytic pathway.liiaitz (1964), Bruce et al (1974), Coles (1973) and Huang (1980)

have produced evidence to suggest that S.mansoni and S.japonicum

are capable of operating a tricarboxylic acid (TCA) cycle and

hexosemonophosphate (HMP) shunt.The enzyme activities measured in the present study indicate

that the pathways, if operational, are running at a low rate. There

fore; their contribution to ATP synthesis may be at best, relatively

small. All TCA enzymes were detectable in S.marqrebowiei but only

citrate synthase, aconitase, and malate dehydrogenase (MDH, oxidising)

were measurable in S.mansoni, suggesting that the TCA cycle may not

be functional in the latter. The significance of the suggestion is

more fully discussed in the incubation section (4.4). It should be noted that the addition of Triton X-100 to samples showed that

normal homogenisation was only liberating about 50% of the TCA

enzymes. Therefore, activities are likely to be approximately twice

the recorded values.The enzyme data for S.mansoni shows close agreement with the

results of Coles (1973b) and Shapiro & Talalay (1982b) although there are a few notable differences. Shapiro & Talalay (1982b) found the

activity of hexokinase (HK) in S.mansoni to be low and suggested

that it may be the main regulatory enzyme of glycolysis. The HK

activities of S.mansoni and S.marorebowiei recorded in the present

study were much higher. Therefore, the enzyme phosphofructokinase

(PFK) may be playing a more important role in regulation.

Phosphoenolpyruvate carboxykinase (PEPCK) activity was also

lower in both species than the values obtained by Bueding & Saz (1968)

and Coles (1973b). It is unfortunate that this enzyme may be subject to pyruvate kinase (PK) interference as this could explain the

variation. The presence of nucleoside diphosphokinase activity in

S.mansoni supports PEPCK activity in the direction of CO^ fixation

(Zammitt & Newsholme, 1976). However, the relative specific

activities of PEPCK and PK suggest that very little phosphoenol-

pyruvate (PEP) participates in CO2 fixation in either species. In

support of this, the failure to detect fumarase suggests that CC^ fixation and a reverse TCA cycle do not operate in S.mansoni.

Shapiro 4 Talalay (1982b) suggested that the presence of MDH

and glycerol-3-phosphate dehydrogenase (GPDH) in S.mansoni indicated

the possible existence of a functional electron shuttle system. The

present study revealed that all species studied showed MDH and GPDH

activity, the latter being particularly active in S.mansoni. The

significance of this high activity and the possible role of MDH are more fully discussed later (section 4.4).

The presence of glucose-6-phosphate dehydrogenase (G-6-PDH)

and 6-phosphogluconate dehydrogenase (6-PGDH) is usually acknow

ledged as indicating the presence of a functional HMP cycle and these enzymes were active in each species examined. In addition, transal

dolase activity was found in S.mansoni but Barrett (1981) points out

that the pathway may terminate at ribulose-5-phosphate formation.

Although U/aitz (1964) has found evidence to suggest that the cycle

is functional in S.mansoni, further work on this area of metabolism

may prove interesting as the cycleTs role as an anaplerotic mech

anism and a source of synthetic molecules has not yet been evaluated.

154

4.3. METABOLITES

#-The difference in mouse liver metabolite levels found between

the data from the present study and Williamson & Brosnan (1974)

may be attributed to the use of different anaesthetics used to

#■ . kill the animals. Killing without anaesthetics can also influence

the results as it may cause the tissue to respond so that it is not

in a resting state. Dickerson (1965) found that anaethesia pro

duced a hepatic shift in schistosomes in mice, thus it is possible

that parasite metabolites will also be influenced by whichever

method is used to kill the host.L-lactate was the most abundant intermediary metabolite in

* S.mansoni and 5.marprebowiei. This supports the idea that schistosomes are heavily dependant on glycolysis as a source of

energy. Oxaloacetate (OAA) levels were also relatively high in

^ males of both species but the metabolic route of formation in theseparasites has yet to be demonstrated. The possible implications of

the high levels of this metabolite are discussed more fully in section

4.4. The inability to detect other TCA cycle intermediates again

suggests that the cycle, if operational, is running at a low rate

and so would not contribute much to ATP synthesis.Dihydroxyacetone phosphate (DHAP) was detected only in S.mansoni

and this is probably due to the activity of glycerol-3-phosphate dehydrogenase (GPDH) which was particularly high in this species.The DHAP could be a precursory step in de novo neutral lipid synthesis or could be participating in a mitochondrial shuttle

AI55

#

*

%

The general levels of metabolites are lower than reported for other parasites eg Hymenolepis (Rahman & Mettrick, 1982) Echinococcus

(McManus & Smyth, 1982) Ligula (Sterry, 1980). Yet the values

do fall within the ranges reported for other invertebrates (Barrett

& Butterworth, 1982), (Beis & Barrett, 1979) and (Beis & Newsholme, 1975). Differences between the data of different workers may be

due to variations in protocols such as methods of wet weight determination or the time taken to recover and freeze the parasites.

The ATP level in male S.mansoni is very close to the value

reported by Bueding & Fisher (1982) for paired worms. The adenylate

charges for S.mansoni indicate that the male and female worms were

in a normal, healthy state prior to removal from the host(s)

(Atkinson, 1971). The higher ATP : AMP ratio and level of ATP

in the male worms indicate that the rate of energy utilisation and

the rate of change of the glycolytic flux is higher than in the females (Beis & Newsholme, 1975). These points suggest that the

females maintain a steadier rate of glycolysis than the males. This may be viewed as a reflection of their probable in vivo life-style in

that they are presumably less mechanically active than the males, who transport the females through the hosts’ mesenteric/portal/urinary

blood vessels. Conversely, the males would require periodic bursts

of energy expenditure to perform this activity and hence they appear to be able to change rapidly from a resting state of glycolysis to

a more active one. This rate of change may be mediated by a marked

sensitivity to FBP which is discussed later (section 4.5.).

system, coupling to a cytochrome electron transport chain.

155

♦

♦

4

4.4. INCUBATIONS

Great care should be exercised when extrapolating the results of in vitro experiments to build up a picture of in vivo biochemistry

as most incubation conditions do not provide an optimal environment for normal metabolism. Variations in media constituents, gaseous phase and the duration of incubations could possibly exert

great influence on the type and quantity of metabolites taken up,

metabolised and excreted/secreted by the parasites. Additionally,

schistosomes contain quantities of host enzymes within their digestive

tracts which could affect assays. However, the assay of certain

gut enzymes in the present study suggests that interference from host enzymes is minimal. This supported by the detection of only

small quantities of radiolabelled CC^ produced by mouse blood during

incubation.L-lactate was the major detectable end-product of glucose

catabolism in each species investigated and this supports the earlier evidence that glycolysis is a major contributor to ATP syn

thesis in these parasites. For the males of each species examined

except 5..haematobium, lactate production stoichiometrically balanced glucose consumption. Thus it seems reasonable to describe these

organisms as essentially lactate fermenters and so.supports the findings of Bueding (1950), Schiller et al (1975) and Shapiro &

Talalay (1982b). In contrast, glucose consumption in S.haematobium exceeded lactate production, indicating that a proportion of glucose must be catabolised via a non-glycolytic pathway. Male S.mansoni

and S.marqrebowiei both produced low levels of CO^ and the former

contained small quantities of glycerol which suggests that alter

native pathways of glucose catabolism are operational.

Bruce et ad (1974), Coles (1973), Huang (1980), Smithers et al

(1965) and liiaitz (1964) presented evidence that S.mansoni and

5.japonicum possess functional tricarboxylic acid (TCA) and hexosemonophosphate (HMP) pathways which may be the mechanisms of CC^ production by male S.mansoni, S.marqrebowiei and S.douthitti.

S.marqrebowiei was found to have a full complement of TCA enzymes and the first two enzymes of the HMP pathway thus suggesting that these

cycles are operational in this species. Male S.mansoni only

exhibited citrate synthase, aconitase and malate dehydrogenase

(MDH, oxidising) activity and the only TCA metabolite detectable

was oxaloacetate (OAA). Therefore, in S.mansoni, the TCA cycle may

not be functioning and CC^ production might be due to HMP shunt

activity or amino acid breakdown (Senft, 1968), (Bruce et al, 1972).

This poses the question as to how OAA is formed by the parasite and

more importantly, what its function is. In both 5,mansoni and

5marqrebowiei OAA levels were higher in males than females. OAA

was not secreted/excreted in appreciable quantities so presumably it is an intermediate metabolite.

If the TCA cycle is functioning, part of the OAA produced would

be condensed with acetyl coenzyme A(ACoA) by citrate synthase in

order to continue the cycle. However, if this is the case, then there would appear to be some sort of restriction of citrate synthase

activity as the data (Tables 15 & 16) shows there is a build up of

□AA. Availability of ACoA could provide a means of limiting citrate

synthase activity. As no pyruvate dehydrogenase (PDH) activity

was detected in S.mansoni the ACoA would have to be derived from

another source eg amino acid or lipid breakdown.The OAA could be an essential component of a transmitochondrial

membrane electron shuttle system. MDH and glutamate-oxaloacetate transaminase (GOT) activity were detected in S,mansoni and these

enzymes could channel aspartate into the cytoplasm. Phosphoenol-

pyruvate carboxykinase (PEPCK) and MDH would provide the malate which

carries electrons into the mitochondria (Lehninger, 1977). A pos

sible function of the shuttle system may be to furnish electrons for

the cytochromes detected in S.mansoni in the present study (Section

3.13). Transaminase activity was also reported by Senft (1963) and

the possibility exists that OAA may be a precursor for amino acid synthesis. Therefore, OAA could possibly be fulfilling a general utility role in metabolism.

It is unfortunate that much of this speculation is dependent

on the cellular distribution of the enzymes involved, of which little

is known. Rotmans (1978) reported that cytoplasmic and mitochondrial MDH showed a twelve fold higher activity for OAA reduction than for

malate oxidation. In addition, the mitochondrial enzyme was found

to be more active than the cytoplasmic form. If this is so then

there would tend to be a build-up of malate in the mitochondrion.

The malate may pass into the cytoplasm for subsequent metabolism by MDH. This mechanism could play a part in providing intramitochondrial NAD+, which in turn could assist in the formation of 2-oxoglutarate

and succinyl coenzyme A. Overall, the proposed MDH activity u/ould

hamper TCA operation unless regulated by some means. Also it may

tend to obstruct the involvement of cytochromes. Therefore it would

probably be more prudent to bear in mind that Rotmans* findings were

for partially purified samples studied under non-physiological

conditions and so the data produced may not be applicable to the

in vivo situation.If the TCA cycle is not operating in S.mansoni then the enzymes

detected may be relics of a redundant cycle. Shutdown of the TCA

cycle may be caused by a decline in availability of ACoA, mediated

via PDH activity. It would be interesting to monitor the activity of this enzyme in cercariae and schistosomulae to determine if there

is a change in activity with development. Citrate synthase, aconitase

and MDH could be participating in a glyoxylate cycle which could

contribute to OAA synthesis but it would require a source of ACoA.

However, the presence of isocitrate lyase and malate synthase has

not been demonstrated in schistosomes. If citrate is not being

produced by S.mansoni in vivo, this may account in part for the apparent insensitivity of phosphofructokinase to this metabolite

(Bueding & Fisher, 1966).As mentioned previously, cytoplasmic PEPCK in male S.mansoni

and S.marqebowiei could catalyse the formation of QAA which could

further be reduced to malate by MDH. PEPCK activity in S.mansoni may be augmented by the action of nucleoside-51-diphosphokinase (NDP)

which would provide inosine -5*-triphosphate (ITP). However, there

150

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♦

is no evidence to support the idea that subsequent metabolism of malate to pyruvate, acetate, alanine or succinate occurs in either species. Also, the relative activities of pyruvate kinase (PK) and

PEPCK would severely limit the amount of phosphoenolpyruvate (PEP) available for fixation. Therefore it seems more likely that

PEPCK may be fulfilling a more anabolic role via gluconeogenesis.

Whatever the physiological role or means of formation of OAA, the

relative levels indicate that it is more important in the metabolism of male parasites.

In view of the available data, it is not known conclusively

whether the strain of male S.mansoni used forrthe present study

possesses fully functional TCA and HMP pathways. However, the evidence does suggest that 5 jnarqrebowiei is capable of at least

operating a TCA cycle. The contribution of the TCA cycle in each species to energy production appears to be minimal in both species.

The female schistosomes studied consumed far more glucose

than could be accounted for by lactate production. L-lactate was

the major end-product detectable and was produced in similar quan

tities as for the males. This suggests that the average rate of

lactate production during incubation was the same for male and female

worms. This contrasts with the work of Bueding & Fisher (1982) which

showed that males had a higher rate of glycolysis over 2h and 18h

incubations. The difference in results could be due to the different media, protocols or parasite strains used. The extra glucose con

sumed by the female parasites could possibly have been catabolised by a non-glycolytic pathway or used in an anabolic process.

OAA may fulfil similar roles in the biochemistry of the female

parasites as previously suggested for the males. The sex related

difference in OAA levels may be linked with the females' apparent

metabolic dependence on the males (Lennox & Schiller, 1972). If OAA is fulfilling the role of a multipurpose metabolite, then the

males could be acting as a store for the female parasites. Cornford

& Huot (1981) observed a similar phenomenon for glucose.As lactate u/as the only major end-product detectable in female

incubates then it seems possible that the extra glucose consumed

by the females may have been used for an anabolic purpose and the

most likely fate would be egg production. S.mansoni and S.marqrebowiei produce an average of 118 (Schiller et al, 1975) and 837 (Southgate

& Knowles, 1977) eggs per day. Sauer & Senft (1972) estimated that

daily egg production by S.mansoni is approximately equivalent to one

tenth of the females' dry body weight. Therefore, egg production

would appear to be an intensely metabolic process and probably requires

large quantities of ATP. This is supported by the fact that S.mansoni oocytes are surrounded by large numbers'of mitochondria (Roberts, 1983). Additionally, the miracidium is thought to be solely dependent on

glycogen as an energy source (Coles, 1973) and so this would probably

be synthesised by the female worm. Smith et al (1971) suggests that free fatty acids may also act as a source of miracidial energy but

it is currently thought that the female worm is not capable of de novo fatty acid synthesis (Meyer et al, 1970). Therefore the excess

glucose could be used to furnish energy and energy storage compounds for the egg and miracidium.

Egg production also requires a source of amino acids for egg

shell formation. Female S.mansoni metabolise several amino acids

(Bruce et al, 1972) and Senft (1963) reported that 6$ of glucose

taken up by worms is converted to alanine. Chappell & Walker (1982) found that glucose did not represent a significant source of protein

amino acids except for alanine, aspartate and glutamate. These

amino acids were found to be incorporated into worm tissue and egg

proteins. In the present study, alanine was not detected in worm

homogenates possibly as the quantities present were outside the

sensitivity range of the assay used. Nevertheless, a small quantity

of glucose was found to have been incorporated into parasite tissue

(Fig. 20). Haemoglobin degradation by the gut proteolytic enzyme may not furnish sufficient amounts of free amino acids (Saver & Senft, 1972) and thus some mechanism of synthesis from other compounds such

as glucose may be of great importance to the parasite. Thus, a

portion of the consumed glucose may go to form structural components for the adult parasites and for egg formation.

Little is known concerning general lipid metabolism in schisto

somes. Meyer et al (1970) reported that paired S.mansoni were incap

able of d£ novo synthesis of saturated fatty acids but retained the

ability to manufacture complex lipids from exogenous sources of fatty

acids and sterols. Venous blood could presumably act as a source of

these lipids. Lipid is a major constituent of schistosomes, being

one third of their dry weight (Huang, 1981). Smith & Brooks (1969) found that free sterols and triglycerides are the major neutral lipid

fractions in S.mansoni and Fried et al (1981) reported that S.mansoni

released free sterols, fatty acids, di and triglycerides into an

incubation medium within 2h. These products may simply be the

result of a desaturation and breakdown mechanism (Meyer et al, 1970).

Yet it is interesting to note that both sexes of S.mansoni contained

small quantities of glycerol. This could have been of host origin

but does not help to explain the relatively high levels of glycerol-

3-phosphate dehydrogenase (GPDH) activity and dihydroxyacetone phosphate (DHAP) that were found in the present study. GPDH activity

and DHAP levels in male and female S.marqrebowiei were lower and no

glycerol was detected in this species. Shapiro & Talalay (1982b) found a very low activity for GPDH in paired S.mansoni and it may be

possible that the strain of S.mansoni used for the present study is capable of glycerol synthesis. Hence, a proportion of the excess

glucose could be channelled along this route. Glycerol could sub

sequently be linked to fatty acid synthesis which in turn may be

central to the metabolism of vitellogenesis in female worms. GPDH

and DHAP may also be important in terms of electron transfer into

the mitochondria for subsequent coupling to electron transport chains. This may be of particular importance in female S.mansoni as they appear to be more dependent on oxidative phosphorylation than the

males (Section 3.14.).Renewal of the schistosome tegument might be another means by

which the excess glucose could be metabolised. Wilson & Barnes

(1977) reported that the large number of discoid granules present in the tegumental cytoplasm contain mucopolysaccharides. . Hockley

(1973) and Morris & Threadgold (1968) reported that non-glycogenic

PAS positive carbohydrate is present on the surface membranes of

S.mansoni. Wilson & Barnes (1977) also estimated that the schist

osomes tegument has a half-life renewal of 2-3 hours therefore,

glucose incorporated into tegumental macromolecules could be shed

into an incubation medium and so remain undetected. No data on the relative quantities of glucose present in the tegument or relative

membrane turnover rates of male and female worms are presently available. If large amounts of incorporated glucose are present in

the tegument then membrane turnover may account for the apparent disappearance of glucose. Kusel & Mackenzie (1975) also found that

fragments of schistosome tegument were sloughed off during incubation.

If tegumental renewal is a main cause of the glucose discrep

ancy then it follows that the females appear to have a higher turn

over rate than the males. This seems odd, as the males would pre

sumably present a greater surface area for potential immune attack

by the host system. It is apparent the further experiments should

be geared to the quantitative recovery and analysis of shed membrane

fragments on a comparative gender or species basis.Nash et aL (1974) and Deelder et al (1976) reported that schis

tosomes released circulating- antigens which were substantially carbohydrate in nature. The quantity of carbohydrate was estimated to be approximately 1% of the lyophilised worm weight for paired worms. This is another potential outlet for the excess consumed

glucose. The antigens are thought to be released as a means of

evading the host immune response.

165

Differences in the glucose : lactate (G:L) ratios between male and female parasites may simply be a reflection of their potentially

greater anabolic nature. It is difficult to assess the relative contributions made by male and female worms to the integrated

metabolism of the adult pair as it is not known to what extent they

are dependant on one another.

Glycolysis appeared to be the major detectable means of ATP

synthesis in the schistosomes studied and the presence or absence of

a functional TCA cycle in each of these particular species remains

to be confirmed. Evaluation of the energy contribution made by electron transport chains is now essential, particularly the means of

coupling to redox reactions, as this mechanism of energy production

may be of great singificance in the biochemistry of the female parasites. The ultimate fate of glucose, especially in female meta

bolism, appears to be potentially manyfold and therefore these worms should not be regarded as solely homolactic fermenters.

4.5. PYRUVATE KINASE

The study of pyruvate kinase (PK) activity in S.mansoni at

saturating assay conditions, indicates that the male enzyme is

susceptible to regulation by FBP and ATP. The female enzyme, while

activated by FBP, is far less sensitive to inhibition by ATP. This is in direct contrast to the findings of Brazier & Jaffe (1973) who reported that paired S.mansoni PK was insensitive to FBP activation

in the range 0.1 - O^mM and ImM ATP inhibited activity by 10p only

and so they concluded that the enzyme was not regulatory. Clearly,

the enzyme in the present study shows different characteristics and

this may be due to strain variation or to the fact that the male

and female enzymes were examined separately.

The results suggest :that male and female PK may be important

in controlling glycolytic flux but the method of control is different

in each sex. The sigmoidal character of the male enzyme and its modulation by FBP and ATP indicate that it is a normal, positively co-operative enzyme. The apparent negative co-operativity of the

female enzyme might be an adaptation to maintain a more steady rate

of glycolysis. Conway & Koshland (1968) have suggested that negative

co-operativity tends to insulate an enzyme from fluctuations in

metabolite concentration. These points are supported by the ATP

and AMP levels in these parasites, the significance of which is

discussed in the metabolite section (4.3). Modulation of the

parasite enzymes occurred within the physiological concentration

ranges determined from the metabolite levels.

With regard to the other species investigated, all the PK saturation curves were sigmoidal and positively co-operative except

S.bovis, for which the data was incomplete. It should be borne in

mind however, that co-operativity does not necessarily indicate regulatory capabilities and the high Km values of the enzymes cast

doubt on their regulatory capacities. Nevertheless, it would seem

that the strain of female S.mansoni used in the present study is

exceptional in its regulation of glycolysis. The apparent KrmKT)

for the enzyme is higher than that found by Brazier & Jaffe (1973)

157

while the male K ’ is lower,cthus stressing the difference in proper

ties between the enzymes.A number of points should be borne in mind when transposing

in vitro data to an in vivo situation. Firstly, the present study

deals only with crude homogenates and the assay system may have been

subject to extraneous enzyme interference but this must be similar

to the system which operates in vivo. Analysis of enzyme activity

in crude homogenates also makes no allowance for possible internal

compartmentalisation of enzymes. It may be that isoenzymes with

different kinetic properties have a differential specific distri

bution throughout the parasite and normal analysis would only yield an average of these activities. Secondly, homogenates will contain

small quantities of endogenous metabolites which will alter assay concentrations but the available data indicates that the maximum

error will only be approximately 2%. Additionally, the measurement of enzyme activity at saturating conditions of reactants will cause

the enzyme to behave in an ’’unphysiological” manner thus creating

the possibility of drawing incorrect conclusions regarding the manner

of its ijn vivo function.

Determination of enzyme.activity at ’’physiological” levels of

metabolites is at least a closer approximation of the in vivo func

tioning of the enzyme but requires a preliminary study of the metabolites involved. There are still a number of problems that must be

considered when performing this type of analysis. The measurement of the intracellular concentration of participating metabolites can

only be an approximation due to the inability to remove all the

extracellular fluid that is present. Also, the schistosome gut

contains host (erythrocyte) enzymes which may be active. However,

the present study indicates that interference from this source is minimal (Section 3.11.5.1.).

Another important factor to consider is that sigmoidal curve does not automatically imply a co-operative or regulatory nature.

A two substrate enzyme mechanism could also account for a sigmoidal

shape (Price & Stevens, 1982). In S.marqrebowiei, male and female

PKs showed co-operative kinetics and were sensitive to modulation

by FBP and ATP. The effective range of ATP inhibition for the male

enzyme was between 1nM - 1jjM which is well below the range for male

S.mansoni PK. The steady state levels of ATP were not measured in

S.marorebowiei but as both sexes produced more lactate during incubation than S.mansoni and showed similar glycolytic enzyme activities, it seems reasonable to suggest that ATP levels in the

parasites would be higher than Ijjffl. The female enzyme was inhibited

within the 1pM - ImM ATP range and thus shows better agreement with the estimated endogenous levels.

Male 5.marqrebowiei PK was more significantly activated by

FBP than the female enzyme from 1nM - 1(jM but the endogenous levels

in males and females were 14 jjM and 28 jjM respectively. In this range

(1 jjM - ImM) the female enzyme was more significantly activated than

the male. The male worms do not appear to require a greater rate of

glycolysis than^.the females. This may be because they are less motile

in vivo than male S.mansoni or they may have other means of generating

ATP such as the utilisation of a more active TCA cyiJe..Measurements of the "physiological” behaviour of S.mansoni PK

showed that FBP activation occurred in both sexes but activation of

the male enzyme was significantly higher than the PK from the females

by a factor of 8. Female PK was not significantly inhibited by ATP

in the absence of FBP and this finding is similar to the result

obtained from the assays performed under saturating metabolite con

ditions. However, in the presence of FBP, which is more representative of the in vivo situation, female PK activity was inhibited to the same extent as the male enzyme. This highlights the potential dangers

of drawing incorrect conclusions from results obtained under non-

physiological conditions. As no sigmoidal relationship was established

for either enzyme it was impossible to determine the extent of co-

operativity.The greater effect of FBP on male PK may be seen as a reflection

of the probable in vitro mode of life of the adult parasites. Male

worms normally carry the female parasites along the hosts’ mesenteric/ portal/urinary blood vessels, sometimes against the flow of blood.

Therefore, it seems reasonable that the males would be required to

produce periodic bursts of energy which could be provided by a large increase in glycolytic rate which in turn could be mediated via FBP.In addition, the greater rate of glycolysis in the males could well be the reason why they contain greater glycogen levels than females.In contrast, the adult females have to perform comparatively little mechA

nical activity and so would not need to alter their glycolytic rate and

170

s

*

m

&

♦

The data regarding ATP and AMP levels in S.mansoni also indicates

that the females have a lomer, steadier rate of glycolysis than the males. The higher ATP levels and ATP : AMP ratio in the male parasites indicate that the rate of energy utilisation and rate of change

of the glycolytic flux is higher than in the females (Beis & Nemsholme

1975). This information supports the idea that male morms mould

require periodic bursts of energy production to hold the females

mithin the gynecophoric canal and to transport them about the hosts1

blood system.

The presence of FBP in assay measurements is a more accurate

representation of the in vivo situation and under such conditions ATP

inhibited male PK activity uncompetitively and female PK competitively.

This suggests a gender based difference in glycolytic control but

as the data did not conform precisely to the normal Linemeaver/Burk transformations, further mork is necessary to confirm this finding.

Male and female PK shomed no preference for Mg or Mn mhereas

Brazier & Jaffe (1973) found that Mn"mas half as affective as Mg’

in terms of enzyme activity. The Mg concentration used in the assay

(5mM) mas reasonably close to the physiological level (approximately

3mM) reported by Sham & Erasmus (1983). In contrast, the PKs from other parasites such as the daughter sporocysts of Microphallus

similis (McManus & James, 1975), Dirofilaria immitis (Brazier &

Jaffe, 1973) and Moniezia expansa (Bryant, 1972) do exhibit a pre-~H* 4fference for Mg or Mn. Yet these preferences may be related to the

so mould require lomer glycogen stores than the males.

in vitro assay conditions as little is knomn concerning the inorganic

1 I I

metabolism of these parasites. The preferred cation may not even be

available in vivo to the parasite enzyme. These observations high

light the difference between the enzyme in the present study and

the enzyme examined by Brazier & Jaffe (1973). Apart from strain

variation, differences in buffer systems and pHs could also account,

in part, for this variation.

Electrophorectic analysis revealed that PK exists in identical

fprms in male and female worms although the number of separated

bands, which may represent discrete isoenzymes, varied according, to the technique employed. Each method of analysis indicates that there is one major form of the enzyme in both sexes with a number of

minor isoenzymes. Mammalian tissues contain at least two distinct

PK isoenzymes which possess different kinetic characteristics (Tanaka

et al 1965, 1967). Type L PK is activated by FBP, is sensitive to

ATP inhibition (Tanaka et al, 1967) (li/eber, 1967) and is predominant

in the liver. Type M PK is found in skeletal muscle, is not activated

by FBP and is far less sensitive to ATP inhibition (Tanaka et_ al,

1967) (Vijayvarqiya et al 1969). Brazier & Jaffe (1973) found that

paired S.mansoni PK resembled Type M PK but the enzymes in the present

study resemble Type L. It is not known if the isoenzymes (sub-units) found in the present study have different kinetic properties but the

isoelectric point measurements will serve as a baseline for the isolation and purification of the enzyme in any subsequent study.

Regulation of glycolysis in S.mansoni is thought to be mediated via hexokinase (HK) (Bueding & Makinnon, 1955a) (Shapiro & Talalay,

1982b), PFK (Bueding & Fisher, 1966) and possibly glucosephosphate

172

#

isomerase (GPI) (Shapiro & Talalay, 1982a). HK and PFK are the

classical regulators of glycolysis, typically catalysing non- equilibrium reactions in vivo and have relatively low specific activities (Price & Stevens, 1982). Traditionally the role of PK

in regulation of glycolysis has been doubtful due to its relatively

high specific activity (Price & Stevens, 1982). Nevertheless, in the

present study the mass action ratios and kinetic study has shown that

PK can control glycolytic flux. HK and PFK ultimately control the

rate of entry of glucose along the pathway but PK would regulate the

rate of lactate formation, assuming that lactate dehydrogenase is not

allosterically controlled. This would be important in terms of energy

synthesis and will be discussed below. Also, the control of PK may

be essential to maintain sub-toxic levels of lactate in the tissues.

PK could also help to maintain cytoplasmic redox balance and it may also allow a small proportion of phosphoenolpyruvate (PEP) and pyruvate to be converted to amino acids (Conn & Stumpf, 1976) or

the former to participate in CD^ fixation reactions.

PK is an extremely important enzyme in glycolysis as it determines the net ATP synthesis by the pathway. Fructose-6-phosphate

(F-6-P) requires ATP for the phosphorylation step to FBP and assuming

that endogenous glucose also requires ATP to be phosphorylated by

HK, then the synthesis of ATP from 1, 3-diphosphoglycerate cleâge will result in an overall balance. To gain energy (ATP) from the

system, the clevage of phosphate from PEP is necessary and for a parasite so heavily dependant on glycolysis, regulation of the net gain step in the system via PK would seem to be essential. A further gain

173

of ATP can be achieved by catabolising glucose-1-phosphate derived

from glycogen breakdown but the relative rates of endogenous and

exogenous glucose utilisation are not known.In summary, the PKs from the strain of S.mansoni used for the

present study, appear to be capable of regulating glycolysis.Control of enzyme activity in both sexes is mediated via the same

modulators although the male enzyme shows an enhanced response,

which is probably correlated to its in vivo life style. A comparative

kinetic study of purified PKs from male and female worms may afford a greater insight as to the mechanism of regulation, providing

"physiological” levels of metabolites are employed. Interspecific

comparisons may also be valuable as differences in control mechanisms

may offer the possibility of exploitation by chemotherapy.

4.6. OXYGEN UPTAKE AND CYTOCHROMES

The physiological role of oxygen in schistosome metabolism has

been the subject of debate for a number of years. Schiller et al

(1975) found that oxygen was necessary for egg production, presumably

for the tanning of egg shells (Seed et al, 1978). Bueding & Charms (1952) failed to find sufficient quantities of cytochrome C and

cytochromecC oxidase in S.mansoni to account for more than one tenth of the parasites’ oxygen uptake. Bueding (1950) found that male and

female worms took up quantities of oxygen which could account for

only 3% of the total glucose utilisation. Hence, this evidence suggests that oxidative phosphorylation is operating negligibly, if

at all in S.mansani.

In contrast, Coles (1972b) reported that oxidative phosphory

lation provides at least one quarter of the worms’ metabolic energy. He also suggested that this was an underestimate of the in vivo

capacity. It is unfortunate that this data refers only to paired

schistosomes as the study of separate oxygen uptake rates for male

and female worms’ would facilitate a better estimate of the contri

bution made by egg production to oxygen uptake.

The present study revealed that female S.mansoni took up eight

times as much oxygen as the male parasites. The values were higher

than those obtained by Bueding (1950) and Bueding & Charms (1952)

and this is probably due to the more physiological medium and more

sensitive polarographic techniques used in this study. 20 mM KCN completely inhibited oxygen uptake by the males but only inhibited

the females’ uptake by 64%. Ross & Bueding (1950) found that com

plete inhibition of uptake in both sexes occurred in the presence

of ImM KCN. Therefore, assuming that KCN is only uncoupling

cytochrome oxidase, then it appears that both sexes can utilise

oxidative phosphorylation for energy production. The presence of

KCN greatly reduced the worms’ motility but did not kill them.

Coles (1972b) reported a concomitant increase in lactate production

in the presence of cytochrome chain uncouplers. The females’

continuing ability to take up oxygen in the presence of KCN could be consistent with a requirement for egg shell production. Another possibility is that the female parasites possess an alternative electron transport chain that is cyanide insensitive. The data

175

w*dL\Cccfe,$ that ATP synthesis by oxidative phosphorylation important to the female parasites than to the males. Morris &

Threadgold (1968) reported that in both sexes the majority of the

mitochondria are found in the uiorms1 parenchyma. Numerous mitochondria are found surrounding oocytes (Roberts, 1983), therefore

oxidative phosphorylation in the female worms may be used to provide

energy for egg synthesis as well as for general metabolism.

Until the present study it has been generally accepted that

measurement of the cytochrome spectra of adult schistosomes was not

possible due to interference by the parasites' gut contents (Coles,

1982). Cheah (1975) used nitrate in an attempt to make a clearer

distinction between Ascaris cytochrome spectra and absorbance due

to haemoglobin. The present study revealed that haematin (a haemoglobin degradation product) and not whole mouse blood is the major

contaminant in cytochrome scans. Also, by performing assays at

-196°C there is an intensification factor of 7 for cytochromes only

and hence is more sensitive than room temperature assays (Wilson, 1967). The present investigation has shown that the cytochrome

spectra of schistosomes can be measured.

Comparisons of parasite spectra with those from rat liver shows

that male and female S.mansoni have identical spectra corresponding

to cytochromes a^, C, and C^. Male parasites also possessed a 'b* type cytochrome which was not detectable in the females. This may

have been due to insufficient material being used in the assays.

However, the absence of cytochrome b in female schistosomes, together

with the degree of KCN insensitivity that was found by oxygen uptake

measurements may indicate a branched respiratory chain. Other para

sites eg A.lumbricoides, Moniezia expansa, Haemonchus contortus and Fasciola hepatica are thought to operate branched cytochrome chains,

with an 0 type cytochrome acting as a terminal oxidase (Barrett,

1981).

The quantities of cytochromes present in both sexes of S.mansoni

are similar therefore if the females are more dependent on oxidative

phosphorylation then some form of restriction or control must occur

in the males. Bryant (1970) found that an oxygen tension of 5 mm Hg

was necessary for normal electron transport and the oxygen tension

of venous blood (49 - 66 mm Hg) is well above this value (Smyth, 1976). Oxygen would presumably be available to the parasites from digested

haemoglobin. Hence, as the male digestive tract contains smaller quantities of digested material than the female, it is possible that

limitation of quantity of oxygen available is a reason for the males'

apparent lower rate of electron transport. However, in vitro, it may

be possible that transtegumental absorption of oxygen occurs in which

case another means of regulation may apply..

Control of electron flux into the mitochondria could be anothermechanism for regulating oxidative phosphorylation. It is not knownhow the transport chains in these parasites are linked to redox balance.

Coupling of the chain may be effected by a malate-asparate shuttle,glycerol

a glycerol shuttle or a TCA cycle. The ' shuttle system, would result in a slightly reduced ATP output as tMs would presumably link at the ubiquinone level and so lose a site 1 phosphorylation.

The feasibility of the existence of these systems in schistosomes

is discussed in the incubation section (4.4).

Electron chain coupling via a TCA cycle must remain a doubtful prospect as the existence of a functional cycle has yet to be clearly

demonstrated in the parasite strains used in the present study.

S.marqrebowiei appears to be capable of operating a cycle (see section

4.2) and even if S.mansoni has a functional pathway, the estimated rates of cycle turnover in these species indicates that it would not

make a significant contribution to ATP synthesis in these worms.

Clearly, the mechanism of cytochrome coupling to redox reactions in

these parasites warrants further investigation.

Huang (1980) found that S.japonicum relied substantially on

oxidative phosphorylation as a means of energy synthesis. He also

found that oxygen consumption was markedly affected by the composi

tion of the incubation medium used for the experiments. The presence

of serum was found to be essential to maintain the worms in an active state for longer than 2 - 3 hours. Rogers (1976) found that

S.douthitti was capable of KCN sensitive and insensitive aerobic metabolism and so, electron transport mechanisms may be more wide

spread and varied in schistosomes than has previously been suggested. The quantities of cytochromes found in 5,mansoni are similar to those

found in Ascaris and Moniezia (Cheah, 1972) and interestingly, are

higher than those found in highly aerobic sea urchin sperm (Wilson &

Epel 1978). Cercarial cytochromes were measured by Coles (1972a)

and Coles & Hill (1972) who detected cytochromes a/a^, b, C and

possibly 0 but quantitative analyses were not performed. The spectral

peaks are different to those obtained in the present study because

the assays were performed at room temperature.If a more active TCA cycle was operating in S.mansoni, then as

6 moles of oxygen are required for the complete oxidation of 1 mole

of glucose, the oxygen uptake rates would account for the oxidation

of 0*1 and 0*7 moles of glucose for males and females respectively. These values would account for the "excess" glucose consumption shown particularly by the female worms. However, all the data indicates that

TCA operation in these parasites is at best, minimal. Nevertheless,

this does not necessarily mean that glucose consumption is not linked to the electron transport chain. A malate or glycerol shuttle opera

ting at a high turnover could be equally as efficient in supplying

electrons for oxidative phosphorylation. As glyceraldehyde-3-phosphate

dehydrogenase (GPBB) activity in S.mansoni was far higher than PEPCK

activity, a glycerol shuttle mechanism would seem more appropriate for

chain coupling than a malate shuttle supplied via CO^ fixation. This

would mean that part of the glucose flux would be diverted from lactate production into synthesis of dihydroxyacetone phosphate (DHAP) and

glycerol-3-phosphate (G-3-P). If a large proportion of the "excess"

glucose is being metabolised in this manner then it would result in relatively high levels of these intermediates. This is supported by the fact that DHAP levels were relatively higher than levels of other

glycolytic intermediates in female S.mansoni. A similar situation did not exist for DHAP levels in male S.mansoni and hence this fits the overall picture of the females being more dependant on oxidative

phosphorylation than the males.

DHAP levels alone in female S.mansoni can account for 20% of the

"excess" glucose (0«6 pmoles) consumed by these worms. Determination

of glycerol-3-phosphate levels in the females may yield similar

values. Therefore the levels of intermediates necessary to operate

a glycerol shuttle could account for nearly half the extra glucose

consumption. Hence a significant proportion of the glucose consump

tion by female S.mansoni not channelled into lactate production could

be accounted for by oxidative phosphorylation coupled via a shuttle

mechanism.Further work is necessary to substantiate this idea but it is

evident from the levels of cytochromes and oxygen uptake rates of

these parasites, that electron transport chains^play an important role in energy production in S.mansoni whatever the means of redox

coupling is. Other species of schistosome with greater levels of

"excess" consumed glucose eg S.haematobium could well be even more

dependent on oxidative phosphorylation than S.mansoni. An inter

specific comparison of this aspect of metabolism may yield interesting

results. The pathway suggested for S.mansoni females does not appear

to fit the females of S.marqrebowiei as DHAP levels were not detectable

in the latter. This species however, appears to be capable of TCA

cycle activity and so a different means of chain coupling could exist

in this case which could even augment other mechanisms of redox

coupling.

If schistosomes are capable of aerobic metabolism then a Pasteur effect should be demonstrable. Bueding (1950), Bueding & Fisher (1966)

and Schiller et al (1975) could find no evidence of this but such

corroborating data was found by Coles (1972b). Again, the choice

of medium or parasite species/strain may lead to different results

but it is obvious that the potential for aerobic metabolism by some, if not all schistosomes, cannot be ignored.

4.7. SUMMARY

Studies of the glucose, glycogen, protein and wet weight levels in

both sexes of S.mansoni and S.marqrebowiei indicated that with the

exception of male S.marqrebowiei, the parasites had levelled off

their growth rates by 56 - 70 days post infection. Male S.marqrebowiei continued to grow and this may be due to the increasing synthesis and

storage of glycogen within the worm.Incubation studies, metabolite and enzyme analyses indicate

that glycolysis is a major pathway of ATP synthesis in schistosomes.The evidence for the functioning of a TCA cycle in S.mansoni is

inconclusive but the data suggests that S.marqrebowiei does operate a cy cle. However, the studies showed that the TCA cycle contributes little,

if at all, to ATP production. HMP shunt enzymes were present in both

species but again, there is no.data to suggest that the pathway is fully functional in these parasites.

The incubation work revealed that whilst male S.mansoni and S.marqrebowiei are possibly homolactic fermenters, the females channel approximately half their glucose consumption into other

metabolic pathways. Part of the excess glucose may be channelled into egg production, tegument formation, synthesis of circulating

antigen or possibly neutral lipid synthesis. Alternatively a; pro

portion of the glucose may be linked to oxidative phosphorylation mechanisms.

Oxygen uptake measurements and cytochrome spectra analyses

indicate that both sexes of S.mansoni are^capable of oxidative phos=« phorylation. The females, however, appear to be more reliant on this

process than the males. Metabolite and specific enzyme analysis suggest that the cytochrome chain is coupled to the redox balance via

a glycerol shuttle mechanism. Additionally,.female S.mansoni also

showed a degree of cyanide insensitivity which could mean that the

oxygen is possibly being used for egg shell tanning or that a branched

cytochrome chain is present.

Enzyme kinetic studies, supported by metabolite and electrophoretic analyses, indicated that the pyruvate kinases from male and

female S.mansoni are structurally similar and exert a regulatory influence on the parasites’ glycolytic flux in vivo. The enzyme

from the males was 8 times more sensitive to FBP activation than the female enzyme. This may be necessary to allow the males to meet the

changes in energy demand placed on them by their in vivo life style.

The PKs of both sexes was inhibited by ATP to the same degree.

Studies on the PKs from S.margrebowiei and 5.japonlcum revealed that

the enzymes are positively co-operative but it is not certain if they are capable of regulating glycolysis in these species.

In general, the schistosomes studied, particularly the males, appeared to be heavily dependent on anaerobic glycolysis for energy

132

production. However, the females of certain species (and both sexesof 5.haematobium) appear capable of other means of ATP synthesis. It

is obvious that female S.mansoni have a large potential for aerobic

energy production. The same remains to be demonstrated in otherAspecies. This may prove to be an important coîderation when exam

ining new approaches to the chemotherapy of schistosomiasis.

♦

*►

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ENERGY METABOLISM OF SCHISTOSOMES by Clement Antonio … · 2016-07-29 · ENERGY METABOLISM OF SCHISTOSOMES by Clement Antonio Earle B.Sc (Hons). A thesis submitted for the degree