Carbon Dioxide Utilisation, and the Regulation of Respiratory Metabolic Pathways in Parasitic Helminths

Carbon Dioxide Utilisation, and the Regulation of Respiratory Metabolic Pathways in Parasitic Helminths C. BRYANT

Department of Zoology, Australian National University, Canberra, AustraIia

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I. Introduction ................. ........ .......... ..... ....... ....................... ..... 11. Carbon Dioxide Fixation .... ,...... ,....................... ............. .... . ...... A. General ....................................................................................... B. The Enzymes of Carbon Dioxide Fixation .......................................... C. The Enzymes in Parasitic Helminths ,................... ........ ... .... ........ 111. The Roles of PEPCK and ME ............................................................... A. General ....................................................................................... B. The Roles of PEPCK and ME in Parasitic Helminths ..... ...... ............. IV. PEPCK/PK Ratios and the Path to Lactate ............,............ .............. .... V. Metabolic Regulation in Parasitic Helminths ...... .... .,....... ............. . ....... A. General ..................................................................................... .. B. Regulatory Enzymes in Parasitic Helmimths ... ..... ...... ..... . ...... ....... C. Metabolic Regulation in Ascaris ...................................... .............. D. Conclusions ................................................................................. Acknowledgements ........ .... . ........ ... . .. .... ... . ..... ... . ........... References .................................... . . . . .. .. . . . ..... ..... .

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36 37 37 39 40 42 42 46 50 56 56 58 60 62 63 63

Note: The following abbreviations are used in the text: ATP, ADP, AMP: adenosine tri-, di- and monophosphates CoA: coenzyme A CO,: carbon dioxide CTP: cytosine triphosphate GTP, GDP; guanosine tri-and diphosphates ITP, IDP; inosine tri- and diphosphates ME: “rnalic enzyme” (E.C. 1.1.1.39 and 1.1.1.40) NAD, NADH :oxidised and reduced nicotinamide adenine dinucleotide NADP, NADPH : oxidised and reduced nicotinamide adenine dinucleotide phosphate PEP :phosphoenolpyruvate PEPCK: phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32) PK: pyruvate kinase (E.C. 2.7.1.40) 3

35

36

C. BRYANT

I. INTRODUCTION Metabolic regulation is one aspect of biochemistry which today occupies the attention of many workers. Its concepts derive from cybernetics and information theory. The organism is considered to be an adaptive control system, an interrelated set of subsystems between which information transfer occurs. Integration is achieved by means of feed-back and feed-forward repression and activation mechanisms. Such mechanisms operate at the molecular, genetic and physiological levels ; cues for a particular subsystem may originate in the environment, in other subsystems within the organism or from the state of the subsystem which itself is being modulated. In a review of this type it is tempting to speculate, but speculation must be tempered by the relative paucity of data available on the enzymology of parasitic helminths. Although the comparative biochemistry of parasites has emerged from its infancy it still lags well behind the study of the biochemistry of vertebrates, for example. There is a wealth of factual information (von Brand, 1966) but few attempts have been made to codify it. However, one international symposium has been devoted solely to the comparative biochemistry of parasites (van den Bossche, 1972) and it is becoming clear that the impetus given to metabolic regulation in recent years is being felt among parasitologists. This review dwells perforce on those relatively few parasites which have been intensively studied, and from which conclusions have been drawn which may or may not have general application. Such conclusions are examined critically. The treatment of the subject matter covers two related areas; that of CO, hation, with its implications for respiration in parasites, and the mechanisms by which the latter is regulated. A grave disadvantage encountered in such an essay is that not all stages of the life history of a parasite are equally well studied. Research into Haemonchus contortus larvae, for example, is well advanced; little is known about the adult. Adult Ascaris lumbricoides has recently been the object of very close scrutiny, but the biochemistry of larval forms is not nearly so well known. Problems also arise with individual animals. Thus, the anterior portion of Moniezia expansa has been investigated, but conclusions valid for the scolex may not necessarily apply to the remaining parts of the same individual at the same time. A further limitation is that information is often deficient about those aspects of the environment of the parasite which are of paramount importance to it. Blood parasites fall into this category. For example, a number of generalisations may be made about intestinal environments, which are usually rich in CO,, poor in oxygen and in which an abundant supply of small molecules is available for the nutrition of the parasite. On the other hand, there are aspects of the nutritional physiology of the microiilariae and the schistosomes which are not clear, and views conflict as to which pathway of metabolism is employed in glucose oxidation. It would be too much to hope for a complete synthesis of such heterogeneous material in the present review. However, a number of features emerge

C A R B O N DIOXIDE UTILISATION I N P A R A S I T I C HELMINTHS

37

which are common to many parasites, and even more important, points of divergence are identified. The significance of these is discussed as fully as the data and the space available permit. 11. CARBON DIOXIDE FIXATION A. GENERAL

It has long been recognised that bicarbonate is an important constituent in the maintenance and cultivation media of many parasites. A detailed discussion of its various roles as a buffer and as an essential nutrient in in vitro systems is considered in a book on the subject of the cultivation of parasites by Taylor and Baker (1968). The experiences of subsequent years have not invalidated the view that CO, is an essential component for the growth of many parasitic helminths. A recent review (Silverman and Hansen, 1971) reaffirms the value of bicarbonate/CO, buffered media and emphasises the importance of pC0, as a trigger to activate the development of many parasites. The fixation of CO, by many parasitic helminths, and the subsequent involvement of carbon from this source in intermediary metabolism, has been well established for many years although the actual route of fixation has been resolved only relatively recently. Rogers and Lazarus (1949) reported a net disappearance of CO, from the anaerobic bicarbonate medium in which Nematodirus spp. and Ascaridia galli were incubated. Glocklin and Fairbairn (1952) showed that glycogen usage by the nematode Heterakis gallinae was doubled in a CO, free environment. In a subsequent paper, Fairbairn (1954) demonstrated that H. gallinae fixed CO, extensively under anaerobic conditions, and that much of the carbon appeared in propionic and, probably, succinic acids. The fact that the normal environment of the parasite, the fowl caecum, is essentially CO, free, and that even under fixation conditions the nematode displayed a small net production of the gas, led Fairbairn to suggest that the worm makes efficient use of metabolically produced CO,. Saz and Vidrine (1959) extended these observations to Ascaris lumbricoides, except that the glycogen sparing effect reported by Glocklin and Fairbairn (1952) was absent (von Brand, 1966). The utilisation of CO, has since been demonstrated (by authorities here cited) in a range of parasites which includes the trematodes Entobdella bumpusi (Hammen and Lum, 1962), Fasciola hepatica (Prichard and Schofield, 1968a); the nematodes Haemonchus contortus (Ward et al., 1968b), Trichinella spiralis (Ward et al., 1969), Ascaris mum (van den Bossche, 1969), Dictyocaulus viviparus (Vaatstra, 1969), Nippostrongylus brasiliensis (D.K. Saz et al., 1971) and probably Syphacia muris (van den Bossche et al., 1971); the cestodes Hymenolepis diminuta (Prescott and Campbell, 1965), Echinococcus granulosus (Agosin and Repetto, 1963) and Moniezia expansa (Bryant, 1972a); and the acanthocephalans Moniliformis dubius (Graff, 1965) and Echinorhynchus gadi (Beitinger and Hammen, 1971).

38

C . BRYANT

In all the cases cited above, radiocarbon from CO, has been detected in the end products of respiratory metabolic pathways. It is also possible that CO, is involved in gluconeogenesis. Thus, Prescott and Campbell (1965) reported that Hymenolepis diminuta incorporated radiocarbon from NaH14C03 into polysaccharide. Corroboration was obtained by Read (1967), who found that gluconeogenesisand glucose uptake in the tape worm were stimulated by the presence of 5 % CO, in the gas phase of the maintenance medium, especially when oxygen was present. A short research note by McDaniel et al. (1968) established that the cestodes Lacistorhynchus tenuis, Calliobothrium verticillatum, Orygmabothrium dohrnii and Tetrabothrium erostris, and the trematode Cryptocotyle lingua, exhibited increased glucose incorporation in the presence of CO,, suggesting that polysaccharide synthesis was taking place. One other CO, fixation pathway requires brief consideration before passing on to an examination of respiratory metabolism. It is the possible contribution of the carbamyl phosphate synthetase reaction in parasitic helminths. An important initial reaction in the urea cycle is the formation of carbamyl phosphate according to the following equation: NH4++ CO, +2ATP4- H,O 0

-

carbamyl phosphate synthetase

-

+

il

H2N - C - 0 -PO:-+

2ADP3-+ P:-+ 3H+

Rogers (1952) showed that nematodes may excrete urea, and urea is also found as an end product amongst other nitrogenous end products in a large number of other helminths (von Brand, 1966). Campbell (1963) explored its formation in Hymenolepis diminuta, and found that incorporation of radiocarbon from bicarbonate into urea occurred. He did not, however, assay carbamyl phosphate synthetase activity in the worm. Rijavec (1965) and Rijavec and Kurelec (1965, 1966) assayed the enzymes of the urea cycle in the liver fluke Fasciola hepatica, and concluded that they were all present. Dicrocoelium lanceatum was also reported to have a complete cycle, but Paramphistomum cervi and Moniezia benedeni were found to lack arginine synthetase. Janssens and Bryant (1969) looked for urea cycle enzymes in five parasites, none of which possessed the full complement. While traces of carbamyl phosphate synthetase were found in Fasciola hepatica, its activity in Moniezia expansa, Taenia pisformis and Echinococcus granulosus was below the levels of detection for the assay method. Kurelec (1972) subsequently confirmed that Fasciola hepatica, as well as Paramphistomum cervi and Moniezia benedeni, were deficient in this enzyme. Thus, Hymenolepis diminuta remains the only helminth for which there is circumstantial evidence for the presence of carbamyl phosphate synthetase, and it clearly needs re-examination. It would seem reasonable to conclude that the incorporation of CO, by the urea cycle route may not occur in parasitic helminths and can be disregarded. It is important, therefore, to determine the point in intermediary metabolic pathways at which CO, incorporation takes place. The next section is concerned with the evidence which implicates specific enzymes.

CARBON D I O X I D E U T I L I S A T I O N I N P A R A S I T I C HELMINTHS

38

B. THE ENZYMES OF CARBON DIOXIDE FIXATION

There are several enzymes which might be involved in CO, fixation in parasites. They have been well characterised in vertebrate, plant and microbial systems, but only in the last few years have parasitologists looked for them in helminths. Much of the literature is confused by the use of synonyms and trivial names which must be defined in current biochemical usage. The following list is derived from Barman (1969). 1. Pyruvate carboxylase (Pyruvate: carbon dioxide ligase (ADP). E.C.6.4.1.1.) Pyruvate carboxylase catalyses the fixation of CO, into pyruvate in the presence of ATP. The product is oxaloacetate, and the reaction proceeds according to the following equation:

+

+

+

ATP +pyruvate CO, H,O = ADP +Pi oxaloacetate. The reaction is reversible and occurs in most animal tissues, The animal enzyme needs acetyl CoA for activity. 2. “Malic enzyme“ (ME) “Malic enzyme” and its synonym “malate dehydrogenase-decarboxylating” have been used as trivial names to describe at least three distinct enzyme activities (Dixon and Webb, 1964). The first need not concern us here, as it has not been found in animal tissues. The second, which is not listed in Barman (1969), has the systematic name L-malate: NAD oxidoreductase (decarboxylating) E.C.1.1.1.39. It has only been found in Ascaris lumbricoides (Saz and Hubbard, 1957). It catalyses the following reaction:

+

L-malate NAD = pyruvate + CO, +NADH.

It is unusual in that it does not decarboxylate added oxaloacetate. The third “malic enzyme” is L-malate: NADP oxidoreductase (decarboxylating) E.C.l.l.l.40. It is widespread in animal tissues, and is specific for NADP, which may be substituted for NAD in the above reaction. It decarboxylates added oxaloacetate. Both enzymes require Mn2+ for activity. In the remainder of this review, the term “malic enzyme” will be used to refer to the NADP-dependent enzyme. Where the NAD-dependent enzyme is indicated, reference will be made to cofactor specificity. 3. Phosphoenolpyruvate carboxykinase (PEPCK) PEPCK is often referred to in the literature as “phosphopyruvate carboxylase”. PEPCK is preferred here. Its systematic name is GTP: oxaloacetate carboxylyase (transphosphorylating) E.C.4.1.1.32. PEPCK catalyses the reaction:

+

GTP oxaloacetate = GDP +phosphoenolpyruvate+ CO,.

The reaction is reversible; ITP can replace GTP, and Mn2+ is required for optimal activity.

40

C. B R Y A N T

It is important to distinguishbetween the enzymes in the following sections, when each of the major groups of parasitic helminths will be considered from the point of view of CO, utilisation. C.

THE ENZYMES IN PARASITIC HELMINTHS

Among the earlier papers that led to speculation that, in Ascaris lumbricoides, the enzyme responsible for CO, fixation was an NAD-linked malic enzyme (NADP was one third as effective), are those of Saz and Hubbard (1957) and S a z and Vidrine (1959). It appeared that succinate was formed by fixation of CO, into pyruvate, followed by the reduction of the malate so produced. This, however, proved to be a rather simplistic view, as the partially purified enzyme was found to react more rapidly in the direction of decarboxylation. Later, Saz and Lescure (1967) demonstrated the presence of a second enzyme capable of fixing CO, in Ascaris muscle. It was detected in supernatant extracts of both adult muscle strips and larvae. It required either IDP or GDP for activity, suggesting strongly that the enzyme was a true PEPCK, and this conclusion was later confirmed by a rigorous examination of the properties of the enzyme in Ascaris muscle extracts (Bueding and Saz, 1968). Independent codrmation of the significance of PEPCK was provided by van den Bossche (1969), who worked with muscle from A. suum. The enzyme from this source has a pH optimum of 7.2, Mn2+ is a better activator than Mg2+, and ADP has little effect. In Ascuris, therefore, the situation was that two enzymes involved in CO, metabolism were present. Saz and Lescure (1969) investigated this system further and found that the half maximum activity of PEPCK at saturating concentrations (Michaelis constant, Km) of PEP was about one seventh of that for oxaloacetate. This observation suggested strongly that PEP CK was acting in the direction of CO, fixation, whereas ME acted in the direction of decarboxylation (Saz and Hubbard, 1957). Such a situation would lead to a futile, energy-consuming cycle unless the enzymes were situated in different compartments within the cell. Studies of the distribution of the enzymes showed that PEPCK was located almost exclusively in the cytosol fraction, whereas the greater portion of the NAD-dependent ME was found in the mitochondria. About 20% only was soluble (Saz and Lescure, 1969), but its presence in the cytosol could well be due to leakage from damaged mitochondria. The differentialdistribution of the two enzymes led Saz and Lescure (1969) to propose a hypothesis for the control of end product formation which will be discussed in a later section. However, one additional observation may be noted here: Papa et al. (1970) examined ME from Ascaris muscle mitochondria and found that it reacted with both NAD and NADP. Saz and Lescure (1969) do not state whether the mitochondria1 enzyme used in their study reduces NADP, and further clarification is required. Other nematodes have not been examined in such great detail. Haemonchus contortus larvae possess both PEPCK and ME (Ward et al., 1968a). Like the Ascaris enzyme, H . contortus PEPCK shows marginally greater activity

C A R B O N DIOXIDE UTILISATION I N PARASITIC HELMINTHS

41

with IDP than with GDP. ADP is much less effective than either, and Mn2+ is required. It differs from Ascaris in that some 30% of activity is associated with the mitochondrial fraction. The intracellular localisation of ME was not determined, but, unlike Ascaris, it proved to be NADPdependent. However, as it was assayed in crude homogenates the authors were not able to determine whether it could use NAD, because of interference from malic dehydrogenase. Thus, little reliance can be placed on this observation. Finally, two other enzymes for which PEP is the substrate and which have not previously been mentioned, phosphoenolpyruvate carboxylase (E.C.4.1.1.3 1) and phosphoenolpyruvate carboxytransphosphorylase (E.C.4.1.1.38), could not be detected. The last two enzymes are probably also absent from Trichinella spiralis larvae (Ward et al., 1969). Their absence is hardly surprising as they are characteristic of plants and micro-organisms. T. spiralis possesses both PEPCK, with normal requirements, and NADP-dependent ME. Finally, there is a group of nematodes in which only PEPCK has been demonstrated; ME is either absent or present in activities below the level of detection. Vaatstra (1969) reports the presence of PEPCK in supernatant fractions from Dictyocaulus viviparus, but was unable to find ME. Srivastava et al. (1970a), in an investigation of glycolysis in Litomosoides carinii, found PEPCK but did not assay for ME. Similarly PEPCK, but not ME, is known from Obeliscoides cuniculi (Lee and Fernando, 197l), Nippostrongylus brasiliensis (D. K. Saz et al., 1971) and Syphacia muris (van den Bossche et al., 1971). Hymenolepis diminuta is the cestode which has been most closely studied. Prescott and Campbell (1965) found both PEPCK and NADP-dependent ME in this tapeworm. The former enzyme was present primarily in a supernatant fraction from the worm; only 7 % was associated with the pellet produced after centrifugation at 20000g. O n the other hand, ME was associated with the particulate fraction. Saz et al. (1972) subsequently confirmed these findings and showed that the mitochondria1ME had an absolute specificity for NADP. They were not, however, able to determine whether or not the enzyme occurred in the cytosol. Scheibel and Saz (1966) found that the main fate of radiocarbon from I4CO2was incorporation into succinate; the enzyme responsible proved to be PEPCK (Bueding and Saz, 1968). Echinococcus granulosus scoleces have been studied by Agosin and Repetto (1965). Three distinct enzyme activities were detected; a fourth (CTPdependent pyruvate carboxylase) is probably a manifestation of PEPCK, which is very active in this animal, and the properties of which are similar to those of other PEPCK. The fact that PEP alone supported a low level of CO, uptake suggested to the authors that a PEP carboxylase other than PEPCK was present. In the absence of further corroboration, and in view of the high activity of PEPCK in E. granulosus, this suggestion seems unlikely. Moniezia expansa also possesses an active PEPCK, but NADP-dependent ME activity is very low. The latter enzyme does not show the same absolute specifcity for NADP as that from Hymenolepis diminuta, and although it is largely mitochondrial, a substantial proportion of the activity occurs in the

42

C . BRYANT

cytosol fraction (Bryant, 1972a; Behm and Bryant, 1974). A low level of pyruvate carboxylase, requiring ATP, acetyl CoA and Mga+ for optimal activity, was found, but its activity was one tenth that of PEPCK. PEPCK itself exists in two forms; one is cytosolic, and the other mitochondrial. They have different cofactor requirements; the soluble one is activated by both Mg2+ and Mn2+,whereas the particulate enzyme is specific for Mn2+(Behm and Bryant, 1974.) Of the trematodes, only Fasciola hepatica has been explored in any depth. Prichard and Schofield (1968a) showed that PEPCK was responsible for the fixation of CO, into organic acid end products in F. hepatica, that it did not differ from the enzyme obtained from other sources, and that it was located mainly in the cytosol fraction. NADP-dependent ME was also present, and was distributed evenly between supernatant and mitochondrial fractions. Pyruvate carboxylase was not found. The existence of ME was subsequently confirmed by Sturm et al. (1969), who reported that it acted mainly in the direction of decarboxylation, and that of a carboxylating PEPCK was confirmed by de Zoeten el al. (1969). The only other reports of the existence of PEPCK in trematodes are those of Bueding and Saz (1968), who detected its presence in male and female Schistosoma mansoni, and Kohler and Stahel (1972) and Kohler (1972) who found it in Dicrocoelium dendriticum. The Acanthocephala are likewise a neglected group; Moniliformis dubius is the only representative commonly studied. In this animal also PEPCK is the predominant CO, fixing enzyme; traces of NAD and NADP-dependent ME are present in both larvae and adults (Horvath and Fisher, 1971). A more complete study by Korting and Fairbairn (1972) confirmed the presence of PEPCK, but could not detect ME in the adult, and found traces only in cystacanths . The distribution of the enzymes which mediate carbon dioxide incorporation is summarised in Tables I and 11. They show clearly that PEPCK is present in all helminths so far studied, and that the status of ME, whether NAD- or NADP- dependent, needs further elucidation. 111. THE ROLES OF PEPCK A.

AND

ME

GENERAL

At this point it is appropriate to summarise the roles that PEPCK, ME and pyruvate carboxylase play in intermediary metabolism in better studied systems, and then to contrast them with their roles in parasites. Liver pyruvate carboxylase is the first step in the gluconeogenic pathway from pyruvate, and, under certain conditions, has been shown to be rate limiting (Williamson et al., 1969a,b; Sijling et al., 1968). The control of the enzyme is complex, as the rate of the reaction it catalyses is affected by substrate concentrations (pyruvate, MgATP2-), activators (acetyl CoA, /?-hydroxy butyryl CoA) and inhibitors (PEP, acetoacetyl CoA) (Utter and Fung, 1971). The absolute requirement for acetyl CoA as a modulating activator is important because while pyruvate can traverse the mitochondria1

TABLB 1

Malic enzyme ( M E ) and lactate dehydrogenase ( L D H ) activities in some parasitic helminths

Parasite

Preparation

Developmental stage

NEMATODA Ascaris lumbricoides

excised muscle

adult

whole animal cytosol

adult

Nippostrongylus brasiliensis Diroflaria immitis Obeliscoides cuniculi

Strongyloides ratti Trichinella spiralis Haemonchus contortus Syphacia muris Litomosoides carinii Chandlerella hawkingi Ascaridia galli

CESTODA Hymenolepis diminuta TREMATODA Fasciola hepatica Schistosoma mansoni Schistosoma japonica Dicrocoelium dendriticum

adult adult? adult 2 4th stage larvae whole animal cytosol 3rd stage larvae (free-living) infective larvae free-living adults whole animal parasitic adult 9 1st stage larvae larvae whole animal cytosol .. whole homogenate (ME), 3rd stage larvae cytosol (LDH) whole animal cytosol adult 9 whole animal cytosol adults whole animal cytosol microfilaria whole animal cytosol mean adult ?+ 8 whole animal cytosol

sonicated mitochondria

(ME)

whole animal cytosol (LDH) whole homogenate (ME) whole animal cytosol whole animal cytosol whole animal cytosol

ACANTHOCEPHALA Moniliformis dubius

+

adults

whole animal cytosol

adults mean adult ?+ 8 mean adult ?+ .~ d adults

{~ ~ ~ ~ & n t h s

ME activity LDH, activity (nmoles/min/mg protein) (nmoles/min/mg protein

+ (NAD dependent;

References

143

1.2

127

3

N.A N.A N.A N.A N.A

3247 19.5 (pH 8.5) 13 (PH 7.0) 29 (pH 8.5) 20 (PH 8.5) 12 (pH 7.0) 8 (pH 8.5 11 (pH 7.0)

4

0

130 67 28 114 399 47

mainly mitochondrial) N.A

0 0 0

53 5.4 N.A N.A N.A N.A

38.3 1365 1900 (pyr +lad) 38 (lact 'pyt) 6.1

124 OOO

353

+

N.A N.A N.A 0

1.2

23 1145 815

5

6 7 8,9 10 11

12 13

2, 14 15, 16

2

2

463

17

577 590)

18

N.A = not assayed; = present, specific activity unknown References: 1 , Sar and Lescure 1969.2 Buediw and Saz 1968: 3, D. K. Saz et al. 1971. 4 Hutchison and McNeill 1970. 5 Lee and Fernando 1971' 6 KBrting and Fairbairn?1971; 7, Ward et ab, 1966; 8, Wdd and Schofield, i967a; 9, Ward et al., 1968b: lO.;an den Bossche eta!., 197;; 11, &vastava et al., 1970;; 12.8ri;astava et al., 1968; 3, Snvastava et al., 1970b; 14, Saz et al., 1972; IS. 16. hchard and Scbofield, 1968a,c; 17, Kohler, 1972; 18, KBrtmg and Fairbalm, 1972.

TABLE I1 Pyruvate kinaselphosphoenolpyruvatecarboxykinase activity ratios (PKIPEPCK) in some parasitic helminths Parasite

Preparation

Developmental stage

NEMATODA Ascaris Iumbricoides excised muscle Nippostrongylus whole animal cytosol brasiliensis whole animal cytosol

Obeliscoides cuniculi whole animal cytosol

Strongyloides rutti Trichinella spiralis Haemonchus contortus Syphacia muris Litomosoides carinii

whole animal

CESTODA whole animal cytosol Hymenolepis diminuta TREMATODA whole animal cytosol Dicrocoelium dendriticum whole animal cytosol Schistosoma mansoni ACANTHOCXPHALA Moniliformis whole animal cytosol dubius

PEPCK. (nmoles/min/ mg protein)

PK/PEPCK 004 1.86

adult adult

7 160

168 86

adult adult b+d

8 453*

N.A

1182 1383 1098 2576

55 78 87 39

Adult 9 [adult 8 { 4th stage larvae 3rd stage free-living [larvae (infective larvae free-living adults parasitic adult 9 1st stage larvae larvae

whole animal cytosol whole homogenate (PEPCK) cytosol (PK) whole animal cytosol whole animal cytosol. whole animal lyophilised extract whole animal cytosol

PK activity (nmoleslmin mg protein)

Major end product

h

References

succinate. etc. succinate and lactate

43

2 3 4 19

5 J

2.2

43

31 32 51 25 140

0-31

succinate, etc.

14

23

0.62

?

9,20

adult 9 adults adults

6 137 76'

438 49 158

001

?

10

adults

323*

170

1.9

adults

105

583

0.18

adults

13

"approx. 254"

~0.05

1150 2030

119 408

z:!j'

}lactate

31 8

90 68

0.34 0.12

some }lactate, succinate.

3rd stage larvae

{g f k k n t h s

69 56 7

40

N.A = not assayed. * = pyruvate kinase. known to be activated by fructose1 ddiphosphate References: 1-18 ash Table I; 19, Brazier and Jaffe, 1973; 20, Ward ef a/..1968;; 21, Kohler and Stahel, 1972

1.8

1 7

0.14 ( ' 1.6 J

succinate, some lactate lactate, propionate, succinate, acetate

6 7

2

17,21 2 18

c1

m

w .e 5 2

1

C A R B O N DIOXIDE UTILISATION I N PARASITIC HELMINTHS

45

membrane, acetyl CoA cannot. Pyruvate carboxylase is a mitochondrial enzyme, and hence gluconeogenesis, which takes place in the cytosol, is made sensitive to the rate at which oxidative metabolism proceeds in the mitochondria. This mechanism illustrates the great importance of compartition in metabolic regulation. Thus, under conditions of ample substrate the oxidation of fatty acids or pyruvate yields acetyl CoA and sufficient ATP to meet tissue demands. The reduction level of the electron transfer system is increased and acetyl CoA accumulates. The accumulation accelerates the pyruvate carboxylase reaction causing the formation of oxaloacetate. Reducing conditions within the mitochondria favour the rapid conversion of oxaloacetate to malate, which then leaves the mitochondria1 compartment for the cytosol. When insacient substrate is available, the level of acetyl CoA falls and the pyruvate carboxylasereaction is inhibited. Oxidative metabolism is accelerated. Under gluconeogenic conditions, PEPCK plays an important role in vertebrates. Malate, derived from the mitochondria in the manner described above, is oxidised to oxaloacetate. This then serves as a substrate for PEPCK and, in the presence of GTP or ITP and Mn2+, is decarboxylated to yield PEP and CO,, and with the formation of GDP or IDP. The sequence of reactions is summarised in Fig. 1, which is a very much simplified version of a scheme initially proposed by Lardy et al. (1965). It illustrates two points which are of relevance when considering the nature of the enzymes from GLUCOSE

PYRUVATE

-

PHOSPHOENOL PY RUVATE

co2d

PYRUVATE

FATTY ACID OXIDATION

+ - - -

4

- ACETY L CnA

phosphoenolpyruvate carboxykinase (PEPCK)

OXA LOAC ETAT E

I-1

MALATE

OX. .LOACETATE

MALATE

Ro. 1. A simplifiedview of gluconeogenesisin some mammals, showing the role of PEPCK. The dashed arrow symbolises the activation of pyruvate carboxylase by acetyl CoA. The mitochondrion is represented by the double-lined box. Reactions written outside the box occur in the cytosol.

46

C. BRYANT

parasitic helminths. The first is that PEPCK in vertebrates operates primarily in the direction of decarboxylation;and the second, that pyruvate carboxylase is a key enzyme in the system. ME has no role to play in gluconeogenesis. There still remains a large number of puzzling observations. Thus, in rat liver, for example, there are cytosolic and mitochondria1 PEPCKS with similar kinetic and physical properties, yet immunological comparison suggests they are distinct enzymes (Ballard and Hanson, 1969). ME (NADP-dependent)in mammalian adipose tissue is a cytosolic enzyme, and its function appears to be to provide a source of reduced NADP for fatty acid synthesis. In muscle, the distribution of the enzyme varies. Heart muscle from rat, guinea pig and rabbit contains far more intra- than extramitochondrial enzyme. This observation is also true for the seagull, but not for the pigeon. Other tissues also are variable with respect to the intracellular localisation of ME; rat and guinea pig liver do not possess the intramitochondrial enzyme at all (Nolte et al., 1972). These workers suggest that, as pyruvate carboxylase is absent from muscle (Utter, 1959; Bottcher et aZ., 1969), ME provides a route for the synthesis of oxaloacetate. Thus, in non-hehinth systems, there is evidence for the activity of ME in both the CO, king, and in the decarboxylating directions. The direction in which ME and PEPCK operate in parasitic helminths is important to ascertain, and is best achieved by examining the end products of carbon dioxide incorporation. This will be done in the next section. B. THE ROLES OF PEPCK AND ME IN PARASITIC HELMIINTHS

It is often stated that parasitic helminths produce a wide range of metabolic end products. The range includes, in many cases, lactic and succinic acids, fatty acids and even ethyl alcohol (von Brand, 1966). It would be a redundant exercise to review all the papers which describe respiratory endproduct formation in nematodes. This section will be restricted to some of the more recent observations in relation to the fate of radiocarbon from CO,. In addition, consideration will be given to the possibility that a proportion at least of the carbon contributes to gluconeogenesis. Many of the early papers of Saz and co-workers drew attention to the fact that CO, fixation commonly resulted in the formation of succinate and subsequent metabolic products in Ascaris. Only relatively recently has attention been directed at alternative routes for the metabolism of carbon from this source. Thus, Saz and Lescure (1967) showed that both larvae and muscle strips from adult A . Zumbricoides were capable of utilising NaH14C0, in the synthesis of glycogen and that this activity depended on the presence of GTP or ITP, clearly implicating PEPCK. Whether the enzyme was primarily concerned with PEP or oxaloacetate synthesis in this animal could not be ascertained. The evidence, however, seemed to suggest that it was capable of filling both roles. The possibility that two forms of the enzyme exist, in different cellular compartments, could not be ruled out. Further exploration of the fate of radiocarbon from CO, led Saz and Lescure (1969) to propose an hypothesis to account for the formation of respiratory end products in

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47

Ascaris. It was found that CO, stimulated the formation of pyruvate from PEP in Ascaris muscle homogenates, and that, as remarked earlier, the Km of PEPCK for PEP was only one seventh of that for oxaloacetate. The greater a f i t y for PEP therefore suggested that PEPCK operated in the carboxylation direction, in contrast to mammalian systems. The additional fact that ME is primarily mitochondrial, whereas PEPCK is found in the cytosol, suggested that the pathway of glucose oxidation is by glycolytic reactions to the level of PEP, at which point PEPCK catalyses the formation of oxaloacetate, which is then reduced to malate. It was then suggested that malate passes through the mitochondrial membrane; evidence of this process was obtained by Papa et al. (1970) who found that butylmalonate, an inhibitor of malate transport across mitochondrial membranes, diminished the reduction of nicotinamide nucleotides and flavoproteins within Ascaris mitochondria. Malate is thought to undergo a dismutation inside the mitochondrion, one portion being converted to pyruvate and acetate, the second portion being converted to fumarate, succinate and propionate in accordance with the accompanying reaction scheme (Fig. 2). FATTY ACIDS

c

$(+HI

1

ACETATE

(-HI

PYRUVATE

-

NADH ------+ NAD

I

PYRUVATE A

IfCo2

NAD-

NADH

I

MALATE

NAD +-NADHJ----J SUCCINATE I

(+ATP)

I

FIG.2. The anaerobic oxidation of glucose by Ascaris showing the roles of PEPCK and ME (modified after Saz, 1972). The mitochondrion is represented by the double-lined box Reactions written outside the box occm in the cytosol.

48

C.

BRYANT

There is considerable merit in this hypothesis. Saz and Lescure (1969) performed experiments with Ascaris muscle mitochondria which showed that radiocarbon from malate was distributed more or less equally between the pathways. The stoichiometry of the system with respect to oxidised and reduced NAD is good. Zee and Zinkham (1968) have reported that multiple malate dehydrogenases occur; three distinct forms are in the cytosol fraction and only one is present in mitochondria. An elegant study by FischerovL and KubistovL (1968) showed that Ascaris muscle strips were capable of maintaining ATP levels in the presence of malate when the glycolytic pathway was inhibited, and Saz and Lescure (1969) and Saz (1971b) have confirmed that malate-supported phosphorylation takes place in Ascaris mitochondria, an observation necessary to the establishment of the validity of the proposal. A similar pathway probably occurs in Haemonchus contortus (Ward et al., 1968b), in Syphacia muris (van den Bossche et al., 1971) and in Trichinella spiralis larvae (Ward et al., 1969). Saz (1970, 1971a, 1972) has discussed this scheme at length. However, while the pathway outlined here is of frequent occurrence in nematodes, it is not universal. Thus, in Dictyocaulus viviparus, Vaatstra (1969) was unable to detect ME. The presence of most of the enzymes of the tricarboxylic acid cycle (the oxoglutarate dehydrogenase complex and fumarate hydratase were absent) led him to suggest a metabolic pathway in which CO, is fixed via PEPCK into oxaloacetate, which then undergoes conversion to malate and fumarate. Fumarate is converted to succinate by a flavo-protein dehydrogenase (for a review of electron transport systems in helminths see Bryant, 1970) which may then either be excreted, or may enter the mitochondrion where it may be oxidised further. In another nematode, Ancylostoma caninurn, Warren and Poole (1970) found yet another point of divergence from the one which is rapidly becoming the classic Ascaris model. Although propionate was a major end product of glucose oxidation, the branched chain fatty acids were apparently derived from amino acid metabolism. If Ascaris lumbricoides is the classic nematode, Hymenolepis diminuta is rapidly becoming the doyen of cestodes. Reference has already been made to the work of Prescott and Campbell (1965) which established the distribution of the carbon dioxide metabolising enzymes in this tapeworm. Subsequent studies have confirmed that H. diminuta excretes both lactate and succinate; these products have been identified in protonephridial canal fluid (Webster, 1972). Scheibel, Saz, Bueding and co-workers have also provided considerable information which supports the view that, in H. diminuta, there exists the anaerobic respiratory pathway, involving malate penetration of mitochondria, which has been described for Ascaris. Scheibel and Saz (1966) found that CO, fixation was primarily in the direction of succinate formation, although some gluconeogenesis occurred, and that the tricarboxylic acid cycle and the pentose phosphate shunt made little contribution to the overall metabolism of the parasite. In addition, Saz et al. (1972) detected a transhydrogenase capable of reducing NAD in the presence of NADPH. This is an important observation as the fumarate reductase system is NAD-linked. In

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49

the absence of a transhydrogenase, it would be difficult to see how malic enzyme, which is NADP-dependent, could make a contribution to the energy producing system in H. diminuta. It therefore seems that the anaerobic mitochondrion of H.diminuta, like that of Ascaris, has the function of catalysing a malate-dependent, electrontransport associated phosphorylation (Fig. 3). A similar pathway may obtain LACTATE

2 /

1

PY R U V ATE

t

I I

I

I

I

I

J-

MALATE

ACETATE

PYR UVATE

malic

enzyme (ME)

MALATE

transhydrogenase

F UMAR AT E NADH -NAD SUCCINATE

(+ATP)

FIG.3. The anaerobic oxidation of glucose by Hymenolepis showing the role of the transhydrogenase. The path to malate is as indicated in Fig. 2. The mitochondrion is represented by the double-lined box. Reactions written outside the box occur in the cytosol.

in Echinococcus granulosus scoleces (Agosin and Repetto, 1965; Dicowsky et al., 1968). However, Moniezia expansa may have a greater resemblance to DictyocauIus viviparus, as ME was barely detectable in whole homogenates (Bryant, 1972a), and even in more carefully prepared cytosol and mitochondrial fractions its activity is very low (Behm and Bryant, 1974). In Fasciola hepatica, the incorporation of radiocarbon into propionate from CO, has been noted by Lahoud et al. (1971a), although the acetate

50

C. B R Y A N T

produced was unlabelled. The absence of label suggests that acetate is not derived from the pyruvate formed from malate. Further work by this group indicated that, like Ancylostoma caninum, many of the branched-chain fatty acids normally produced by Fasciola hepatica are derived from the dissimilation of amino acids (Lahoud et al., 1971b). Prichard and Schofield (1968b) and Buist and Schofield (1971) found that the tricarboxylic acid cycle and pentose phosphate shunt were unimportant in the liver fluke. Although many enzymes of the cycle were present, aconitase and fumerase activities were absent from the mitochondria (although the latter enzyme occurred in the cytosol), and NAD-specific isocitrate dehydrogenase was absent from both mitochondria and cytosol. Many of these observations have been confirmed by de Zoeten et al. (1969), and two schemes for respiratory metabolism in the liver fluke have been published. The scheme of de Zoeten et al. (1969) and de Zoeten and Tipker (1969) is similar to the one proposed for Dictyocaulus viviparus. It involves fixation of CO, into PEP to give oxaloacetate, malate and fumarate. Fumarate is converted to succinate by an NADH-dependent fumarate reductase. It is not clear from the papers where this occurs. As fumarate reductase activity is associated only with mitochondria (de Zoeten and Tipker, 1969), the formation of succinate in the cytosol, as their figure suggests, is precluded. In any event, succinate once formed is either excreted or is converted to succinyl CoA in the mitochondrion. Two possible routes are now available for its further metabolism: decarboxylation to propionate, or partial oxidation. A flaw in the scheme is that no role is proposed for malic enzyme. Prichard and Schofield (1968a) have put forward an alternative suggestion which bears a strong resemblance to those discussed earlier for Hymenolepis (Fig. 3). If malate can be shown to penetrate Fasciola mitochondria and if a transhydrogenase exists, all requirements for the proposal are met. Few other trematodes have been studied. The schistosomes constitute a special case and will be considered in the next section. In Dicrocoelium dendriticum, the end products of glucose oxidation have been identified as lactic, acetic and propionic acids, with small amounts of succinate and CO,; such information as is available suggests that respiratory metabolism takes place according to the pathway suggested by Prichard and Schofield (1968a) for Fasciola (Kohler and Stahel, 1972; Kohler, 1972). Little has been accomplished with the Acanthocephala. Monilformis dubius excretes both ethanol and succinate (Crompton and Ward, 1967a), and lactate and succinate are excreted by Polymorphus minutus (Crompton and Ward, 1967b). The pathway to succinate in these helminths probably follows one of the routes involving PEPCK and malic enzyme (Horvath and Fisher, 1971; Korting and Fairbairn, 1972).

Iv. PEPCK/PK RATIOSAND

THE

PATH TO LACTATE

The foregoing sections have concentrated on the role of the CO, fixing enzymes, and the subsequent fate of radio-carbon from this source. Many features common to a number of parasites have been noted, but emphasis on

C A R B O N DIOXIDE U T I L I S A T I O N IN P A R A S I T I C HELMINTHS

51

these similarities leads to a rather simplistic view of parasite metabolism. The real situation is more complex. The main problem centres around the metabolism of PEP. There is an important alternative route for its metabolism which is similar to the pathway of glycolysis found in mammalian muscle, for example. Thus, under the influence of pyruvate kinase, and in the presence of ADP, PEP is converted to pyruvate with the generation of a molecule of ATP. Pyruvate may subsequently be converted to lactate in the presence of NADH. Lactate is a frequent excretory product of parasitic helminths. In terms of energy yield the pyruvate kinase reaction is the equal of the PEPCK pathway, in which a molecule of ITP is formed which presumably serves to phosphorylate ADP. In each case, a subsequentreaction (oxaloacetateto malate, or pyruvate to lactate) reoxidises NADH produced in the earlier part of the glycolytic pathway. Very often, the relative proportions of the terminal products of respiration depend on whether parasites are maintained under aerobic or anaerobic conditions. Thus, von Brand et al. (1968) reported a marked depression in the amount of succinate produced by Taenia taeniaeformis under aerobic conditions when glucose was present. At the same time, lactate production increased. Bryant (1972a) pointed out a conflicting situation in Moniezia expansa. Thus, under aerobic conditions and in the absence of added glucose, much more radioactivity was incorporated from NaHl4CO3into succinate than into lactate. When glucose was present, much more lactate was formed. More recent studies, in which absolute metabolic pool size was measured, indicate that under both aerobic and anaerobic conditions, lactate is a major product and that its production increases under anaerobiosis (Behm and Bryant, 1974). Saz (1971a), in a review of anaerobiosis in invertebrates, suggested that an important control point in the respiration of parasitic helminths existed at the level of PEP, and that the nature of the end product formed depended on the competition for PEP by the two enzymes, PEPCK and pyruvate kinase (PK). The latter enzyme is well known to be regulated in mammals by a number of allosteric effectors, including fructose-1,6-diphosphateand ATP/ ADP ratios (Bailey et al., 1968; Garber and Ballard, 1970). Moreover, it exists in two characteristic forms, one of which, associated with the liver, is activated by fructose-l,6-diphosphate;and the other, a muscle form, is not so affected. In many parasites, therefore, it seems probable that competition for substrate by PEPCK and PK could account for the apparent shift in the proportions of succinate and lactate formed when oxygen is present in the maintenance medium (see, for example, D. K. Saz et al., 1971). However, there is a group of parasites (which need not be taxonomically related) which have been described as homolactate fermenters. Schistosoma mansoni, which possesses PEPCK, belongs in this group (Bueding and Saz, 1968). Homolactate fermenters produce lactate as a sole end product of respiration, but there is, for practical purposes, a continuum between this group and the succinate producers in which the relative proportions of these two end products change.

52

C . BRYANT

In order to test the competition hypothesis of Saz, ratios of the activities of PK to those of PEPCK have been determined in a number of parasitic helminths (Table 11). Bueding and Saz (1968) first determined the ratio in Ascaris muscle; its value is 0.04, suggesting that PEPCK is by far the most active enzyme, and thus accounting for the heavy emphasis on the production of succinate and its derivatives in this nematode. Other values that have been obtained for nematodes include, for example, 1986 for Nippostrongylus brasiliensis (D. K. Saz et al., 1971). This would suggest a greater emphasis on lactate production; indeed, the total amount of lactate produced by this worm from glucose under both aerobic and anaerobic conditionsis variable, sometimes exceeding the amount of succinate, sometimes less. However, when conditions are changed there is usually a greater change in the lactate pool than in the succinate pool. In Obeliscoides cuniculi, the ratio was found to vary slightly between developmental stages (Lee and Fernando, 1971). For the adult female the value is 21.5; for the adult male, 17.7; for the fourth stage larva, 12.6; and for the free-living third stage larva, 65.8. It would appear reasonable to expect lactate as an excretory product of 0. cuniculi. Unfortunately, the activity of LDH in this animal is low, so that the fate of the pyruvate produced by pyruvate kinase is not known. A similar study, carried out on Strongyloides ratti (Korting and Fairbairn, 1971), produced values of 2.2 for infective larvae, 1-8 for free-living adults, 0.14 for parasitic females and 1.6 for first stage larvae. Clearly there is a marked difference between the parasitic females and the other We-cycle stages which led the authors to suggest that the parasitic female was anaerobic. In larvae of Trichinella spiralis the PK/PEPCK ratio was 0-31 (Ward et al., 1969), and in Haemonchus contortus larvae the value obtained was 0-62(Ward et al., 1968a,b). The former parasite excretes a wide range of acid endproducts, but little lactate (von Brand, 1966); the enzyme levels in the latter are consistent with the pathway previously shown to operate in Ascaris. Finally, the ratio found for the two enzymes in Syphacia muris by van den Bossche et al. (1971) is 0.01; and in Litomosoides carinii by Srivastava et al. (1970a) is 2.8. The excretory products of the former are not known; those of the latter are primarily lactate and acetate. Amongst the trematodes, Kohler (1972) and Kohler and Stahel (1972) reported a ratio of 20 for Dicrocoelium dendriticum, and in the Acanthocephala, Korting and Fairbairn (1972) found that Moniliformis dubius gave values of 0.34 (adults) and 0.12 (cystacanths). On the whole, the general hypothesis that Saz (1971a) put forward appears to be substantiated. However, a note of caution in interpreting PK/PEPCK ratios must be sounded. It has already been pointed out earlier in this section that, in mammals, PK is an enzyme subject to regulation, and that at least two forms are known to exist. Some recent papers have begun to explore the possibility that helminth PKs may also be subject to regulation. Prichard and Schofield (1968a) found PEPCK in the liver fluke but were

C A R B O N DIOXIDE U T I L I S A T I O N I N P A R A S I T I C HELMINTHS

53

unable to detect PK. Lee and Vasey (1970), however, detected this enzyme in extracts from Fasciola hepatica, and showed that it needed Mga+ or Mna+, high PEP concentrations, and a pH of 7.5. PEPCK was optimally active under different conditions. Lee and Vasey (1970) concluded that the situation in Fasciola resembled that in Hymenolepis diminuta. This conclusion, however, may be premature. H. diminuta excretes primarily succinate, and Bueding and Saz (1968) found that the PK/PEPCK ratio was 0.18, an excellent correlation. However, a recent note by Carter et al. (1972) describes no fewer than five isozymes of PK in this parasite. Although only two were modulated by FDP and ADP, and most of the activity was associated with the non-modulated isozymes, the value of the ratio may be modilied by activation. The situation can thus become very complex and interpretation very difficult. Brazier and Jaffe (1973), for example, report a ratio of 10.5 for crude extracts of male and female Dirofilaria immitis, but showed that the activity of PK from this source could be nearly doubled in the presence of 0.25 m~ FDP. Similarly, the ratio of 2.8 reported by Srivastava et al. (1970a) may be variable, as Brazier and Jaffe (1973) found that the enzyme from Litomosoides carinii had similar properties to that of the Dirofilarial enzyme. In Schistosoma mansoni, PK/PEPCK ratios of 5.0 and 9-7 have been reported for males and females respectively (Bueding and Saz, 1968). This animal produces lactate exclusively, even in the larval stage (Coles, 1972). No FDP modulation of PK was detected by Brazier and Jaffe (1973), although there was some sensitivity to changing ATP levels. In many of the cases quoted in Table 11, where the value for the PK/ PEPCK ratio is below 0.5 or above 5.0, modulation of PK activity probably would not affect the dominance of one pathway over the other except in degree. For ratios between these values, there is a real danger that changing the conditions of assay may have implications for the direction in which metabolism proceeds. Bryant (1972a,b) examined some of the properties of PK from Moniezia expansa. There are at least two, one of which is similar to the mammalian hepatic form, the other to the muscle form (Table 111). TABLE III

Variations in the activity of pyruvate kinase from Moniezia expansa with conditions of assay. (From Bryant, 1972b) Conditions of assay

+ Mg2+ + Mnz+ + Mgz + FDP (0.4 m ~ ) + Mna++ FDP (0.4 m ~ ) + Mn2 + FDP (0.4 m ~ ) + malate (1-0m ~ ) +

+

m

40 % (NH4)2S04fraction 50 % (NH4),S04 fraction (optimum pH 6.5) (optimum pH 7.0)

8 32 17 94

4 44

4 44

45

44

e activity expressed as nmoles/min/mg protein in fraction

54

C . BRYANT

Different ratios can be calculated under different conditions of PK assay. Thus, a value of 0.15 is obtained when PK is assayed in the presence of Mgz+ and no FDP; 0-28with FDP; 0-91when Mn2+is substituted for Mgz+in the absence of FDP; and 1.6 when MnP+and FDP are both present. To complicate matters further, malate, an intermediate which has not previously been implicated as an allosteric effector for PK, inhibits the FDP activated enzyme. This situation presumably reflects the fact that, in Moniezia expansa, considerable quantities of lactate are produced at all times. In the production of lactate by intestinal helminths, two additional points must be borne in mind. The first is that, in order to produce lactate, PK is not essential. Thus, if the scheme of Saz and Lescure (1969) is correct and malate is decarboxylated within mitochondria, the pyruvate so formed could act as a substrate for lactate dehydrogenase, and lactate would thus be derived from the following sequence of reactions: glycogen PEP --f oxaloacetate --f malate --f pyruvate --f lactate. A priori, the two major prerequisites for this metabolic scheme would be possession, by the parasite, of malic enzyme and lactate dehydrogenase. A survey of the literature shows that the situation with respect to the possession by parasites of lactate dehydrogenase at high activities is variable. Thus, in the following parasitic helminths the activity of lactate dehydrogenase is comparable to or greater than PK : Nippostrongylus brasiliensis, Litomosoides carinii, Diroflaria immitis, Trichinella spiralis larvae, Ascaridia galli, Ascaris lumbricoides muscle, Syphacia muris, Strongyloides ratti, Fasciola hepatica, Schistosoma mansoni, Dicrocoelium dendriticum, Hymenolepis diminuta and Moniliformis dubius. Only in Haemonchus contortus larvae and Obeliscoides cuniculi are the levels of PK considerably higher. Of the parasites listed here, malic enzyme has been shown to be present in Ascaris lumbricoides, Haemonchus contortus larvae, Trichinella spiralis larvae, Hymenolepis diminuta, Fasciola hepatica and possibly Moniliformis dubius (see Tables I and 11). Langer and Smith (1971) have recently examined the implications of the presence of lactic dehydrogenase in three parasites : Ascaris mum, Oesophagostomum radiatum and Haemonchus contortus. They determined the for the various substrates (lactate, NAD, apparent Michaelis constants (Km) pyruvate and NADH) and specific activities for the enzyme derived from each parasite. These results are presented in Table IV. Assuming that the observed specific activities approximated to the maximum velocities (omax) of the reactions, they calculated the concentration of lactate which would yield a rate of oxidation comparable with the rate of reduction of pyruvate. They further assumed the following relationship between v,,, and K, for the reaction :

-

v,,, u,,,

(pyruvate) - K,X,(pyruvate) (lactate) (lactate)

Thus, if the ratio of the maximum velocities for the reaction is multiplied by K , (lactate), a rate-corrected, apparent affinity constant is obtained. It may

TABLE IV Michaelis constants (K,,,) for the substrates and cofactors and the specific activities with lactate andpyruvate, of the lactic dehydrogenases of three nematodes. (From Langer and Smith, 1971) ~

Enzyme source

Lactate

K, (molar) for reactants NAD ~

Ascaris mum (6) Ascaris suum (9) Oesophgostomum radiatum Haemonchus contortus

~

~~~

Pyruvate

NADH

~~

~

~

~~

~

Specific activity for substrates (moles NAD(H) oxidised (reduced)/ng protein/min at 37") 0 Lactate Pyruvate 2:

~~

10.8 x 10-2 1.8 x 10-2 6-7 x

1 . 2 10-3 ~ 1.6 x 10-3 1.2 x 10-3

2.8 x 10-4 5.4 x 10-4 4.3 x 10-5

6.3 x 10-5 24 x 10-5 1 . 2 10-5 ~

112.2 88.6 173.0

48.4 38.0 5.0

1.1 x 10-3

1.5 x 10-3

1.2 x 10-3

5.5 x 10-6

147.5

3.3

56

C. B R Y A N T

then be compared with the observed K , for pyruvate. The results are summarised in Table V. Langer and Smith (1971) interpret the data as follows. For Haemonchus contortus, the calculated K , is one order lower than the observed value; a reaction rate for lactate oxidation equal to half the maximum rate of pyruvate reduction is therefore obtained with a lactate concentration one tenth that of pyruvate. Thus, little lactate should be produced by this organism. The calculated values for Oesophagostomum radiatum and Ascaris mum males and females are much greater than the observed values, so that the lactate pool size should be greater than that of pyruvate and lactate would probably be excreted. No data are available for 0. radiatum, but Ascaris does in fact produce lactate (von Brand, 1966). TABLE V Observed K, (pyruvate) compared with the calculated K, (pyruoate)for the lactic dehydrogenases from three nematodes. (Data from Langer and Smith, 1971) 0bserved K , (pyruvate) A . suum (male) A . s u m (female) H. contortus 0. radiatum

2.8 x 5.4 x 1.2 x 4.3 x

10-4 10-4

Calculated

K,,, (pyruvate) 4.6 x 7 x 10-3 2.4 x 10-4 1.9 x 10-3

10-3 10-6

This sort of treatment of enzyme kinetics is valuable as an aid to rationalising the ways in which metabolic pathways in parasites function, but the approach of Langer and Smith (1971) is probably too simple. Thus, any effect that NAD and NADH may have on the binding of lactate and pyruvate to the enzyme is not allowed for, nor is the fact that the authors themselves show that lactate dehydrogenase exists in multiple forms in each of the parasites. Kinetic data were derived from total lactate dehydrogenase; compartition within the worm may alter markedly the behaviour of the metabolic pools. However, it is easy to criticise; Langer and Smith (1971) are to be praised for bringing a quantitative approach to the study of the regulation of parasite respiration. In any case, the existence of multiple forms of lactate dehydrogenase is not necessarily the rule among parasites, as Burke et al. (1972) have found only a single enzyme in Hymenolepis diminuta. V. METABOLIC REGULATION A.

IN

PARASITIC HELMINTHS

GENERAL

A recent article by Atkinson (1971) summarises the philosophy which underlies the study of metabolic regulation. In particular, it emphasises the point that metabolic sequences are stoichiometrically coupled, and that

C A R B O N D I O X I D E U T I L I S A T I O N IN P A R A S I T I C H E L M I N T H S

57

coupling is achieved by the relative requirements of pathways for ATP, or by their capacity for producing it. Thus, respiratory systems which produce ATP are linked to biosynthetic systems which utilise it. The energy resident in adenine nucleotides may be considered to be an energy charge, which Atkinson defines as : [ATPI+&[ADPI charge = [ATP]+ [ADP]+AMP] Contributions are made to the energy charge by respiratory processes, and synthetic processes draw from it. Clearly, such processes are inversely rehted. The reaction rates of sequences which synthesise ATP should be inhibited by increased charge, and the rates of those that use ATP should increase with increased charge. Sensitivity of control is heightened if the responses are concentrated in one charge region, and not manifested equally over the whole range of energy charge values. An example of how such a system may operate in v i m is provided by the important gluconeogenicenzyme, fructose-1, 6-diphosphatase.It is inhibited by high concentrations of ADP, which would normally imply that the concentration of ATP, and hence the energy charge, within the cell had decreased. The formation of fructose-6-phosphate from fructose-l,6-diphosphateis thus an important regulatory step in glycogen synthesis. The above example is a case where an adenine nucleotide acts directly as a modifier of activity. A second situation involves alternative high energy compounds (acetyl CoA, for example) or compounds acting, as it were, at one remove from the ATP system. Thus fructose-1,6 diphosphate, the concentrations of which are regulated by the level of the energy change, is itself a modulator of pyruvate kinase activity. For this reason, and also because it is essential to know whether the reaction catalysed by a particular enzyme is at equilibrium or whether it is markedly displaced from its equilibrium point (which might implicate it as a regulator of the pathway of which it is part), it is imperative to have a knowledge of metabolic pool sizes in the tissue or organism which is being studied. Without this knowledge, no satisfactory account of the regulatory processes operating in the organism in question can be put forward. An additional complicationis that there are, in eukaryote organisms, quite distinct compartments within the cell. The same compounds may occur in each compartment, and their concentrations must be individually determined, and metabolic processes must be instantaneously arrested to enable determination. Gumaa et al. (1971) have illustrated these points in a recent essay, and offer methods for calculating ratios of substrates and products in a number of reactions occurring in different subcellular compartments in mammalian liver. Parasitic helminths present even more complex problems. Only in Ascaris, from which muscle strips can be derived relatively easily, has it been really practical to isolate a specific tissue. Generally, the starting material for studies of parasite biochemistry has been the whole animal, which thus has a further dimension of intercellular compartition. This fact must be remembered when reading the remainder of this section.

58

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If it is accepted that an essential requisite for a complete understanding of regulatory processes in parasites is a knowledge of metabolic pool sizes, then uptake of nutrients which play a role in the maintenance of pool sizes must also be considered. Helminth nutrition is an area of research which has been intensively studied, and the interested reader is directed to reviews by Rothstein and Nicholas (1969) and Jennings (1968). It is worth noting, however, that even the processes of uptake of nutrients may be subject to activation which may be allosteric. MacInnis et al. (1965) provided evidence for the mediated transport of purines and pyrimidines in Hymenolepis diminuta and noted that thymine stimulated uracil transport. Subsequently MacInnis and Ridley (1969) established that thymine was acting as an allosteric activator for uracil uptake. The kinetics of glycerol uptake by H. diminuta resemble those for uracil (Pittrnan and Fisher, 1972). In Fasciola hepatica, acetate absorption is increased by the presence of citrate in the maintenance medium (Isseroff and Walczak, 1971). This is by no means an exhaustive list. The few examples given here are chosen to illustrate a common phenomenon. Another area of considerable importance in the regulation in mammalian metabolic systems is the effect of hormones. Studies of hormone action in parasites are still in their infancy; the results obtained are problematical, and a lengthy review of this work would be, to say the least, premature. A few examples will suffice to point out the conflicting nature of the data. Mansour (1962) and Mansour and Mansour (1962) showed that homogenates of Fasciola hepatica responded by increased glycolysis to the addition of 5-hydroxytryptamine and adenosine3’,5’-phosphate.The target enzyme is ph.osphofructokinase.A similar effect was observed in homogenates of Taenia pisiformis, Schistosoma mansoni and Ascaris lumbricoides. Pantelouris (1964) used a histochemical method to demonstrate that liver flukes incubated in media to which insulin had been added contained less glycogen than controls incubated in the absence of the hormone. Isseroff and Read (1968) and Buist and Schofield (1971) failed to observe any effect of insulin, but Hines (1969) confirmed the results of Pantelouris by different techniques, though only in whole animals in which the oral suckers had been tied. Esch (1969) demonstrated a stimulation of carbohydrate metabolism in larval Taenia crassiceps during long-term incubations. Hutton et al. (1972) found no significant effects of thyroxine, histamine, epinephrine, nor-epinephrine, progesterone, testosterone or hydrocortisone on intact animals or homogenates of Fasciola hepatica. However, they agreed with the earlier observations of Mansour and Mansour (1962) that 5-hydroxytryptamine was active. B.

REGULATORY ENZYMES IN PARASITIC HELMINTHS

In this section, the enzymes which have been implicated in respiratory metabolic pathways, and which have been formally studied with a view to elucidating their importance in metabolic control, will be discussed. Only the more recent work will be described, as modern techniques have necessitated a reappraisal of older data. Ascaris will be the subject of a final section.

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In determining the value of much of the data that follows, a few words of preliminary explanation are required. In many instances, activitiesfor parasite enzymes are quoted in the original literature. They have not been reproduced here, for several reasons. The first is that, in order to give a meaningful estimate of specific activity, the peculiar characteristics of each enzyme must first be known. Such studies have not usually been carried out, and in most cases, activities have been determined with the use of assay techniques which have been developed for other, unrelated, organisms. Second, activities should be determined with purified enzymes which are free from possible inhibitors. Third, activities of enzymes are usually expressed as moles substrate utilised (or product formed)/mg worm protein/min. This is a seemingly objective standard, but makes comparison between different compartments in the helminth unreliable (the cytosol may contain more protein than the mitochondrion) and comparisons between helminths virtually impossible. Thus, an enzyme reported to have a very low activity may, in fact, be so distributed within the cell as to have a high local concentration and very high activity indeed. The value of lists of enzymes present in a particular parasite thus seems mainly to reside in giving an indication of what pathways of metabolism could be present, and perhaps in offering some possibility for internal comparison of enzyme activities within a single preparation. Ward and Schofield (1967a,b) have examined Haemonchus contortus larvae to determine whether the enzymes of the glycolytic sequence and the tricarboxylic acid cycle were present. All the enzymes of glycolysis were found, although PK activity required further work to establish its presence (Ward et al., 1968a). Ward and Schofield (1967a) did not consider that the enzymes differed markedly in activity from those of rat liver. Similarly, all the enzymes of the tricarboxylic acid cycle were found. The activity of citrate synthetase was much greater than that of rat liver; and whereas the NADP-dependent isocitrate dehydrogenase activity was lower, the NAD-dependent enzyme was active and was modulated by ADP. Thus, Ward and Schofield (1967b) concluded that the tricarboxylic acid cycle could operate under aerobic conditions. It is also of interest to note that, with the exception of the succinate oxidase system, the enzymes were found both in cytosol and particulate fractions; whether this represents the true situation, or whether it may be ascribed to the fact that methods for the preparation of parasite mitochondria are not nearly so advanced as those for mammalian mitochondria, is not clear. Glycolytic and tricarboxylic acid cycle enzymes have also been demonstrated in DirofiIaria immitis (Hutchison and McNeill, 1970; McNeill and Hutchison, 1971). While the activities of the glycolytic enzymes are consistent with the view that lactate could be an end product of respiratory metabolism in this nematode, low levels of aconitase and isocitrate dehydrogenase, and the fact that malate dehydrogenase activity could only be demonstrated in the direction of the malate formation, suggest that the tricarboxylic acid cycle does not function. These observationsindicate that a pathway similar to that described for Ascaris (see Section 111) may operate in DirofIaria immitis. Studies on Ascaridia galli (Srivastava et al., 1970b), Litomosoides carinii

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(Srivastava et ul., 1970a) and Chandlerella hawkingi (Srivastava et al., 1968) have also demonstrated the presence of glycolytic enzymes in these nematodes, suggesting that lactate is an end product of metabolism. In Fasciola hepatica, all the enzymes of glycolysis are present (Prichard and Schofield, 1968c; Lee and Vasey, 1970), although activity of lactate dehydrogenase is considerably lower than that of rat liver. Phosphofructokinase was also present at very low activities, which suggests that it is involved in the regulation of the glycolytic pathway as in mammalian tissues. The sensitivity of this enzyme to 5-hydroxytryptamine and 3’-5’AMP (Mansour and Mansour, 1962) is corroborative evidence.Although all enzymes of the tricarboxylic acid cycle are present (Prichard and Schofield, 1968b), the low levels of aconitase and NADP-specific isocitrate dehydrogenase, and the absence of the NAD-dependent enzyme, suggest that the cycle is of minor importance in the liver fluke. The presence of many of these enzymes has been confirmed by Sturm et al. (1969). In Dicrocoelium dendriticum all the glycolytic enzymes were detected by Kohler (1972), with rate-limiting steps catalysed by hexokinase and phosphofructokinase;as in Fasciola hepatica, pyruvate kinase and lactate dehydrogenase activities were low. Many of the glycolytic enzymes of Moniliformis dubius have been assayed (Korting and Fairbairn, 1972; Horvath, 1972); all the enzymes except PK were detected by the latter author, although it was felt that this could be an artefact. However, Korting and Fairbairn (1972) detected PK in adults and cystacanths. There is a marked discrepancy in the specific activities reported in the two papers: these were felt by Horvath (1972) to be due to different experimental techniques employed in each study. Such an observation points to the pitfall mentioned earlier. Little attempt has been made to characterise each enzyme. The roles of pH, activators or natural inhibitors need to be investigated before definitive specific activities can be obtained. C.

METABOLIC REGULATION IN

Ascaris

Although some of the enzymes of glycolysis in Ascaris have been found by a number of workers it is only very recently that an exhaustive study of this pathway has been carried out. Barrett and Beis (1973a) conducted investigations into the activities of glycolytic and associated enzymes in muscle. In addition they measured the steady-state content of phosphorylated glycolytic intermediates in freeze-clamped tissue. Freeze-clamping tissue has the advantage of bringing to an instantaneous halt all reactions in the tissue, and especially the unwanted degradations which often occur when less abrupt methods are used. In this study, worms were brought to the laboratory, muscle strips rapidly removed and flattened in Wollenberger clamps precooled in liquid nitrogen. An active glycolytic sequence of enzymes was present in muscle extracts. Their activities were comparable to those found in other tissues which carry out glycolysis, and were present in approximately the same ratios. There was little glucose-6-phosphatase activity, but fructose-1,6-diphosphatase(previously described in Ascuris by Saz and Lescure (1967) ) was present and may

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be involved in gluconeogenesis. Unlike mitochondria from Moniezia (Cheah, 1971), mitochondria1 a-glycerophosphate dehydrogenase activity was absent from Ascaris muscle, so that the electron-transport mediated phosphorylation system involving the oxidation of a-glycerophosphate which Cheah (1971) found, does not occur. If mass action ratios for the glycolytic enzymes are calculated from the steady-state concentrations of glycolytic intermediates in freeze-clamped muscle and compared with the reported equilibrium constants for the reactions, it is possible to detect which reaction steps are far removed from their equilibrium positions. These are the reactions which are the most likely candidates for regulation. The reactions catalysed by phosphofructokinase and PK are the only ones to conform to this requirement, as a further study (Barrett and Beis, 1973b) showed that glyceraldehyde-3-phosphatedehydrogenase and phosphoglycerate kinase were also in equilibrium. Phosphofructokinase and PK are important regulators in mammalian systems (see, for example, Gumaa et al., 1971); they also fill the same role in Moniezia expansa (Behm and Bryant, 1974.). As the rate-limiting step in Ascuris muscle is phosphofructokinase, and as phosphorylase is much more active than hexokinase, Barrett and Beis (1973a) suggest that, if the phosphorylase were acting at optimum rates, glycogen utilisation would approximate to 10 g/h/lOO g muscle at 30". The reserves were found to be 13 g/lOO g muscle, so that they would suffice for relatively short periods only. It is suggested that glycogen serves as an energy source during periods of increased activity and that under other circumstances glucose, which must first be phosphorylated by the much less active enzyme, hexokinase, is utiIised. There remain two rather mystifying features about glycolysis in Ascaris. The part played by PK remains obscure. Its activity is low, and the nonequilibrium of the reaction suggests that it should be regulatory; but glycolysis does not lead to the production of pyruvate. The alternative route to oxaloacetate, mediated by PEPCK, is far more active; this reaction also is not at equilibrium and must therefore be considered a candidate for regulation. In an extension of this study, Barrett and Beis (1973b) used similar techniques to determine the redox state of the free nicotinamide adenine dinucleotide couple in Ascaris muscle. The free NAD/NADH ratio gives a measure of that fraction of the dinucleotide which participates in dehydrogenase reactions, especially those in the respiratory pathway. In aerobic organisms and tissues this ratio has a value of the order of 1OOO; under anaerobic conditions it decreases markedly. Although attempts have been made to measure dinucleotide levels directly, the lability of the system renders such measurements unreliable. A more satisfactory method is to compute a value from measurements of concentrations of substrate and product of dehydrogenase reactions which approach equilibrium (Williamson et al., 1969~).Barrett and Beis (1973b) selected four enzymes; three were cytosolic (glyceraldyde-3phosphate dehydrogenase, malate dehydrogenase and lactate dehydrogenase). The ratios obtained were 785, 1393 and 2214 respectively. Malic enzyme, which is primarily in mitochondria, gave a value of 0.072.

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Clearly, these data show that the cytosol is relatively highly oxidised compared with the mitochondria, even though the animal is largely anaerobic. As Ascaris depends on glycolysis for much of its energy requirements it is important that the NAD/NADH ratio in the cytosol should be maintained at a high level. On the other hand, it is considered that the very low oxidation level in mitochondria is necessary for the reduction of fumarate to succinate, which is an NADH-dependent, energy yielding reaction. Ballard and Phillippidis (1971) described an experiment in which the cytosol and mitochondrial NAD/NADH ratios were investigated in livers from neonatal rats maintained in air and under nitrogen for 20 min. The cytosol NAD/NADH ratio was 614 in the control; it dropped to 139 in anoxic rats. Similarly the mitochondrial value dropped from 29.7 to 2.4. It would thus seem that Ascaris, which is able to maintain a relatively high cytosol NAD/ NADH ratio, is well adapted to its anaerobic habit. In the same experiment Ballard and Phillippidis (1971) calculated the adenylate energy charge according to the method ofAtkinson (1971). It fell from 0.82 under aerobic conditions to 0-598 under anaerobiosis. Barrett (1973) provides sufficient data to calculate an adenylate energy charge for Ascaris muscle. The value of 0.80 compares favourably with the value obtained for aerobic neonatal rat liver, suggesting that Ascaris is capable of maintaining a high level of ATP even though it lacks the classical mitochondrial oxidative phosphorylative system. In freeze-clampedAscaris muscle, Barrett (1973) found substantial quantities of ATP, ADP, AMP and GTP. In spite of the dependence of this tissue on PEPCK with a requirement for IDP or GDP, these were present only as traces. However, an extremely active nucleotide diphosphate kinase could rapidly catalyse the transfer of high energy phosphate from ITP or GTP to ADP, so that effective levels of TDP or GDP may be maintained. The relatively high concentration of GDP suggests that this is the nucleotide involved, rather than ITP. D.

CONCLUSIONS

The study of metabolic regulation in parasitic helminths is clearly rudimentary when compared with the state of the science in other organisms. However, there is an increasing awareness of the need for much more rigorous analysis in order to understand the ways in which metabolic pathways function in helminths, and recent papers reflect this need (e.g. Dedman et al., 1973). A relatively clear idea of how Ascaris metabolism is controlled is beginning to emerge, but many questions still remain to be answered. Of the other helminths, only Fasciola hepatica and Hymenolepis diminuta have been studied in anything like the same depth. It is plain that there is no necessary uniformity amongst parasites in the details of their metabolic pathways. Generalisations of any validity are almost impossible to make, since the size of the sample of different species of parasites investigated is disconcertingly small. Comparisons are equally difficult to achieve, because superficial similarities may mask quite different metabolic mechanisms. An example is the production of lactate which may occur by metabolic routes in which PK is present or absent. A fruitful area of comparison could be with organisms

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which are not parasitic. Intertidal molluscs possess metabolic pathways which have a similarity to those described in this review. The pathways are related not t o parasitism, but to enforced, temporary anaerobic conditions. It follows that many of the respiratory (and other) mechanisms of parasites reflect a response t o an environment with specific characteristics, and that the term “parasite” is without real meaning for the organism thus classified.

ACKNOWLEDGEMENTS I would like t o thank Mrs C. A. Behm for much fruitful discussion; she, together with Mrs A. Chilcott and Miss R. Cornish, attacked the inaccuracies of the manuscript with great enthusiasm, for which I am very grateful. A n y that remain are my responsibility. I would also like t o express appreciation t o the Rural Credits Fund of the Reserve Bank of Australia for financing such of my own work as appears in this review. REPERENCES

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