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The tricarboxylic acid cycle and associated reactions in Moniezia expansa (Cestoda)
Comp. Biochem. Physiol., 1969, Vol. 31, pp. 503 to 511. Pergamon Press. PHnted in Great Britain
THE TRICARBOXYLIC ACID CYCLE AND ASSOCIATED REACTIONS IN M O N I E Z I A E X P A N S A (CESTODA) R. A. DAVEY and C. BRYANT Department of Zoology, School of General Studies, The Australian National University, Canberra, A.C.T., Australia (Received 14 March 1969) A b s t r a c t - - 1 . Experiments with radioactive substrates have provided evidence
for the presence of the tricarboxylic acid cycle in Moniezia expansa. 2. Incorporation of 1'CO2 probably involves both "malic enzyme" and phosphoenol pyruvate carboxykinase.
INTRODUCTION ALTHOUGH Cheah & Bryant (1966) demonstrated that a major respiratory pathway in preparations from Moniezia expama utilized fumarate as an electron acceptor, the possibility remained that a small proportion of respiration involved molecular oxygen. Subsequently, Cheah (1968) showed that, while the major cytochrome component of M. expansa was implicated in the fumarate-succinate transformation, it was also possible to detect an orthodox cytochrome system which possessed very low concentrations of a cytochrome oxidase. It was therefore assumed that this pathway was partially responsible for the small oxygen uptake observed when mitochondrial preparations from M. expama were incubated with succinate, a-glycerophosphate or NADH. Prichard & Schofield (1968a) successfully demonstrated the presence of enzymes of the tricarboxylic acid cycle in Fasciola hepatica. Protoscolices of Echinococcus granulosus and Ascaris muscle have also been shown to have an active cycle (Agosin & Repetto, 1963; Oya et al., 1965). Prior to this work the existence of the tricarboxylic acid cycle in parasitic helminths was problematical as the accumulation of succinate, and hence the anaerobic component of respiration, were of more immediate significance. The only cestode hitherto investigated which has been shown to possess an active tricarboxylic acid cycle is E. granulosus (Agosin & Repetto, 1963). Scheibel & Saz (1966) investigated intermediary metabolism in Hymenolepis diminuta and concluded that the tricarboxylic acid cycle was not active. Other cestodes have also been shown to accumulate succinic acid, and as early as 1933 this was demonstrated in M. expama (yon Brand, 1933, 1966). It was therefore of considerable interest to determine whether the cytochrome oxidase system of M. expama was associated with tricarboxylic acid cycle activity. 503
504
R. A. DawY AND C. BRYA~rr
MATERIALS AND METHODS Adult M. expansa were obtained from slaughtered lambs at the Queanbeyan Abattoirs, N.S.W., and stored on ice in Ringer's solution. They were used within 4 hr of collection. The worms were washed thoroughly in distilled water and in Ringer's solution to remove intestinal contents, and a known wet weight of worm was homogenized in a Teflon-pestled hand homogenizer with the appropriate volume of buffer solution (for further details see tables). The homogenate was stored on ice for 15 min, which allowed a lipid layer to separate. The lower, relatively lipid-free fraction was used in the experiments. Supplies of [1:4-1*C~]-succinate (specific activity= 8"8mc/mM), U-I~C malate (specific activity = 22.6 mc/mM) and U-14C-glutamate (specific activity-~ 14"9 mc/mM) and NaH14COz (specific activity = 54.5 mc/mM) were obtained from the Radiochemical Centre, Amersham, Bucks, England. NAD, NADH2, NADP, NADPH~, acetyl coenzyme A, coenzyme A, ADP, A T P and pyridoxal phosphate were supplied by the Sigma Chemical Corporation, U.S.A. All other reagents were analytical grade and glass-distilled water was used throughout. Reduced and oxidized pyridine nucleotides in M. expansa were measured by the method of Slater & Sawyer (1962). The general procedures adopted for the incubation of M. expansa with radioactive substrates, and the subsequent chromatography and identification of spots, were those of Smith & Moses (1960). Radioactive counting was carried out as described by Bryant & Janssens (1969). In all the radioactive experiments except those with NaH14CO3, 0"1 ml of the buffered homogenate was used for each incubation. Details of individual experiments are given with the tables. In the experiments with NaH14CO ~ the incubations were carried out in 1 '0-ml disposable hypodermic syringes in order to eliminate the gaseous phase from the incubation chamber. At 15, 30 and 120 rain one-third of the contents of the syringes was expelled into 0"5 ml absolute ethanol and treated as above.
RESULTS Table I showsthat the concentrations of NAD, NADH~, NADPand NADPH2 i n whole M. expansa are very m u c h lower t h a n i n rat liver. T h e c o n c e n t r a t i o n of N A D i n M. expansa was a p p r o x i m a t e l y five t i m e s greater t h a n that of NADH2, a n d t h a t of N A D P H 2 was a p p r o x i m a t e l y one a n d a half t i m e s t h a t of N A D P . A l t h o u g h these p r o p o r t i o n s differ f r o m those i n rat liver, the figures are similar i n that, i n each case, N A D a n d N A D P H 2 are i n t h e h i g h e r c o n c e n t r a t i o n s . TABLE 1--LEVELS OF REDUCEDAND OXIDIZEDPYRIDINENUCLEOTIDESIN M. expansa
M. expansa Pyridine nucleotide NAD NADH~ NADP NADPH2
Rat liver
Number of estimations
Range
Mean
Standard deviation
Mean
Standard deviation
30 30 30 30
40.0-115.0 7.3-23.0 2-8- 8"8 8"2- 12.7
65.9 13.5 6"0 10.9
+_18.4 _ 3.9 + 1.5 + 1.1
549 171 39 309
+ 29 _ 5 + 4 +20
Results expressed as/zg/g wet wt. of tissue. Rat liver data from Slater & Sawyer (1962).
THE TRICARBOXYLIC ACID CYCLE IN MONIEZIA ~EXPANSA
505
T h e results obtained when radioactive succinate was incubated for 20 m i n with homogenates of M. expansa are shown in T a b l e 2. Radioactivity was recovered in four acids of the tricarboxylic acid cycle, fumarate, malate, citrate and a-ketoglutarate. Incorporation into citrate was increased f r o m 500 to 13,100 counts/min in the presence of N A D + acetyl CoA. I n the additional presence of N A D P the radioactivity in malate decreased f r o m 28,700 to 1500 counts/min. TABLE 2--THE METABOLISM OF [ 1 : 4 - ~ 4 C ~ ] - S U C C I N A T E BY HOMOGENATES OF M . expansa-INCORPORATION OF RADIOCARBON INTO SOLUBLE INTERMEDIATES AFTER 2 0 r a i n (RESULTS EXPRESSED AS counts/min x 10 -3)
+ NAD
+ NAD and acetyl CoA
+ NAD, NADP and acetyl CoA
561.9 33"7 25.1 0"5 0"7
501 "2 34'9 34"8 2"6 1"6
556"0 40"8 28"7 13'1 2'7
521"1 38"9 1 "5 5"1 1"2
Aspartate Glutamate Glutamine ?-Amino hutyrate Alanine
0'9 0.6 0.9 0 52.7
3"2 0-9 1"4 0"3 47"8
9'1 0-7 1"4 0 48"9
4"6 0"6 1"6 1"1 44.4
Lactate Glycolytic intermediates Unknown
90"0"
90"1
86-7
105"9
1"4 0
0"6 23 "4
3"2 18 "7
Metabolic intermediates Succinate Fumarate Malate Citrate a-Ketoglutarate
No additions
0-7 0
A 1 : 1 homogenate of M. expansa was prepared in 0"25 M glycyl glycine buffer. Incubation medium contained 0"1 ml homogenate, 1/zc (approximately 0"1/zmole) radioactive succinate and, where present, 0"1 /zmole NAD and NADP, and 0"05 /zmole acetyl CoA in a total volume of 0"15 ml. Incubations were carried out in stoppered glass tubes at 37°C with air as the gas phase. Reaction was stopped by the addition of 0"2 ml absolute ethanol. Succinate initially present = 1665"0 x l0 s counts/rain. * Approximate value only. Radioactivity was recovered in five amino acids. T h e s e were aspartate, glutamate, glutamine, alanine, and in the presence of N A D , and of N A D + N A D P + acetyl CoA, radioactively labelled y-amino butyric acid was detected. Incorporation of radiocarbon f r o m succinate into aspartate was increased in the presence of cofactors and was at a m a x i m u m with N A D + acetyl CoA. I n addition, considerable amounts of radioactivity were detected in lactate, and small amounts in other glycolytic intermediates (probably phospho-enol pyruvate and phospho-glycerate). I n the presence of N A D + N A D P + a c e t y l CoA appreciable incorporation into an unidentified product occurred.
506
R. A. DAVEY AND C. BRYANT
T a b l e 3 shows t h e m e t a b o l i s m o f u n i f o r m l y l a b e l l e d m a l a t e b y h o m o g e n a t e s of M. expansa. R a d i o c a r b o n f r o m m a l a t e was r e c o v e r e d in citrate, a - k e t o g l u t a r a t e , s u c c i n a t e a n d f u m a r a t e , all of w h i c h are i n t e r m e d i a t e s in t h e t r i c a r b o x y l i c a c i d TABLE 3 - - T H E METABOLISM OF [U-14C]-MALATE BY HOMOGENATES OF M. expansa-INCORPORATION OF RADIOCARBON INTO SOLUBLE INTERMEDIATES AFTER 10 m i n (RESULTS EXPRESSED AS counts/min x 10 -8)
Metabolic intermediates
No additions
+ NAD
+ Acetyl CoA
Malate Citrate ~-Ketoglutarate Succinate Fumarate
174" 5 5"1 5"7 283'1 56"7
123"6 4" 1 1"1 272"7 37"8
104"4 23"3 2"1 238"6 48"3
Aspartate Glutamate Alanine
11 "6 1"4 270' 5
7"2 1"1 280.8
7"4 1 "0 304" 1
Lactate
16'3
62"4
20"3
x4CO~ (absorbed on K O H paper)
61"9
89-8
144.4
A 3 : 1 homogenate of M. expansa was prepared in 0"2 M glycyl glycine buffer, p H 7"4. Incubation medium contained 0.1 ml homogenate, 1/~c (approximately 0"04/zmoles) radioactive malate and, where present, 0"05/zmole N A D and 0"005/~mole acetyl CoA in a total volume 0"14 ml. Incubations were carried out and the reaction stopped as described in Table 2 except that K O H traps were attached to each tube. Malate initially present = 1200"0 x 103 counts/min. TABLE 4 - - - T H E METABOLISM OF [U-14C]-GLUTAMATE BY HOMOGENATES OF M. expansa-INCORPORATION OF RADIOCARBON INTO SOLUBLE INTERMEDIATES AFTER 60 m i n (RESULTS EXPRESSED AS counts/rain x 10 -3)
Metabolic intermediates
+ NAD
+ N A D and pyridoxal phosphate
+ Oxaloacetate and pyridoxal phosphate
799"5
761"5
742"7
442'5
124'2 38"7 9' 4
128"5 43"7 11' 2
128"9 46'1 12-7
277"8 17"2 11" 3
No additions
Glutamate ~-Ketoglutarate Succinate Fumarate
A 1 : 1 homogenate of M. expansa was prepared in 0"2 M glycyl glycine buffer, p H 7"4. Incubation medium contained 0"1 ml homogenate, 1/xc (approximately 0"07/xmole) radioactive glutamate and, where present, 0'1/xmole N A D , 0"02/zmole pyridoxal phosphate and 4"0/zmole oxaloacetate in a total volume of 0"15 ml. Incubations were carried out and the reaction stopped as described in Table 2. Glutamate initially present = 1738 x 103 counts/rain.
507
THE TRICARBOXYLICACID CYCLE IN MONIEZIA EXPANSA
cycle. The utilization of malate was increased both by NAD and by acetyl CoA, and the amount of radioactivity in citrate was increased from 5100 to 23,300 counts/rain by acetyl CoA. Radiocarbon was also recovered in the amino acids aspartate, glutamate and alanine, and in lactate. Incorporation into lactate was increased fourfold by NAD. An increased production of 14CO2 was also observed in the presence of NAD and of acetyl CoA. When uniformly labelled glutamate was used as a substrate, radioactivity was detected only in a-ketoglutarate, suceinate and fumarate. NAD and N A D + pyridoxal phosphate had no effect on the distribution of radiocarbon, but with oxaloacetate+ pyridoxal phosphate the amount of radioactivity in a-ketoglutarate was doubled, the incorporation of radiocarbon into succinate was reduced, and that into fumarate was not affected (Table 4). The incorporation of radiocarbon from NaH14CO3 into the soluble intermediates of M. expansa is presented in Table 5. Only three acids of the tricarboxylic acid cycle were labelled, namely malate, fumarate and succinate. Incorporation into succinate was almost doubled by the addition of pyruvate to the incubation medium, and the concomitant addition of ATP resulted in a further TABLE 5 - - T H E UTILIZATION OF NaH14CO3 BY HOMOGENATES OF M . expansa--THE DISTRIBUTION OF RADIOCARBONAMONGST SOLUBLE INTERMEDIATESAFTER 2 hr (RESULTS EXPRESSEDAS counts/min x 10 -3)
No additions
+ Pyruvate
+ Pyruvate and ATP
Malate Fumarate Succinate
44.5 9"3 252"8
73"0 47"5 413 "4
66"8 81"1 805 "6
Aspartate
15"1
10"9
27"6
Alanine Lactate Pyruwte
353"5 34"7 37"2
498"7 23"5 66"9
604"3 13.6 96"5
Unknown
51"9
174"6
81"0
Metabolic intermediates
A 3 : 1 homogenate of M . expansa was prepared in 0"2 M glycyl glycine buffer, p H 7-4. Incubation m e d i u m contained 0.69 ml homogenate, 300/zc (approximately 6/zmoles) radioactive NaH14CO3 and, where present, 0"3/zmole pyruvate and 0"075/~mole A T P in a total v o l u m e of 0"99 ml. Incubations were carried out in 1.0-ml syringes at 37°C in the absence of a gas phase. T h e reaction was stopped by squirting 0'33-ml aliquots into 0"5 ml absolute ethanol.
doubling. Pyruvate and pyruvate + ATP also caused increases in the incorporation of radiocarbon from NaH14COs into malate and fumarate. Radiocarbon was also detected in aspartate and in alanine. As in the case of succinate, addition of pyruvate and p y r u v a t e + A T P caused substantial increases in the amount of t7
508
R. A. DAVEY AND C. BRYANT
radioactivity present in alanine. Other labelled intermediates detected were lactate and pyruvate. T h e additions caused an increase of incorporation of radiocarbon into pyruvate, but a decrease in lactate. An unidentified intermediate was also present. Figure 1 shows the rate of incorporation of radiocarbon from NaHI~CO3 into succinate. In the absence of additives, incorporation of radioactivity was linear for 30 rain, after which there was a slight decline. With pyruvate, the radioactivity in succinate increased over the 2 hr of the experiment. However, in the presence of pyruvate + A T P the initial rate of incorporation was maintained for the duration of the experiment. Thus, although there is no appreciable difference in the initial rate of incorporation of radiocarbon from NaH14COa into succinate, with pyruvate and p y r u v a t e + A T P the amounts of 14CO2 incorporated are eventually much greater.
C. (+
800
i
7
o
pyruvate
and ATP)
7o0
a
600 5OO
"~.
400
--
~
B. (+
~wj~wm
pyruvate)
o 300
2OO
~
o
~
e
A.
(no
additions)
100 I
I
15 30
I
I
60
120 Incubation
time
in
mins
FXG. 1. The utilization of 14CO2 by homogenates of M . expansa: the accumulation of radiocarbon from NaHI4CO3 in succinate over 2 hr, in the presence of pyruvate and pyruvate + ATP. Experimental conditions as described in Table 5.
THE TRICARBOXYLIC ACID CYCLE I N M O N I E Z I A E X P A N S A
509
DISCUSSION The results described here indicate that there is a strong probability that M. expansa possesses all the enzymes of the tricarboxylic acid cycle. The appearance of radiocarbon from the labelled dicarboxylic acids in fumarate, malate, citrate and a-ketoglutarate (Tables 2 and 3) suggests the presence of succinic dehydrogenase, fumarase, malic dehydrogenase, condensing enzyme, aconitase and isocitric dehydrogenase. The appearance of radiocarbon in aspartate indicates that oxaloacetic acid must have been formed and acted as an amino group acceptor. This observation, combined with the stimulatory effect of acetyl CoA on citrate recovery, provides good additional evidence for the presence of condensing enzyme. The appearance of radiocarbon from malate in succinate (Table 3) cannot, however, be ascribed to the patency of the cycle, as Cheah & Bryant (1966) have demonstrated that the back reaction, in which fumarate is converted to succinate in M. expansa, is probably more active than the forward reaction. Further confirmation is obtained from 14CO2 incorporation studies (Table 5) during which succinate also accumulates, whereas no radiocarbon is detected in citrate or c~-ketoglutarate. Although an experiment with labelled citrate was attempted no metabolism occurred. This agrees with the resuks obtained with homogenates of E. granulosus protoscolices (Agosin & Repetto, 1963), with Turbatrix aceti (Ells & Read, 1961) and with Macracanthorhynchus hirudinaceus (Dunagan & Scheifinger, 1966) and may be due to the fact that citrate of external origin cannot easily achieve the appropriate steric configuration with the enzymes concerned with its further metabolism. Thus, the case for the conversion of a-ketoglutarate to succinate rests upon the results obtained with radioactive glutamate (Table 4). Glutamate is readily converted to a-ketoglutarate ; in the presence of an amino group acceptor (oxaloacetate) and pyridoxal phosphate, the amount of ~-ketoglutarate formed is doubled. It is energetically unlikely that succinate could be formed from a-ketoglutarate by reversing the tricarboxylic acid cycle; in any event, one would expect to detect radiocarbon in citrate, alanine, aspartate and malate under these circumstances. Rasero et al. (1968) have recently shown that glutamate could be converted to succinate in M. expansa homogenates by way of 7-aminobutyrate and succinic semialdehyde. However, they showed that the initial step, the decarboxylation of glutamate, does not take place at pH 7.4. As this was the pH chosen for the present experiment, and as no radioactive 7-aminobutyrate was formed from the uniformly labelled glutamate, the 7-aminobutyrate shunt can be excluded as a possibility in the present case. Thus, the evidence strongly suggests that the formation of succinate was mediated by a-ketoglutarate dehydrogenase. It is apparent that this enzyme may be one of the rate-limiting steps in the tricarboxylic acid cycle in M. expansa homogenates. Addition of NAD or NAD + pyridoxal phosphate did not increase the formation of labelled succinate, and when the concentration of ~-ketoglutarate was effectively raised by the addition of pyridoxal phosphate and oxaloacetate, the formation of labelled succinate was diminished. However, the latter effect could be due to the formation of a large pool of unlabelled succinate from oxaloacetate which might well result in the
510
R.A. DAVEYAND C. BRYANT
inhibition of the dehydrogenase. The results, however, suggest that either the activity of c~-ketoglutarate dehydrogenaseor its concentration in the homogenates is very low, especiallyas attempts at spectrophotometric assay proved negative. The behaviour of several of the pools of labelled intermediates in the presence or absence of added cofactors is worth noting. Table 1 shows that the concentrations of the oxidized and reduced pyridine nucleotides in M . expansa were much lower than those in the rat. Therefore, although relatively concentrated homogenates were employed in the experiments, it was considered advisable to fortify them with added cofactors. Addition of NADP resulted in a decrease in the radiocarbon recovered in malate (Table 2), which suggests that an NADP-dependent decarboxylase is present. The presence of a decarboxylase is further substantiated by Table 5 and Fig. 1. The pattern of incorporation of 14CO2 into intermediates in homogenates of M . expansa is consistent with the activity of "malic enzyme". The maintenance effect of pyruvate (Fig. 1) supports the malic enzyme hypothesis, and the even greater effect of pyruvate + ATP suggests that an initial conversion of pyruvate to phosphoenol pyruvate is possible, followed by CO 2 fixation by phosphoenol pyruvate carboxykinase. Similar systems have been described in an acanthocephalan (Graft, 1965), cestodes (Agosin & Repetto, 1965; Prescott & Campbell, 1965), nematodes (Fairbairn, 1954; Saz & Hubbard, 1957; Saz & Vidrine, 1959), and trematodes (Hammen & Lum, 1962; Prichard & Schofield, 1968b). Thus, there is a considerable body of evidence which suggests that in M. expansa all the enzymes necessary for tricarboxylic-acid-cycle activity are present. However, it is possible that some are at very low activities, and that under in vitro conditions their contribution to the overall metabolism of preparations from the worm is slight. In vivo its significance is even less clear. The cytochrome oxidase system cannot function efficiently at an oxygen tension of less than 5 mm Hg (Hill, 1936), so that one might expect to encounter tensions of this order in the sheep gut. In fact, Rogers (1949) has recorded oxygen tensions as high as 30.2 mm Hg. It is, however, apparent that the anaerobic component of respiration, as exemplified in the present paper by the CO2 fixation experiments, is a major pathway. Acknowledgements--The authors gratefully acknowledge a generous grant towards the cost of this work from the Rural Credits Fund of the Reserve Bank of Australia.
REFERENCES AGOSlNM. 8¢REPETTOY. (1963) Studies on the metabolism of Echinococcus granulosus--VII. Reactions of the tricarboxylic acid cycle in E. granulosus scolices. Comp. Biochem. Physiol. 8, 245-261. ACOSlNM. & REPETTOY. (1965) Studies on the metabolism of Echinococcusgranulosus--VIII. The pathway to succinate in E. granulosus scolices. Comp. Biochern. Physiol. 14, 299-309. BRYANT C. • JANSSENS P. A. (1969) Intermediary metabolism in a terrestrial planarian Geoplana caerulea (Moseley). Comp. Biochem. Physiol. 3~}, 841-857. CHEAHK. S. (1968) The respiratory components of Moniezia expansa (Cestoda). Biochim. biophys. Acta 153, 718-720.
TI-IB T R I C A R B O X Y L I C ACID CYCLE I N M O N I E Z l A E X P A N S A
511
CHEAH K. S. & BRYANT C. (1966) Studies on the electron transport system of Moniezia expansa (Cestoda). Comp. Biochem. Physiol. 19, 197-223. DUNAGANT. T. & SCHEIFINCERC. C. (1966) Studies on the T C A cycle of Macracanthorhynchus hirudinaceus (Acanthocephala). Comp. Biochem. Physiol. 18, 663-667. ELLS H. A. & READ C. P. (1961) Physiology of the vinegar eel worm, Turbatrix aceti ( N e m a t o d a ) - - I . Observations on respiratory metabolism. Biol. Bull. mar. biol. Lab., Woods Hole 120, 326-336. FAIRBAIRN D. (1954) The metabolism of Heterakis gallinae--II. Carbon dioxide fixation. Exptl Parasitol. 3, 52-63. GRAFF D. J. (1965) T h e utilization of 14COa in the production of acid metabolites by Moniliformis dubius (Acanthocephala). J . Parasit. 51, 72-75. HAMMEN C. S. & LUM S. C. (1962) Carbon dioxide fixation in marine invertebrates--III. T h e main pathway in flatworms. J . biol. Chem. 237, 2419-2422. HILL R. (1936) Oxygen dissociation curves of muscle haemoglobin. Proc. R. Soc. B 110, 472-483. OYA H., KIKUCHI G., BANDO T. & HAYASHI H. (1965) Muscle tricarboxylic acid cycle in Ascaris lumbricoides var. suis. Expl Parasitol. 17, 229-240. PRESCOTT L. M. & CAMPBELL J. W. (1965) Phosphoenolpyruvate carboxylase activity and glycogenesis in the flatworm, Hymenolepis diminuta. Comp. Bioehem. Physiol. 14,491-511. PRICHARD R. K. & SCHOFIELD P. J. (1968a) A comparative study of the tricarboxylic acid cycle in Fasciola hepatica and rat liver. Comp. Biochem. Physiol. 25, 1005-1020. PRICHARD R. K. & SCHOFmLD P. J. (1968b) Phosphoenolpyruvate carboxykinase in the adult liver fluke, Fasciola hepatica. Comp. Biochem. Physiol. 24, 773-785. RASERO F. S., MONTEOLIVA M. & MAYOR F. (1968) Enzymes related to 4-aminobutyrate metabolism in intestinal parasites. Comp. Biochem. Physiol. 25, 693-701. ROGERS W. P. (1949) On the relative importance of aerobic metabolism in small nematode parasites of the alimentary t r a c t - - I . Oxygen tensions in the normal environment of the parasites. Aust.J. scient. Res. B 2, 166-174. SAZ H. J. & HUBBARD J. A. (1957) T h e oxidative decarboxylation of malate by Ascaris lumbricoides. J. biol. Chem. 225, 921-933. SAZ H. J. & VIDRINE A. (1959) T h e mechanism of formation of succinate and propionate by Ascaris lumbricoides muscle. J. biol. Chem. 234, 2001-2005. SCHEmEL L. W. & SAZ H. J. (1966) The pathway of anaerobic carbohydrate dissimilation in Hymenolepis diminuta. Comp. Biochem. Physiol. 18, 151-162. SLATER T. F. & SAWER B. (1962) A colorimetric method for estimating the pyridine nucleotide content of small amounts of animal tissue. Nature, Lond. 193, 45 a, A,56. SMITH M. J. H. & MOSES V. (1960) Uncoupling reagents in metabolism--I. T h e effects of salicylate and 2:4-dinitrophenol on the incorporation of 14C from labelled glucose and acetate into the soluble intermediates of isolated rat tissues. Bioehem. J. 76, 579-585. VON BRAND T. (1933) Untersuchungen iiber den Stoffbestand einiger Cestoden brad den Stoffwechsel yon Moniezia expansa. Z. vergl. Physiol. 18, 562-596. y o n BRAND T. (1966) Biochemistry of Parasites. Academic Press, New York and London.
Key Word Index--Tricarboxylic acid cycle; Moniezia expansa; cestode biochemistry (TCA); malic enzyme; phosphoenol pyruvate carboxykinase.
THE TRICARBOXYLIC ACID CYCLE AND ASSOCIATED REACTIONS IN M O N I E Z I A E X P A N S A (CESTODA) R. A. DAVEY and C. BRYANT Department of Zoology, School of General Studies, The Australian National University, Canberra, A.C.T., Australia (Received 14 March 1969) A b s t r a c t - - 1 . Experiments with radioactive substrates have provided evidence
for the presence of the tricarboxylic acid cycle in Moniezia expansa. 2. Incorporation of 1'CO2 probably involves both "malic enzyme" and phosphoenol pyruvate carboxykinase.
INTRODUCTION ALTHOUGH Cheah & Bryant (1966) demonstrated that a major respiratory pathway in preparations from Moniezia expama utilized fumarate as an electron acceptor, the possibility remained that a small proportion of respiration involved molecular oxygen. Subsequently, Cheah (1968) showed that, while the major cytochrome component of M. expansa was implicated in the fumarate-succinate transformation, it was also possible to detect an orthodox cytochrome system which possessed very low concentrations of a cytochrome oxidase. It was therefore assumed that this pathway was partially responsible for the small oxygen uptake observed when mitochondrial preparations from M. expama were incubated with succinate, a-glycerophosphate or NADH. Prichard & Schofield (1968a) successfully demonstrated the presence of enzymes of the tricarboxylic acid cycle in Fasciola hepatica. Protoscolices of Echinococcus granulosus and Ascaris muscle have also been shown to have an active cycle (Agosin & Repetto, 1963; Oya et al., 1965). Prior to this work the existence of the tricarboxylic acid cycle in parasitic helminths was problematical as the accumulation of succinate, and hence the anaerobic component of respiration, were of more immediate significance. The only cestode hitherto investigated which has been shown to possess an active tricarboxylic acid cycle is E. granulosus (Agosin & Repetto, 1963). Scheibel & Saz (1966) investigated intermediary metabolism in Hymenolepis diminuta and concluded that the tricarboxylic acid cycle was not active. Other cestodes have also been shown to accumulate succinic acid, and as early as 1933 this was demonstrated in M. expama (yon Brand, 1933, 1966). It was therefore of considerable interest to determine whether the cytochrome oxidase system of M. expama was associated with tricarboxylic acid cycle activity. 503
504
R. A. DawY AND C. BRYA~rr
MATERIALS AND METHODS Adult M. expansa were obtained from slaughtered lambs at the Queanbeyan Abattoirs, N.S.W., and stored on ice in Ringer's solution. They were used within 4 hr of collection. The worms were washed thoroughly in distilled water and in Ringer's solution to remove intestinal contents, and a known wet weight of worm was homogenized in a Teflon-pestled hand homogenizer with the appropriate volume of buffer solution (for further details see tables). The homogenate was stored on ice for 15 min, which allowed a lipid layer to separate. The lower, relatively lipid-free fraction was used in the experiments. Supplies of [1:4-1*C~]-succinate (specific activity= 8"8mc/mM), U-I~C malate (specific activity = 22.6 mc/mM) and U-14C-glutamate (specific activity-~ 14"9 mc/mM) and NaH14COz (specific activity = 54.5 mc/mM) were obtained from the Radiochemical Centre, Amersham, Bucks, England. NAD, NADH2, NADP, NADPH~, acetyl coenzyme A, coenzyme A, ADP, A T P and pyridoxal phosphate were supplied by the Sigma Chemical Corporation, U.S.A. All other reagents were analytical grade and glass-distilled water was used throughout. Reduced and oxidized pyridine nucleotides in M. expansa were measured by the method of Slater & Sawyer (1962). The general procedures adopted for the incubation of M. expansa with radioactive substrates, and the subsequent chromatography and identification of spots, were those of Smith & Moses (1960). Radioactive counting was carried out as described by Bryant & Janssens (1969). In all the radioactive experiments except those with NaH14CO3, 0"1 ml of the buffered homogenate was used for each incubation. Details of individual experiments are given with the tables. In the experiments with NaH14CO ~ the incubations were carried out in 1 '0-ml disposable hypodermic syringes in order to eliminate the gaseous phase from the incubation chamber. At 15, 30 and 120 rain one-third of the contents of the syringes was expelled into 0"5 ml absolute ethanol and treated as above.
RESULTS Table I showsthat the concentrations of NAD, NADH~, NADPand NADPH2 i n whole M. expansa are very m u c h lower t h a n i n rat liver. T h e c o n c e n t r a t i o n of N A D i n M. expansa was a p p r o x i m a t e l y five t i m e s greater t h a n that of NADH2, a n d t h a t of N A D P H 2 was a p p r o x i m a t e l y one a n d a half t i m e s t h a t of N A D P . A l t h o u g h these p r o p o r t i o n s differ f r o m those i n rat liver, the figures are similar i n that, i n each case, N A D a n d N A D P H 2 are i n t h e h i g h e r c o n c e n t r a t i o n s . TABLE 1--LEVELS OF REDUCEDAND OXIDIZEDPYRIDINENUCLEOTIDESIN M. expansa
M. expansa Pyridine nucleotide NAD NADH~ NADP NADPH2
Rat liver
Number of estimations
Range
Mean
Standard deviation
Mean
Standard deviation
30 30 30 30
40.0-115.0 7.3-23.0 2-8- 8"8 8"2- 12.7
65.9 13.5 6"0 10.9
+_18.4 _ 3.9 + 1.5 + 1.1
549 171 39 309
+ 29 _ 5 + 4 +20
Results expressed as/zg/g wet wt. of tissue. Rat liver data from Slater & Sawyer (1962).
THE TRICARBOXYLIC ACID CYCLE IN MONIEZIA ~EXPANSA
505
T h e results obtained when radioactive succinate was incubated for 20 m i n with homogenates of M. expansa are shown in T a b l e 2. Radioactivity was recovered in four acids of the tricarboxylic acid cycle, fumarate, malate, citrate and a-ketoglutarate. Incorporation into citrate was increased f r o m 500 to 13,100 counts/min in the presence of N A D + acetyl CoA. I n the additional presence of N A D P the radioactivity in malate decreased f r o m 28,700 to 1500 counts/min. TABLE 2--THE METABOLISM OF [ 1 : 4 - ~ 4 C ~ ] - S U C C I N A T E BY HOMOGENATES OF M . expansa-INCORPORATION OF RADIOCARBON INTO SOLUBLE INTERMEDIATES AFTER 2 0 r a i n (RESULTS EXPRESSED AS counts/min x 10 -3)
+ NAD
+ NAD and acetyl CoA
+ NAD, NADP and acetyl CoA
561.9 33"7 25.1 0"5 0"7
501 "2 34'9 34"8 2"6 1"6
556"0 40"8 28"7 13'1 2'7
521"1 38"9 1 "5 5"1 1"2
Aspartate Glutamate Glutamine ?-Amino hutyrate Alanine
0'9 0.6 0.9 0 52.7
3"2 0-9 1"4 0"3 47"8
9'1 0-7 1"4 0 48"9
4"6 0"6 1"6 1"1 44.4
Lactate Glycolytic intermediates Unknown
90"0"
90"1
86-7
105"9
1"4 0
0"6 23 "4
3"2 18 "7
Metabolic intermediates Succinate Fumarate Malate Citrate a-Ketoglutarate
No additions
0-7 0
A 1 : 1 homogenate of M. expansa was prepared in 0"25 M glycyl glycine buffer. Incubation medium contained 0"1 ml homogenate, 1/zc (approximately 0"1/zmole) radioactive succinate and, where present, 0"1 /zmole NAD and NADP, and 0"05 /zmole acetyl CoA in a total volume of 0"15 ml. Incubations were carried out in stoppered glass tubes at 37°C with air as the gas phase. Reaction was stopped by the addition of 0"2 ml absolute ethanol. Succinate initially present = 1665"0 x l0 s counts/rain. * Approximate value only. Radioactivity was recovered in five amino acids. T h e s e were aspartate, glutamate, glutamine, alanine, and in the presence of N A D , and of N A D + N A D P + acetyl CoA, radioactively labelled y-amino butyric acid was detected. Incorporation of radiocarbon f r o m succinate into aspartate was increased in the presence of cofactors and was at a m a x i m u m with N A D + acetyl CoA. I n addition, considerable amounts of radioactivity were detected in lactate, and small amounts in other glycolytic intermediates (probably phospho-enol pyruvate and phospho-glycerate). I n the presence of N A D + N A D P + a c e t y l CoA appreciable incorporation into an unidentified product occurred.
506
R. A. DAVEY AND C. BRYANT
T a b l e 3 shows t h e m e t a b o l i s m o f u n i f o r m l y l a b e l l e d m a l a t e b y h o m o g e n a t e s of M. expansa. R a d i o c a r b o n f r o m m a l a t e was r e c o v e r e d in citrate, a - k e t o g l u t a r a t e , s u c c i n a t e a n d f u m a r a t e , all of w h i c h are i n t e r m e d i a t e s in t h e t r i c a r b o x y l i c a c i d TABLE 3 - - T H E METABOLISM OF [U-14C]-MALATE BY HOMOGENATES OF M. expansa-INCORPORATION OF RADIOCARBON INTO SOLUBLE INTERMEDIATES AFTER 10 m i n (RESULTS EXPRESSED AS counts/min x 10 -8)
Metabolic intermediates
No additions
+ NAD
+ Acetyl CoA
Malate Citrate ~-Ketoglutarate Succinate Fumarate
174" 5 5"1 5"7 283'1 56"7
123"6 4" 1 1"1 272"7 37"8
104"4 23"3 2"1 238"6 48"3
Aspartate Glutamate Alanine
11 "6 1"4 270' 5
7"2 1"1 280.8
7"4 1 "0 304" 1
Lactate
16'3
62"4
20"3
x4CO~ (absorbed on K O H paper)
61"9
89-8
144.4
A 3 : 1 homogenate of M. expansa was prepared in 0"2 M glycyl glycine buffer, p H 7"4. Incubation medium contained 0.1 ml homogenate, 1/~c (approximately 0"04/zmoles) radioactive malate and, where present, 0"05/zmole N A D and 0"005/~mole acetyl CoA in a total volume 0"14 ml. Incubations were carried out and the reaction stopped as described in Table 2 except that K O H traps were attached to each tube. Malate initially present = 1200"0 x 103 counts/min. TABLE 4 - - - T H E METABOLISM OF [U-14C]-GLUTAMATE BY HOMOGENATES OF M. expansa-INCORPORATION OF RADIOCARBON INTO SOLUBLE INTERMEDIATES AFTER 60 m i n (RESULTS EXPRESSED AS counts/rain x 10 -3)
Metabolic intermediates
+ NAD
+ N A D and pyridoxal phosphate
+ Oxaloacetate and pyridoxal phosphate
799"5
761"5
742"7
442'5
124'2 38"7 9' 4
128"5 43"7 11' 2
128"9 46'1 12-7
277"8 17"2 11" 3
No additions
Glutamate ~-Ketoglutarate Succinate Fumarate
A 1 : 1 homogenate of M. expansa was prepared in 0"2 M glycyl glycine buffer, p H 7"4. Incubation medium contained 0"1 ml homogenate, 1/xc (approximately 0"07/xmole) radioactive glutamate and, where present, 0'1/xmole N A D , 0"02/zmole pyridoxal phosphate and 4"0/zmole oxaloacetate in a total volume of 0"15 ml. Incubations were carried out and the reaction stopped as described in Table 2. Glutamate initially present = 1738 x 103 counts/rain.
507
THE TRICARBOXYLICACID CYCLE IN MONIEZIA EXPANSA
cycle. The utilization of malate was increased both by NAD and by acetyl CoA, and the amount of radioactivity in citrate was increased from 5100 to 23,300 counts/rain by acetyl CoA. Radiocarbon was also recovered in the amino acids aspartate, glutamate and alanine, and in lactate. Incorporation into lactate was increased fourfold by NAD. An increased production of 14CO2 was also observed in the presence of NAD and of acetyl CoA. When uniformly labelled glutamate was used as a substrate, radioactivity was detected only in a-ketoglutarate, suceinate and fumarate. NAD and N A D + pyridoxal phosphate had no effect on the distribution of radiocarbon, but with oxaloacetate+ pyridoxal phosphate the amount of radioactivity in a-ketoglutarate was doubled, the incorporation of radiocarbon into succinate was reduced, and that into fumarate was not affected (Table 4). The incorporation of radiocarbon from NaH14CO3 into the soluble intermediates of M. expansa is presented in Table 5. Only three acids of the tricarboxylic acid cycle were labelled, namely malate, fumarate and succinate. Incorporation into succinate was almost doubled by the addition of pyruvate to the incubation medium, and the concomitant addition of ATP resulted in a further TABLE 5 - - T H E UTILIZATION OF NaH14CO3 BY HOMOGENATES OF M . expansa--THE DISTRIBUTION OF RADIOCARBONAMONGST SOLUBLE INTERMEDIATESAFTER 2 hr (RESULTS EXPRESSEDAS counts/min x 10 -3)
No additions
+ Pyruvate
+ Pyruvate and ATP
Malate Fumarate Succinate
44.5 9"3 252"8
73"0 47"5 413 "4
66"8 81"1 805 "6
Aspartate
15"1
10"9
27"6
Alanine Lactate Pyruwte
353"5 34"7 37"2
498"7 23"5 66"9
604"3 13.6 96"5
Unknown
51"9
174"6
81"0
Metabolic intermediates
A 3 : 1 homogenate of M . expansa was prepared in 0"2 M glycyl glycine buffer, p H 7-4. Incubation m e d i u m contained 0.69 ml homogenate, 300/zc (approximately 6/zmoles) radioactive NaH14CO3 and, where present, 0"3/zmole pyruvate and 0"075/~mole A T P in a total v o l u m e of 0"99 ml. Incubations were carried out in 1.0-ml syringes at 37°C in the absence of a gas phase. T h e reaction was stopped by squirting 0'33-ml aliquots into 0"5 ml absolute ethanol.
doubling. Pyruvate and pyruvate + ATP also caused increases in the incorporation of radiocarbon from NaH14COs into malate and fumarate. Radiocarbon was also detected in aspartate and in alanine. As in the case of succinate, addition of pyruvate and p y r u v a t e + A T P caused substantial increases in the amount of t7
508
R. A. DAVEY AND C. BRYANT
radioactivity present in alanine. Other labelled intermediates detected were lactate and pyruvate. T h e additions caused an increase of incorporation of radiocarbon into pyruvate, but a decrease in lactate. An unidentified intermediate was also present. Figure 1 shows the rate of incorporation of radiocarbon from NaHI~CO3 into succinate. In the absence of additives, incorporation of radioactivity was linear for 30 rain, after which there was a slight decline. With pyruvate, the radioactivity in succinate increased over the 2 hr of the experiment. However, in the presence of pyruvate + A T P the initial rate of incorporation was maintained for the duration of the experiment. Thus, although there is no appreciable difference in the initial rate of incorporation of radiocarbon from NaH14COa into succinate, with pyruvate and p y r u v a t e + A T P the amounts of 14CO2 incorporated are eventually much greater.
C. (+
800
i
7
o
pyruvate
and ATP)
7o0
a
600 5OO
"~.
400
--
~
B. (+
~wj~wm
pyruvate)
o 300
2OO
~
o
~
e
A.
(no
additions)
100 I
I
15 30
I
I
60
120 Incubation
time
in
mins
FXG. 1. The utilization of 14CO2 by homogenates of M . expansa: the accumulation of radiocarbon from NaHI4CO3 in succinate over 2 hr, in the presence of pyruvate and pyruvate + ATP. Experimental conditions as described in Table 5.
THE TRICARBOXYLIC ACID CYCLE I N M O N I E Z I A E X P A N S A
509
DISCUSSION The results described here indicate that there is a strong probability that M. expansa possesses all the enzymes of the tricarboxylic acid cycle. The appearance of radiocarbon from the labelled dicarboxylic acids in fumarate, malate, citrate and a-ketoglutarate (Tables 2 and 3) suggests the presence of succinic dehydrogenase, fumarase, malic dehydrogenase, condensing enzyme, aconitase and isocitric dehydrogenase. The appearance of radiocarbon in aspartate indicates that oxaloacetic acid must have been formed and acted as an amino group acceptor. This observation, combined with the stimulatory effect of acetyl CoA on citrate recovery, provides good additional evidence for the presence of condensing enzyme. The appearance of radiocarbon from malate in succinate (Table 3) cannot, however, be ascribed to the patency of the cycle, as Cheah & Bryant (1966) have demonstrated that the back reaction, in which fumarate is converted to succinate in M. expansa, is probably more active than the forward reaction. Further confirmation is obtained from 14CO2 incorporation studies (Table 5) during which succinate also accumulates, whereas no radiocarbon is detected in citrate or c~-ketoglutarate. Although an experiment with labelled citrate was attempted no metabolism occurred. This agrees with the resuks obtained with homogenates of E. granulosus protoscolices (Agosin & Repetto, 1963), with Turbatrix aceti (Ells & Read, 1961) and with Macracanthorhynchus hirudinaceus (Dunagan & Scheifinger, 1966) and may be due to the fact that citrate of external origin cannot easily achieve the appropriate steric configuration with the enzymes concerned with its further metabolism. Thus, the case for the conversion of a-ketoglutarate to succinate rests upon the results obtained with radioactive glutamate (Table 4). Glutamate is readily converted to a-ketoglutarate ; in the presence of an amino group acceptor (oxaloacetate) and pyridoxal phosphate, the amount of ~-ketoglutarate formed is doubled. It is energetically unlikely that succinate could be formed from a-ketoglutarate by reversing the tricarboxylic acid cycle; in any event, one would expect to detect radiocarbon in citrate, alanine, aspartate and malate under these circumstances. Rasero et al. (1968) have recently shown that glutamate could be converted to succinate in M. expansa homogenates by way of 7-aminobutyrate and succinic semialdehyde. However, they showed that the initial step, the decarboxylation of glutamate, does not take place at pH 7.4. As this was the pH chosen for the present experiment, and as no radioactive 7-aminobutyrate was formed from the uniformly labelled glutamate, the 7-aminobutyrate shunt can be excluded as a possibility in the present case. Thus, the evidence strongly suggests that the formation of succinate was mediated by a-ketoglutarate dehydrogenase. It is apparent that this enzyme may be one of the rate-limiting steps in the tricarboxylic acid cycle in M. expansa homogenates. Addition of NAD or NAD + pyridoxal phosphate did not increase the formation of labelled succinate, and when the concentration of ~-ketoglutarate was effectively raised by the addition of pyridoxal phosphate and oxaloacetate, the formation of labelled succinate was diminished. However, the latter effect could be due to the formation of a large pool of unlabelled succinate from oxaloacetate which might well result in the
510
R.A. DAVEYAND C. BRYANT
inhibition of the dehydrogenase. The results, however, suggest that either the activity of c~-ketoglutarate dehydrogenaseor its concentration in the homogenates is very low, especiallyas attempts at spectrophotometric assay proved negative. The behaviour of several of the pools of labelled intermediates in the presence or absence of added cofactors is worth noting. Table 1 shows that the concentrations of the oxidized and reduced pyridine nucleotides in M . expansa were much lower than those in the rat. Therefore, although relatively concentrated homogenates were employed in the experiments, it was considered advisable to fortify them with added cofactors. Addition of NADP resulted in a decrease in the radiocarbon recovered in malate (Table 2), which suggests that an NADP-dependent decarboxylase is present. The presence of a decarboxylase is further substantiated by Table 5 and Fig. 1. The pattern of incorporation of 14CO2 into intermediates in homogenates of M . expansa is consistent with the activity of "malic enzyme". The maintenance effect of pyruvate (Fig. 1) supports the malic enzyme hypothesis, and the even greater effect of pyruvate + ATP suggests that an initial conversion of pyruvate to phosphoenol pyruvate is possible, followed by CO 2 fixation by phosphoenol pyruvate carboxykinase. Similar systems have been described in an acanthocephalan (Graft, 1965), cestodes (Agosin & Repetto, 1965; Prescott & Campbell, 1965), nematodes (Fairbairn, 1954; Saz & Hubbard, 1957; Saz & Vidrine, 1959), and trematodes (Hammen & Lum, 1962; Prichard & Schofield, 1968b). Thus, there is a considerable body of evidence which suggests that in M. expansa all the enzymes necessary for tricarboxylic-acid-cycle activity are present. However, it is possible that some are at very low activities, and that under in vitro conditions their contribution to the overall metabolism of preparations from the worm is slight. In vivo its significance is even less clear. The cytochrome oxidase system cannot function efficiently at an oxygen tension of less than 5 mm Hg (Hill, 1936), so that one might expect to encounter tensions of this order in the sheep gut. In fact, Rogers (1949) has recorded oxygen tensions as high as 30.2 mm Hg. It is, however, apparent that the anaerobic component of respiration, as exemplified in the present paper by the CO2 fixation experiments, is a major pathway. Acknowledgements--The authors gratefully acknowledge a generous grant towards the cost of this work from the Rural Credits Fund of the Reserve Bank of Australia.
REFERENCES AGOSlNM. 8¢REPETTOY. (1963) Studies on the metabolism of Echinococcus granulosus--VII. Reactions of the tricarboxylic acid cycle in E. granulosus scolices. Comp. Biochem. Physiol. 8, 245-261. ACOSlNM. & REPETTOY. (1965) Studies on the metabolism of Echinococcusgranulosus--VIII. The pathway to succinate in E. granulosus scolices. Comp. Biochern. Physiol. 14, 299-309. BRYANT C. • JANSSENS P. A. (1969) Intermediary metabolism in a terrestrial planarian Geoplana caerulea (Moseley). Comp. Biochem. Physiol. 3~}, 841-857. CHEAHK. S. (1968) The respiratory components of Moniezia expansa (Cestoda). Biochim. biophys. Acta 153, 718-720.
TI-IB T R I C A R B O X Y L I C ACID CYCLE I N M O N I E Z l A E X P A N S A
511
CHEAH K. S. & BRYANT C. (1966) Studies on the electron transport system of Moniezia expansa (Cestoda). Comp. Biochem. Physiol. 19, 197-223. DUNAGANT. T. & SCHEIFINCERC. C. (1966) Studies on the T C A cycle of Macracanthorhynchus hirudinaceus (Acanthocephala). Comp. Biochem. Physiol. 18, 663-667. ELLS H. A. & READ C. P. (1961) Physiology of the vinegar eel worm, Turbatrix aceti ( N e m a t o d a ) - - I . Observations on respiratory metabolism. Biol. Bull. mar. biol. Lab., Woods Hole 120, 326-336. FAIRBAIRN D. (1954) The metabolism of Heterakis gallinae--II. Carbon dioxide fixation. Exptl Parasitol. 3, 52-63. GRAFF D. J. (1965) T h e utilization of 14COa in the production of acid metabolites by Moniliformis dubius (Acanthocephala). J . Parasit. 51, 72-75. HAMMEN C. S. & LUM S. C. (1962) Carbon dioxide fixation in marine invertebrates--III. T h e main pathway in flatworms. J . biol. Chem. 237, 2419-2422. HILL R. (1936) Oxygen dissociation curves of muscle haemoglobin. Proc. R. Soc. B 110, 472-483. OYA H., KIKUCHI G., BANDO T. & HAYASHI H. (1965) Muscle tricarboxylic acid cycle in Ascaris lumbricoides var. suis. Expl Parasitol. 17, 229-240. PRESCOTT L. M. & CAMPBELL J. W. (1965) Phosphoenolpyruvate carboxylase activity and glycogenesis in the flatworm, Hymenolepis diminuta. Comp. Bioehem. Physiol. 14,491-511. PRICHARD R. K. & SCHOFIELD P. J. (1968a) A comparative study of the tricarboxylic acid cycle in Fasciola hepatica and rat liver. Comp. Biochem. Physiol. 25, 1005-1020. PRICHARD R. K. & SCHOFmLD P. J. (1968b) Phosphoenolpyruvate carboxykinase in the adult liver fluke, Fasciola hepatica. Comp. Biochem. Physiol. 24, 773-785. RASERO F. S., MONTEOLIVA M. & MAYOR F. (1968) Enzymes related to 4-aminobutyrate metabolism in intestinal parasites. Comp. Biochem. Physiol. 25, 693-701. ROGERS W. P. (1949) On the relative importance of aerobic metabolism in small nematode parasites of the alimentary t r a c t - - I . Oxygen tensions in the normal environment of the parasites. Aust.J. scient. Res. B 2, 166-174. SAZ H. J. & HUBBARD J. A. (1957) T h e oxidative decarboxylation of malate by Ascaris lumbricoides. J. biol. Chem. 225, 921-933. SAZ H. J. & VIDRINE A. (1959) T h e mechanism of formation of succinate and propionate by Ascaris lumbricoides muscle. J. biol. Chem. 234, 2001-2005. SCHEmEL L. W. & SAZ H. J. (1966) The pathway of anaerobic carbohydrate dissimilation in Hymenolepis diminuta. Comp. Biochem. Physiol. 18, 151-162. SLATER T. F. & SAWER B. (1962) A colorimetric method for estimating the pyridine nucleotide content of small amounts of animal tissue. Nature, Lond. 193, 45 a, A,56. SMITH M. J. H. & MOSES V. (1960) Uncoupling reagents in metabolism--I. T h e effects of salicylate and 2:4-dinitrophenol on the incorporation of 14C from labelled glucose and acetate into the soluble intermediates of isolated rat tissues. Bioehem. J. 76, 579-585. VON BRAND T. (1933) Untersuchungen iiber den Stoffbestand einiger Cestoden brad den Stoffwechsel yon Moniezia expansa. Z. vergl. Physiol. 18, 562-596. y o n BRAND T. (1966) Biochemistry of Parasites. Academic Press, New York and London.
Key Word Index--Tricarboxylic acid cycle; Moniezia expansa; cestode biochemistry (TCA); malic enzyme; phosphoenol pyruvate carboxykinase.