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Succinate dehydrogenase and cytochrome oxidase in adult and plerocercoid Diphyllobothrium latum
Comp. Biochem. Physiol., 1974, Vol. 49B, pp. 87 to 92. Pergamon Press. Printed in Great Britain
SUCCINATE DEHYDROGENASE AND CYTOCHROME OXIDASE IN A D U L T AND PLEROCERCOID D I P H Y L L O B O T H R I U M LA T U M KALEVI S A L M I N E N
Department of Food Hygiene, College of Veterinary Medicine, H/imeentie 57, 00550 Helsinki 55, Finland (Received 7 September 1973)
Abstract--1. The apparent K~'s with succinate dehydrogenase using phenazine methosulphate and cytochrome c as electron aeceptors and with cytochrome oxidase of mitochondrial fraction of adult and plerocercoid Diphyllobothrium latura were determined. The specific activities of suceinate CoQ oxidoreductase and succinate cytochrome oxidoreductase were also determined. 2. The plerocercoids are able to transfer electrons from succinate to CoQ, but not to cytochrome c. 3. The kinetic constants of adult and plerocercoid succinate dehydrogenase do not show significant differences. 4. The K~ and specific activity of larval cytochrome oxidase are considerably lower than those of the adult cytochrome oxidase.
INTRODUCTION
THE RESPIRATORY chain system of several intestinal helminths seems to differ markedly from that of the vertebrate hosts. It has been shown that the mitochondrial succinate dehydrogenase of Ascaris acts physiologically in a manner opposite to that of mammalian tissues, that is, as a "fumarate reductase" producing succinate (Kmetec & Bueding, 1961). Intramitochondrially formed NADH is used to reduce malate to succinate v~a fumarate with the concomitant formation of ATP (Saz & Lescure, 1969; Saz, 1970). The concepts of the role of cytochromes in the respiration of intestinal helminths are variable. Cytochrome oxidase activity could not be demonstrated in adult Ascaris by Bueding & Charms (1952) and no evidence for cytochrome a+a3 was observed spectrally by Chance & Parsons (1963) and Hill et al. (1971). On the contrary it has been shown that Ascaris and Moniezia have functional b, c and atype cytochromes, and two CO-reactive haemoproteins identified as cytochromes o and a a (Cheah, 1968; Cheah & Chance, 1970). Unlike the classical mammalian respiratory chain both Ascaris and Moniezia modify their respiratory chain systems to adapt to the intestinal environment by having the two mentioned terminal oxidases, cytochromes o and a3. 87
88
KALEVI SALMINEN
T h e b r o a d t a p e w o r m DiphyUobothrium latum is an e x a m p l e of a cestode, t h e v a r i o u s d e v e l o p m e n t a l stages o f w h i c h i n h a b i t g r e a t l y different hosts. T h e p l e r o cercoid inhabits water organisms, whereas the adult worm parasitizes the intestinal t r a c t o f several m a m m a l i a n species i n c l u d i n g m a n . T h e larvae w e r e s h o w n as l o n g ago as 1933 to possess a c y t o c h r o m e s y s t e m ( F r i e d h e i m & Baer, 1933). N o i n f o r m a t i o n o f t h e a d u l t w o r m is available, b u t M . expansa, a n o t h e r large i n t e s t i n a l c e s t o d e was s h o w n to b e a e r o b i c b y t h e e x i s t e n c e o f c y t o c h r o m e as ( C h e a h , 1971). I t was t h e r e f o r e o f i n t e r e s t to d e t e r m i n e w h e t h e r t h e t w o d e v e l o p m e n t a l stages o f D. latum h a v e r e s p i r a t o r y e n z y m e s s i m i l a r to t h o s e of a e r o b i c t i s s u e s a n d w h e t h e r a n y d i f f e r e n c e s o c c u r b e t w e e n t h e t w o stages. MATERIALS AND METHODS
Preparation of the particulate fraction T h e plerocercoid material used in the experiments was recovered from naturally infested pikes. T h e adult worms were obtained from experimentally infested golden hamsters, which were sacrificed by decapitation and autopsied about 2 weeks after the infestation. Immediately after the plerocercoids had been isolated from the fish and the adult worms removed from the test animals, they were washed several times with ice-cold 0"4 M sucrose containing 10 -3 M E D T A , 0"2% heparine, p H 6'9, to remove the remains of the tissue and ingesta. T h e isolated plerocereoids and adult worms were homogenized in a tenfold amount of the sucrose. T h e rough homogenization was carried out with an Ultra-Turrax homogenizer operated for 10 see with a low cutter speed. T h e slurry was further homogenized with a Potter-Elvehjem-type tissue grinder equipped with a tight-fitting Teflon pestle (manufactured by Arthur A. Thomas Company, Philadelphia) at 200 rev/min for 1 min. T h e vessel was kept immersed in ice-water. T h e homogenate was spun for 10 min at 1800 g to remove the nuclei and unbroken cells. T h e supernatant fluid was further centrifuged for 20 min at 25,000 g to sediment the mitochondrial particulate fraction. T h e pellet was washed once. Finally, the sediment was suspended in 0"05 M ethanolamine buffer, p H 7"6, and stored for enzyme assays at a temperature of - 4 5 ° C . Protein determination was carried out according to the method of Lowry et al. (1951) with bovine serum albumin as a protein standard.
Enzyme assays T h e enzyme assays were carried out with a Perkin-Elmer U V - V I S 137 Spectrophotometer with quartz cuvettes of 300/~1 capacity. T h e spectrophotometer was equipped with a Digital Concentration Read-out unit, which allowed a numerical recording of the absorbance at 2-sec intervals. All the chemicals used were commercial products. Succinate dehydrogenase (succinate : (acceptor) oxidoreductase E.C. 1.3.99.1). T h e electron acceptors employed were (1) phenazine methosulphate, (2) cytochrome c and (3) CoQ~. T h e decrease or increase of absorbance was measured at (1) 600 nm, (2) 550 nm and (3) 275 nm, respectively. In addition to the protein, the assay mixtures contained, in a total volume of 300/xl: (1) 45 m o l e s K C N , crystalline bovine serum albumin in a final concentration of 0"1%, 30 nmoles D C P I P * , 0-06-4.8/zmoles succinate, 0"9-36 nmoles P M S and 70/xl 0"02 M phosphate buffer, p H 7"8. * Abbreviations used: DCPIP, 2,6 dichlorphenol-indophenol; PMS, phenazine methosulphate.
SUCCINATE DEHYDROGENASE AND CYTOCHROME OXIDASE I N
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89
The assay mixture was preincubated in the presence of the protein for 5 min at + 38°C. The reaction was initiated by the addition of PMS and DCPIP. (2) 90 nmoles KCN, crystalline bovine serum albumin in a final concentration of 0"1%, 2-20 umoles succinate, 0"045-1"8 #moles cytochrome c and 100 #1 0-02 M phosphate buffer, pH 7"8. (3) Potassium succinate (0"5 M) 30 #1, 1 M potassium phosphate buffer pH 7"0 30 pl, 1 mM E D T A 30#1, 0"3% Triton X-100 10#1, 1"5 mM DCPIP 10#1 and 0"6 mM CoQ6 in methanol 10 #1. The mixture with protein was incubated for 5 min in a waterbath at 38°C. The reaction was initiated by the addition of DCPIP and CoQs. Cytochrome oxidase (ferrocytochrome c : oxygen oxidoreductase E.C. 1.9.3.1). The cytochrome oxidase was assayed by monitoring the oxidation of ascorbate-reduced ferrocytochrome by the enzyme at 550 nm. The reaction mixture contained, in a total volume of 300 #1, 0"9-18 nmoles cytochrome c in a 0"01 M phosphate buffer, pH 7-0. Each experimental value represents the mean of three separate experiments conducted in duplicate. Individual values did not differ from the mean by more than _+10%. The values of Kra were determined as described before (Salminen, 1972). RESULTS T h e c o m m o n artificial electron acceptor P M S acts as an oxidant for succinate dehydrogenase in both the adult and plerocercoid, D. latum. T h e physiological electron acceptor, cytochrome c, on the other hand, acts as an oxidant for succinate dehydrogenase only in the adult w o r m (Table 1). T h e observed values of K ~ "~inate and K PMs of adult and plerocercoid enzymes do not differ significantly. TABLE 1--THE
MEASURED M I C H A E L I S CONSTANTS OF THE SUCCINATE DEHYDROGENASE OF ADULT AND PLEROCERCOID
D. latum
Adult Oxidant Phenazine methosulphate Cytochrome c
K~ptor (M) 4.4 x 10 -s 1"7 x 10 -5
Plerocercoid K~U~nate (M)
8.6 x 10 -4 8"8 x 10 -~
K~ptor (M) 5.5 x 10 -6 --
K~te (M) 9"2 x 10 -4 --
Kr~ values were determined at four to five concentrations over a ten to eightyfold range by double reciprocal plots. I n the mitochondrial electron transport chain, the electron acceptor for succinate dehydrogenase is coenzyme Q. T h e enzymes of the particulate fraction of both the adult and plerocercoid are capable of transferring electrons f r o m succinate to CoQ 8. T h e activity of the adult enzyme is about nine orders of magnitude higher than that of the plerocercoid (Table 2). T h e presence of cytochrome oxidase and plerocercoid was demonstrated as shown in T a b l e 3. T h e activity of the plerocercoid enzyme is about one-sixth, and the observed value of K,~ about one-third of the corresponding value of the adult worm. T h e observed value of K ~fe~r°~yt°ch~°mec is three times higher than the value of the plerocercoid enzyme.
90
KALEVI SALMINEN
TABLE 2 - - T H E
SPECIFIC ACTIVITY OF SUCCINATE C o Q 6 - AND SUCCINATE CYTOCHROME ¢OXIDOREDUCTASE IN ADULT AND PLEROCERCOID D. latum
Electron acceptor
Adult
Plerocercoid
CoQ6 Cytochrome c
15"2 7'8
1 "7 --
The activity is expressed in nmoles succinate oxidized/min per mg protein. TABLE 3--THE SPECIFIC ACTIVITY OF CYTOCHROME OXIDASE AND MEASURED M I C H A E L I S CONSTANTS FOR THE OXIDATION OF FERROCYTOCHROME C BY ADULT AND PLEROCERCOID D .
latum
Enzyme source Adult Plerocercoid
Specific activity
K~,~ (M)
119'0 18'0
2"1 x 10 _5 6"9 x 10 _6
The activity is expressed as nmoles cytochrome c oxidized/min per mg protein. The K m values were determined at five concentrations over a twentyfold range. DISCUSSION T h e kinetic constants of adult and plerocercoid succinate dehydrogenase as determined by the spectrophotometric phenazine assay do not show significant differences. Consequently it seems that the transition from larval to adult stage does not involve any change in succinate dehydrogenase. T h e observed values of K succinate lie within the range of Michaelis constants from various organisms as summarized by Singer (1966), and are close to the values reported for aerobic organisms, even though K,,+ only poorly correlates to the character of metabolism. A more reliable parameter is the ratio of rates of succinate oxidation to fumarate reduction. T h e electron acceptor of succinate of the mammalian respiratory chain is coenzyme Q. Ubiquinone is considered by Ernster et al. (1969) to be essential for the interaction of succinate dehydrogenase, and this interaction is a requisite for the normal functioning of the respiratory chain. On the assumption that there occurs an electron transport system in cestodes similar to that of mammalian tissues, the demonstration of the succinate CoQ oxidoreductase was not surprising. According to Bryant (1970) the actual participation of ubiquinone in the electron transfer system of cestodes has not been unequivocally shown. Succinate cytochrome c oxidoreductase activity was demonstrated in adult D. latum, whereas no activity was measurable in the plerocercoid. T h e r e seems to be a metabolic defect in the plerocercoid between ubiquinone and cytochrome c. T h e cytochrome systems of dscaris and Moniezia differ in somewhat similar manner.
SUCCINATE DEHYDROGENASE AND CYTOCHROME OXIDASE I N DIPHYLLOBOTHR1UM
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Moniezia has the classical mammalian cytochrome b and Cl, neither of which were observed in Ascaris (Cheah, 1972). The specific activity of succinate cytochrome c oxidoreductase of adult D. latum is close to the values reported for Haemochnus contortus larvae (Moon & Schofield, 1967) and Fasciola hepatica (Prichard &
Sehofield, i971). The presence of eytochrome oxidase in adult D. latum indicates that, at least, a part of the substrates can be completely oxidized. Due to the several experimental factors that influence the value of KmfCrr°eyt°chr°mec as outlined by Wainio (1970) the values of Km given can be used for comparison only between the adult and plerocercoid enzymes. The lower value of the plerocercoid enzyme might be indicative of lower cytochrome c content of the larval tissues. The specific activity of the adult cytochrome oxidase is so high that cytochrome oxidase can account for the rate of succinate oxidation actually observed. The absence of succinate cytochrome oxidoreductase, lower specific activities of succinate CoQ oxidoreductase and cytochrome oxidase could express the minor importance of the pathway in the plerocercoid. The present results indicate that adult D. latum has an electron transfer chain capable of oxidizing substrates completely, and which in aerobic systems is similar to the currently accepted mammalian electron transport system. This does not exclude the possibility that in anaerobic systems the worm produces succinate by the succinate oxidase system described by Kmetec & Bueding (1961). This type of metabolism seems to be probable in the plerocercoid, which cannot oxidize succinate by the succinate-cytochrome c pathway. REFERENCES BRYANTC. (1970) Electron transport in parasitic helminths and protozoa. Adv. Parasit. 8, 139-172. BUEDING E. & CHAaMSB. (1952) Cytochrome c, cytochrome oxidase, and succinoxidase activities of helminths. J. biol. Chem. 196, 615-627. CHANCEB. & PARSONSD. F. (1963) Cytochrome function in relation to inner structure of mitochondria. Science, Wash. 142, 1176-1179. CHEAHK. S. (1968) The respiratory components of Moniezia expansa (Cestoda). Biochim. biophys. Acta 153, 718-720. CHEAHK. S. (1971) Oxidative phosphorylation in Moniezia muscle mitochondria. Biochim. biophys. Acta 253, 1-11. CHEAHK. S. (1972) Cytochromes in Ascaris and Moniezia. In Comparative Biochemistry of Parasites (Edited by VANDEN BoSSeHEH.), pp. 417-432. AcademicPress, New York. CHEAHK. S. & CHANCEB. (1970). The oxidase systems of Ascaris muscle mitochondria. Biochim. biophys. Acta 223, 55-60. EaNSTEa L., LEE I.-Y., NOaDLINO B. & PEaSSON B. (1969) Studies with ubiquinonedepleted submitochondrial particles. Eur.J. Biochem. 9, 299-310. FamDHEIM E. A. H. & BAEaJ. G. (1933) Untersuchungen iiber die Atmung yon Diphyllobothrium latum (L.). Biochem. Z. 265, 329-337. HIr.L G. C., PERKOWSKIC. A. & MATrmWSONN. W. (1971) Purification and properties of cytochrome css0 from Ascaris lumbricoides var. suum. Biochim. biophys. Acta 236, 242245. KM~TEC E. & BUEDING E. (1961) Succinic and reduced diphosphopyridine nucleotide oxidase systems of Asearis muscle. J. biol. Chem. 236, 584-591.
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KALEVI SALMINEN
LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MOON K. E. & SCHOFIELDP. J. (1968) Reduction of cytochrome c by Haemonchus contortus larvae. Comp. Biochem. Physiol. 26, 745-748. PRICHARD R. K. & SCHOFIELDP. J. (1971) Fasciola hepatica: eytochrome c oxidoreductases and effects of oxygen tension and inhibitors. Expl. Parasit. 29, 215-222. SALMINEN K. (1972) The oxidation of external N A D H by adult and plerocercoid Diphyllobothrium latum. Comp. Biochem. Physiol. 44B, 283-289. SAZ H. J. (1970) Comparative energy metabolisms of some parasitic helminths. J. Parasit. 56, 634-642. SAZ H. J. & LESCUaE O. L. (1969) T h e functions of phosphoenolpyruvate carboxykinase and malic enzyme in the anaerobic formation of succinate by Ascaris lumbricoides. Comp. Biochem. Physiol. 30, 49-60. SINCER T. A. (1966) Flavoprotein dehydrogenases of the respiratory chain. In Comprehensive Biochemistry (Edited by FLORKIN M. & STOTZ E. H.), Vol. XIV, pp. 127-198. Elsevier Publishing Company, Amsterdam. WAINIO W. (1970) The Mammalian Mitochondrial Respiratory Chain, pp. 285-286. Academic Press, New York.
Key Word Index--Cestode; Diphyllobothrium latum; succinate dehydrogenase; cytochrome oxidase; parasite development; parasite metabolism.
SUCCINATE DEHYDROGENASE AND CYTOCHROME OXIDASE IN A D U L T AND PLEROCERCOID D I P H Y L L O B O T H R I U M LA T U M KALEVI S A L M I N E N
Department of Food Hygiene, College of Veterinary Medicine, H/imeentie 57, 00550 Helsinki 55, Finland (Received 7 September 1973)
Abstract--1. The apparent K~'s with succinate dehydrogenase using phenazine methosulphate and cytochrome c as electron aeceptors and with cytochrome oxidase of mitochondrial fraction of adult and plerocercoid Diphyllobothrium latura were determined. The specific activities of suceinate CoQ oxidoreductase and succinate cytochrome oxidoreductase were also determined. 2. The plerocercoids are able to transfer electrons from succinate to CoQ, but not to cytochrome c. 3. The kinetic constants of adult and plerocercoid succinate dehydrogenase do not show significant differences. 4. The K~ and specific activity of larval cytochrome oxidase are considerably lower than those of the adult cytochrome oxidase.
INTRODUCTION
THE RESPIRATORY chain system of several intestinal helminths seems to differ markedly from that of the vertebrate hosts. It has been shown that the mitochondrial succinate dehydrogenase of Ascaris acts physiologically in a manner opposite to that of mammalian tissues, that is, as a "fumarate reductase" producing succinate (Kmetec & Bueding, 1961). Intramitochondrially formed NADH is used to reduce malate to succinate v~a fumarate with the concomitant formation of ATP (Saz & Lescure, 1969; Saz, 1970). The concepts of the role of cytochromes in the respiration of intestinal helminths are variable. Cytochrome oxidase activity could not be demonstrated in adult Ascaris by Bueding & Charms (1952) and no evidence for cytochrome a+a3 was observed spectrally by Chance & Parsons (1963) and Hill et al. (1971). On the contrary it has been shown that Ascaris and Moniezia have functional b, c and atype cytochromes, and two CO-reactive haemoproteins identified as cytochromes o and a a (Cheah, 1968; Cheah & Chance, 1970). Unlike the classical mammalian respiratory chain both Ascaris and Moniezia modify their respiratory chain systems to adapt to the intestinal environment by having the two mentioned terminal oxidases, cytochromes o and a3. 87
88
KALEVI SALMINEN
T h e b r o a d t a p e w o r m DiphyUobothrium latum is an e x a m p l e of a cestode, t h e v a r i o u s d e v e l o p m e n t a l stages o f w h i c h i n h a b i t g r e a t l y different hosts. T h e p l e r o cercoid inhabits water organisms, whereas the adult worm parasitizes the intestinal t r a c t o f several m a m m a l i a n species i n c l u d i n g m a n . T h e larvae w e r e s h o w n as l o n g ago as 1933 to possess a c y t o c h r o m e s y s t e m ( F r i e d h e i m & Baer, 1933). N o i n f o r m a t i o n o f t h e a d u l t w o r m is available, b u t M . expansa, a n o t h e r large i n t e s t i n a l c e s t o d e was s h o w n to b e a e r o b i c b y t h e e x i s t e n c e o f c y t o c h r o m e as ( C h e a h , 1971). I t was t h e r e f o r e o f i n t e r e s t to d e t e r m i n e w h e t h e r t h e t w o d e v e l o p m e n t a l stages o f D. latum h a v e r e s p i r a t o r y e n z y m e s s i m i l a r to t h o s e of a e r o b i c t i s s u e s a n d w h e t h e r a n y d i f f e r e n c e s o c c u r b e t w e e n t h e t w o stages. MATERIALS AND METHODS
Preparation of the particulate fraction T h e plerocercoid material used in the experiments was recovered from naturally infested pikes. T h e adult worms were obtained from experimentally infested golden hamsters, which were sacrificed by decapitation and autopsied about 2 weeks after the infestation. Immediately after the plerocercoids had been isolated from the fish and the adult worms removed from the test animals, they were washed several times with ice-cold 0"4 M sucrose containing 10 -3 M E D T A , 0"2% heparine, p H 6'9, to remove the remains of the tissue and ingesta. T h e isolated plerocereoids and adult worms were homogenized in a tenfold amount of the sucrose. T h e rough homogenization was carried out with an Ultra-Turrax homogenizer operated for 10 see with a low cutter speed. T h e slurry was further homogenized with a Potter-Elvehjem-type tissue grinder equipped with a tight-fitting Teflon pestle (manufactured by Arthur A. Thomas Company, Philadelphia) at 200 rev/min for 1 min. T h e vessel was kept immersed in ice-water. T h e homogenate was spun for 10 min at 1800 g to remove the nuclei and unbroken cells. T h e supernatant fluid was further centrifuged for 20 min at 25,000 g to sediment the mitochondrial particulate fraction. T h e pellet was washed once. Finally, the sediment was suspended in 0"05 M ethanolamine buffer, p H 7"6, and stored for enzyme assays at a temperature of - 4 5 ° C . Protein determination was carried out according to the method of Lowry et al. (1951) with bovine serum albumin as a protein standard.
Enzyme assays T h e enzyme assays were carried out with a Perkin-Elmer U V - V I S 137 Spectrophotometer with quartz cuvettes of 300/~1 capacity. T h e spectrophotometer was equipped with a Digital Concentration Read-out unit, which allowed a numerical recording of the absorbance at 2-sec intervals. All the chemicals used were commercial products. Succinate dehydrogenase (succinate : (acceptor) oxidoreductase E.C. 1.3.99.1). T h e electron acceptors employed were (1) phenazine methosulphate, (2) cytochrome c and (3) CoQ~. T h e decrease or increase of absorbance was measured at (1) 600 nm, (2) 550 nm and (3) 275 nm, respectively. In addition to the protein, the assay mixtures contained, in a total volume of 300/xl: (1) 45 m o l e s K C N , crystalline bovine serum albumin in a final concentration of 0"1%, 30 nmoles D C P I P * , 0-06-4.8/zmoles succinate, 0"9-36 nmoles P M S and 70/xl 0"02 M phosphate buffer, p H 7"8. * Abbreviations used: DCPIP, 2,6 dichlorphenol-indophenol; PMS, phenazine methosulphate.
SUCCINATE DEHYDROGENASE AND CYTOCHROME OXIDASE I N
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The assay mixture was preincubated in the presence of the protein for 5 min at + 38°C. The reaction was initiated by the addition of PMS and DCPIP. (2) 90 nmoles KCN, crystalline bovine serum albumin in a final concentration of 0"1%, 2-20 umoles succinate, 0"045-1"8 #moles cytochrome c and 100 #1 0-02 M phosphate buffer, pH 7"8. (3) Potassium succinate (0"5 M) 30 #1, 1 M potassium phosphate buffer pH 7"0 30 pl, 1 mM E D T A 30#1, 0"3% Triton X-100 10#1, 1"5 mM DCPIP 10#1 and 0"6 mM CoQ6 in methanol 10 #1. The mixture with protein was incubated for 5 min in a waterbath at 38°C. The reaction was initiated by the addition of DCPIP and CoQs. Cytochrome oxidase (ferrocytochrome c : oxygen oxidoreductase E.C. 1.9.3.1). The cytochrome oxidase was assayed by monitoring the oxidation of ascorbate-reduced ferrocytochrome by the enzyme at 550 nm. The reaction mixture contained, in a total volume of 300 #1, 0"9-18 nmoles cytochrome c in a 0"01 M phosphate buffer, pH 7-0. Each experimental value represents the mean of three separate experiments conducted in duplicate. Individual values did not differ from the mean by more than _+10%. The values of Kra were determined as described before (Salminen, 1972). RESULTS T h e c o m m o n artificial electron acceptor P M S acts as an oxidant for succinate dehydrogenase in both the adult and plerocercoid, D. latum. T h e physiological electron acceptor, cytochrome c, on the other hand, acts as an oxidant for succinate dehydrogenase only in the adult w o r m (Table 1). T h e observed values of K ~ "~inate and K PMs of adult and plerocercoid enzymes do not differ significantly. TABLE 1--THE
MEASURED M I C H A E L I S CONSTANTS OF THE SUCCINATE DEHYDROGENASE OF ADULT AND PLEROCERCOID
D. latum
Adult Oxidant Phenazine methosulphate Cytochrome c
K~ptor (M) 4.4 x 10 -s 1"7 x 10 -5
Plerocercoid K~U~nate (M)
8.6 x 10 -4 8"8 x 10 -~
K~ptor (M) 5.5 x 10 -6 --
K~te (M) 9"2 x 10 -4 --
Kr~ values were determined at four to five concentrations over a ten to eightyfold range by double reciprocal plots. I n the mitochondrial electron transport chain, the electron acceptor for succinate dehydrogenase is coenzyme Q. T h e enzymes of the particulate fraction of both the adult and plerocercoid are capable of transferring electrons f r o m succinate to CoQ 8. T h e activity of the adult enzyme is about nine orders of magnitude higher than that of the plerocercoid (Table 2). T h e presence of cytochrome oxidase and plerocercoid was demonstrated as shown in T a b l e 3. T h e activity of the plerocercoid enzyme is about one-sixth, and the observed value of K,~ about one-third of the corresponding value of the adult worm. T h e observed value of K ~fe~r°~yt°ch~°mec is three times higher than the value of the plerocercoid enzyme.
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KALEVI SALMINEN
TABLE 2 - - T H E
SPECIFIC ACTIVITY OF SUCCINATE C o Q 6 - AND SUCCINATE CYTOCHROME ¢OXIDOREDUCTASE IN ADULT AND PLEROCERCOID D. latum
Electron acceptor
Adult
Plerocercoid
CoQ6 Cytochrome c
15"2 7'8
1 "7 --
The activity is expressed in nmoles succinate oxidized/min per mg protein. TABLE 3--THE SPECIFIC ACTIVITY OF CYTOCHROME OXIDASE AND MEASURED M I C H A E L I S CONSTANTS FOR THE OXIDATION OF FERROCYTOCHROME C BY ADULT AND PLEROCERCOID D .
latum
Enzyme source Adult Plerocercoid
Specific activity
K~,~ (M)
119'0 18'0
2"1 x 10 _5 6"9 x 10 _6
The activity is expressed as nmoles cytochrome c oxidized/min per mg protein. The K m values were determined at five concentrations over a twentyfold range. DISCUSSION T h e kinetic constants of adult and plerocercoid succinate dehydrogenase as determined by the spectrophotometric phenazine assay do not show significant differences. Consequently it seems that the transition from larval to adult stage does not involve any change in succinate dehydrogenase. T h e observed values of K succinate lie within the range of Michaelis constants from various organisms as summarized by Singer (1966), and are close to the values reported for aerobic organisms, even though K,,+ only poorly correlates to the character of metabolism. A more reliable parameter is the ratio of rates of succinate oxidation to fumarate reduction. T h e electron acceptor of succinate of the mammalian respiratory chain is coenzyme Q. Ubiquinone is considered by Ernster et al. (1969) to be essential for the interaction of succinate dehydrogenase, and this interaction is a requisite for the normal functioning of the respiratory chain. On the assumption that there occurs an electron transport system in cestodes similar to that of mammalian tissues, the demonstration of the succinate CoQ oxidoreductase was not surprising. According to Bryant (1970) the actual participation of ubiquinone in the electron transfer system of cestodes has not been unequivocally shown. Succinate cytochrome c oxidoreductase activity was demonstrated in adult D. latum, whereas no activity was measurable in the plerocercoid. T h e r e seems to be a metabolic defect in the plerocercoid between ubiquinone and cytochrome c. T h e cytochrome systems of dscaris and Moniezia differ in somewhat similar manner.
SUCCINATE DEHYDROGENASE AND CYTOCHROME OXIDASE I N DIPHYLLOBOTHR1UM
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Moniezia has the classical mammalian cytochrome b and Cl, neither of which were observed in Ascaris (Cheah, 1972). The specific activity of succinate cytochrome c oxidoreductase of adult D. latum is close to the values reported for Haemochnus contortus larvae (Moon & Schofield, 1967) and Fasciola hepatica (Prichard &
Sehofield, i971). The presence of eytochrome oxidase in adult D. latum indicates that, at least, a part of the substrates can be completely oxidized. Due to the several experimental factors that influence the value of KmfCrr°eyt°chr°mec as outlined by Wainio (1970) the values of Km given can be used for comparison only between the adult and plerocercoid enzymes. The lower value of the plerocercoid enzyme might be indicative of lower cytochrome c content of the larval tissues. The specific activity of the adult cytochrome oxidase is so high that cytochrome oxidase can account for the rate of succinate oxidation actually observed. The absence of succinate cytochrome oxidoreductase, lower specific activities of succinate CoQ oxidoreductase and cytochrome oxidase could express the minor importance of the pathway in the plerocercoid. The present results indicate that adult D. latum has an electron transfer chain capable of oxidizing substrates completely, and which in aerobic systems is similar to the currently accepted mammalian electron transport system. This does not exclude the possibility that in anaerobic systems the worm produces succinate by the succinate oxidase system described by Kmetec & Bueding (1961). This type of metabolism seems to be probable in the plerocercoid, which cannot oxidize succinate by the succinate-cytochrome c pathway. REFERENCES BRYANTC. (1970) Electron transport in parasitic helminths and protozoa. Adv. Parasit. 8, 139-172. BUEDING E. & CHAaMSB. (1952) Cytochrome c, cytochrome oxidase, and succinoxidase activities of helminths. J. biol. Chem. 196, 615-627. CHANCEB. & PARSONSD. F. (1963) Cytochrome function in relation to inner structure of mitochondria. Science, Wash. 142, 1176-1179. CHEAHK. S. (1968) The respiratory components of Moniezia expansa (Cestoda). Biochim. biophys. Acta 153, 718-720. CHEAHK. S. (1971) Oxidative phosphorylation in Moniezia muscle mitochondria. Biochim. biophys. Acta 253, 1-11. CHEAHK. S. (1972) Cytochromes in Ascaris and Moniezia. In Comparative Biochemistry of Parasites (Edited by VANDEN BoSSeHEH.), pp. 417-432. AcademicPress, New York. CHEAHK. S. & CHANCEB. (1970). The oxidase systems of Ascaris muscle mitochondria. Biochim. biophys. Acta 223, 55-60. EaNSTEa L., LEE I.-Y., NOaDLINO B. & PEaSSON B. (1969) Studies with ubiquinonedepleted submitochondrial particles. Eur.J. Biochem. 9, 299-310. FamDHEIM E. A. H. & BAEaJ. G. (1933) Untersuchungen iiber die Atmung yon Diphyllobothrium latum (L.). Biochem. Z. 265, 329-337. HIr.L G. C., PERKOWSKIC. A. & MATrmWSONN. W. (1971) Purification and properties of cytochrome css0 from Ascaris lumbricoides var. suum. Biochim. biophys. Acta 236, 242245. KM~TEC E. & BUEDING E. (1961) Succinic and reduced diphosphopyridine nucleotide oxidase systems of Asearis muscle. J. biol. Chem. 236, 584-591.
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LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MOON K. E. & SCHOFIELDP. J. (1968) Reduction of cytochrome c by Haemonchus contortus larvae. Comp. Biochem. Physiol. 26, 745-748. PRICHARD R. K. & SCHOFIELDP. J. (1971) Fasciola hepatica: eytochrome c oxidoreductases and effects of oxygen tension and inhibitors. Expl. Parasit. 29, 215-222. SALMINEN K. (1972) The oxidation of external N A D H by adult and plerocercoid Diphyllobothrium latum. Comp. Biochem. Physiol. 44B, 283-289. SAZ H. J. (1970) Comparative energy metabolisms of some parasitic helminths. J. Parasit. 56, 634-642. SAZ H. J. & LESCUaE O. L. (1969) T h e functions of phosphoenolpyruvate carboxykinase and malic enzyme in the anaerobic formation of succinate by Ascaris lumbricoides. Comp. Biochem. Physiol. 30, 49-60. SINCER T. A. (1966) Flavoprotein dehydrogenases of the respiratory chain. In Comprehensive Biochemistry (Edited by FLORKIN M. & STOTZ E. H.), Vol. XIV, pp. 127-198. Elsevier Publishing Company, Amsterdam. WAINIO W. (1970) The Mammalian Mitochondrial Respiratory Chain, pp. 285-286. Academic Press, New York.
Key Word Index--Cestode; Diphyllobothrium latum; succinate dehydrogenase; cytochrome oxidase; parasite development; parasite metabolism.