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The oxidation of external NADH by adult and plerocercoid Diphyllobothrium latum
Comp. Biochem. Physiol., 1973, Vol. 44B, pp. 283 to 289. Pergamon Press. Printed in Great Britain
THE OXIDATION OF EXTERNAL NADH BY A D U L T AND PLEROCERCOID D I P H Y L L O B O T H R I U M LATUM KALEVI SALMINEN Department of Food Hygiene, College of Veterinary Medicine, H/imeentie 57, 00550 Helsinki 55, Finland
(Received 16 May 1972) Abstract--1. The apparent Km's and relative rates of mitochondrial NADH dehydrogenase and specific activity of NADH-CoQ e oxidoreductase of adult and plerocercoid Diphyllobothrium latura were determined. 2. As electron accepters for NADH dehydrogenase K-ferricyanide, 2,6dichlorphenolindophenol and cytochrome c were used. 3. The g,, ferrocrtoohromoo of the adult enzyme is the same as that observed for the plerocercoid, whereas the Km NADa of the adult enzyme is only onetwenty-fifth of that of the plerocercoid enzyme. 4. The activity of the adult NADH-CoQ8 oxidoreductase was over twenty times greater than that of the plerocercoid enzyme. INTRODUCTION THE aEACTIONS of the tricarboxylic acid cycle occur in vertebrate tissues in the soluble cytoplasm of the cells in which N A D H is produced. N A D H is unable to diffuse from this compartment into the mitochondria. The diffusion takes place through the glycerol phosphate shuttle and N A D H is further oxidized by the mitochondrial electron transport chain. This respiratory chain-linked N A D H dehydrogenase is amytal and rotenone sensitive (Singer & Gutman, 1971). Studies with mammalian liver mitochondria have shown that there also exists an enzyme system capable of oxidizing external NADH. This enzyme system is located in the outer mitochondrial membrane (Sottocasa et al., 1957). The oxidation of external N A D H by cytochrome c seems to be antimycin A, amytal and rotenone insensitive and it is not coupled to oxidative phosphorylation. The role of these mitochondrial NADH-oxidizing enzymes in helminths is not known. In an aerobic environment N A D H can be oxidized by the mitochondrial electron transport chain, whereas under anaerobic conditions various pathways have been suggested for the dehydrogenation of NADH. Since the various developmental stages of Diphyllobothrium latum inhabit greatly different hosts, this helminth was selected for a comparative study of the NADH-dehydrogenating enzyme systems. The plerocercoid lives in water organisms under virtually anaerobic conditions; the adult worm, on the other hand, inhabits the intestinal tract of 283
284
KALEVI SALMINEN
several mammalianspecies,includingman, wherethere is enoughmolecularoxygen present to permit an aerobic metabolism (Laser, 1944; Hobson, 1948; Berntzen, 1966). MATERIALS AND METHODS
Preparation of the particulate fraction The plerocercoid material used in the experiments was recovered from naturally infested pikes. The adult worms were obtained from experimentally infested golden hamsters, which were sacrificed by decapitation and autopsied about 2 weeks after infestation. Immediately after the plerocercoids had been isolated from the pikes and the adult worms removed from the golden hamsters, they were washed several times with ice-cold 0"4 M sucrose containing 0"001 M E D T A , 0"2% heparin, pH 6"9 to remove the remains of fish tissue and intestinal contents. Homogenization was carried out in a tenfold amount of the sucrose solution. The material to be homogenized was at first cut into small pieces with an Ultra-Turrax homogenizer operated for 10 sec at a low cutter rotation speed. The prehomogenized slurry was further homogenized with a Potter-Elvehjem type tissue grinder equipped with a tightfitting Teflon pestle (Arthur A. Thomas Co., Philadelphia, U.S.A.) at 200 rev/min for 1 min. The vessel was kept immersed in ice-water. The homogenate was centrifuged for 10 min at 1800 g to remove the unbroken cells and nuclei. T h e supernatant was further spun for 20 min at 25,000 g to sediment the mitochondrial particulate fraction. The pellet was washed once. Finally, the pellet was suspended in an 0"05 M triethanol amine buffer, pH 7"6 and stored for enzyme assays at 45 °C. Protein determination was carried out according to the Folin method of Lowry et al. (1951), with bovine serum albumin as the protein standard. -
N A D H dehydrogenase assay T h e enzyme assays were carried out with a Perkin Elmer UV-VI S 139 spectrophotometer with cuvettes of 300 ktl capacity. The spectrophotometer was equipped with a Digital Concentration Read-out unit, which allowed a numerical recording of the absorbance at 2-sec intervals. The enzyme activity was measured by the methods of Minakami et al. (1962). The following were used as electron acceptors (1) ferricyanide, (2) 2,6-dichlorphenolindophenol (DCPIP), (3) cytochrome c and (4) coenzyme Q6- The decrease or increase of absorbance was monitored at the following wavelengths: (1) 420 nm, (2) 600 nm, (3) 550 n m and (4) 275 nm. The assay mixtures contained, in addition to the protein, in a total volume of 300 ~1: (1) 0"02-0"3/~moles K-ferricyanide, 0'018-0"13/xmoles N A D H and 100/L1 0"12M triethanolamine buffer, pH 7"8; (2) 5-25 n-moles DCPIP, 9-90 n-moles N A D H and 100/xl 0"12 M triethanolamine buffer, pH 8"5 ; (3) 5-40 n-moles cytochrome c, 0.9/xmoles KCN, 5"427 n-moles N A D H and 100/.d 0"12 M triethanolamine buffer, pH 8"5. All reactions were initiated by the addition of the substrate. (4) The reaction medium, containing 0"5 M Tris sulphate at pH 8"0 30/xl, 30 m M K C N 10/xl, 6 m M CoQe in methanol 5/xl and protein 5 or 10/xl was preincubated for 2"5 min at 30°C. The reaction was initiated by the addition of 10/xl of 4 m M N A D H . All the chemicals were commercial products.
Calculation of the values of Km and V T h e values of K,~ and V were calculated according to the double reciprocal method. From the successive readings of the individual assays a regression equation characterizing the actual reaction rate was calculated. When calculating the initial reaction rate the readings recorded during the first 20 sec were in most cases used. Occasionally, when the
OXIDATION OF EXTERNAL BY
NADH
DIPHYLLOBOTHRIUM L A T U M
285
course of the reaction was linear during a shorter time than 20 sec, the readings of the linear part of the reaction were chosen for the calculations. The linearity of the regression equations was checked by calculating the regression coefficient, which varied within a range of 0.995-0"980. From reciprocals of the reaction rate and substrate concentration a regression equation was developed giving the values of Km and V. RESULTS T h e Michaelis constants for N A D H dehydrogenase of adult and plerocercoid D. latum are shown in Table 1. T h e artificial electron acceptors ferricyanide and D C P I P can act as electron acceptors in the dehydrogenation of N A D H , as can T A B L E 1 - - T H E MEASURED MICHAELIS CONSTANTS OF THE NADH DEHYDROGENASE OF ADULT AND PLEROCERCOID D . l a t u m : K m VALUES DETERMINED AT FOUR TO FIVE CONCENTRATIONS OVER A FIVE- TO FIFTEEN-FOLD RANGE BY DOUBLE RECIPROCAL PLOTS
Adult
K., acceptor Oxidant Ferricyanide DCPIP Cytochrome c
(M) 8"7 x 10 -5 3"3 x 10 -s 1-1 x 10 -4
Plerocercoid KmNADH
(M) 8-8 x 10 -4 2-2 x 10 -5 1"2 x 10 -s
K~ acceptor (M) 4"6 x 10 -5 1"8 x 10 -4 5.9 x 10 -5
K m NADH
(M) 9"5 x 10 -5 1"3 x 10 -5 3"2 x 10 -4
also the natural electron acceptor cytochrome c. A comparison of the numerical values of K m acc,ptor for adult and plerocercoid enzymes shows that the Km values for ferricyanide and cytochrome c with the adult enzyme are about two orders of magnitude higher than with the plerocercoid enzyme, while for D C P I P the Km with the adult enzyme is only about one-fifth of that with the plerocercoid enzyme. T h e values of Km NADH with adult and plerocercoid enzymes vary considerably; for ferricyanide the K m with the adult enzyme is about nine orders of magnitude and for D C P I P about two orders of magnitude higher but for cytochrome c only one-twenty-fifth of the Km of the plerocercoid enzyme. T h e relative rates (V acc,ptorin terms of oxidant-reduced and NADH-oxidized) of dehydrogenation of N A D H by the previously mentioned electron acceptors relative to ferricyanide are shown in Table 2. In general the rates of cytochrome c reduction and N A D H dehydrogenation by D C P I P and cytochrome c are higher with the adult enzyme, whereas the relative rate of D C P I P reduction with the plerocercoid enzyme is about three times higher than with the adult enzyme. This finding is consistent with the situation regarding Kin, which for D C P I P with the adult enzyme is only about one-fifth of that with the plerocercoid enzyme. T h e activity of N A D H - C o Q 6 oxidoreductase was found to be 8.7 and 0.37 n-moles NADH-oxidized/min per mg protein for the adult and plerocercoid enzyme, respectively. CoQ6 can act as an electron acceptor both for the adult and plerocercoid N A D H dehydrogenase, the activity of the former being over twenty times higher than that of the latter.
286
KALEVI SALMINEN
TABLE 2 - - N A D H DEHYDROGENASE OF ADULT AND PLEROCERCOID D. RATE ( V aeeept°r IN TERMS OF OXIDANT-REDUCED AND N A D H - o x I D I Z E D )
latum: THE RELATIVE OF OXIDATION OF
N A D H BY SOME ELECTRON ACCEPTORS RELATIVE TO FERRICYANIDE (ARBITRARILY SET AT 1 0 0 )
Adult Oxidant
Ferricyanide DCPIP Cytochrome c
V aecept°r
100 15"5 2"9
Plerocercoid V NADH
100 5"2 3"2
V accept°r
V NADtt
100 45"0 1"8
100 3"7 1-1
DISCUSSION The presence of all the enzymes or acids of the tricarboxylic acid cycle has been demonstrated in several parasites, such as Echinococcusgranulosus (Agosin & Repetto, 1963), Ascaridia galli and Nematodirus spp. (Massey & Rogers, 1950), Fasciola hepatica (Thorsell, 1963; Prichard & Schofield, 1968), Trichinella spiralis larvae (Goldberg, 1957), Ascaris lumbricoides muscle (Kmetec & Bueding, 1961, Oya et al., 1965), Moniezia expansa (Davey & Bryant, 1969) and Haemonchus contortus (Ward & Schofield, 1967). The operating TCA cycle includes the transfer of four pairs of electrons. Three pairs of electrons are removed by forming in the cytosol NADH, which must be dehydrogenated in some way in order for the cycle to continue to function. If the analogy between vertebrates and invertebrates holds true, NADH cannot penetrate the mitochondrion and be oxidized in this way. It is therefore necessary to postulate that (a) a shuttle mechanism exists, (b) the mitochondria of invertebrate tissue are permeable to N A D H and (c) a mitochondrial enzyme system capable of transferring electrons to the cytochrome system without a phosphorylating effect exists. There seems to be as yet no data reported for helminths concerning the existence of a shuttle mechanism. A NADH-oxidase system, whether phosphorylating or nonphosphorylating, has been shown for helminths (Cheah, 1967). Kmetec & Bueding (1961) have demonstrated the coupling of NADH oxidation to fumarate reduction under anaerobic condition in a particulate preparation from A. lumbricoides muscle, and have suggested that this transfer is mediated by electron transport enzymes. This finding was further supported by Cheah & Bryant (1966) by showing that in M. expansa the coupling of NADH oxidation to the reduction of fumarate to succinate was mediated by an electron transport system involving a cytochrome of the b type and flavoprotein carriers. Similarly, since many helminths contain very low lactate dehydrogenase activity, the production of succinate has been suggested as a means of dehydrogenating the NADH formed during glycolysis (Bueding, 1963). This mechanism has even been shown in A. lumbricoides to provide the energy-rich bonds in the form of ATP (Seidman & Entner, 1961). On the other hand, an active phosphoenolpyruvate carboxykinase has been demonstrated in H. diminuta (Prescott & Campbell, 1965). The oxalacetate so formed was
OXIDATION OF EXTERNALNADH BY DIPHYLLOBOTHRIUM LATUM
287
then reduced by an active malate dehydrogenase, this reoxidizing the NADH produced during glycolysis (Simpson & Awapara, 1966). In conclusion we can state that the particulate fraction of several parasites is able to dehydrogenate NADH, and that of A. lumbricoides, in addition, is able to couple ATP production to both aerobic and anaerobic dehydrogenation of NADH. The problem of NADH penetration into mitochondria has not been considered. The present studies have shown that both adult and plerocercoid D. latum mitochondria are able to transfer electrons from NADH to CoQ and cytochrome c. In mammals the evidence strongly supports the hypothesis that CoQ mediates the electron transport between NADH dehydrogenase and cytochrome system (Ernster et al., 1969). In the present study the nonphysiological reduction of exogenous CoQ was assayed. However, in all preparations which reduce external CoQ, endogenous CoQ and phospholipids are present. In fact it seems very likely that in such systems external CoQ accepts electrons from endogenous CoQ, rather than from the dehydrogenase directly (Machinist & Singer, 1965). The question of NADH penetration remains open, but a shuttle mechanism function does not seem to be involved, since only the particulate fraction without any cytosol components was utilized. The possibility that D. latum mitochondria might be permeable to NADH, is however, not excluded. The mitochondria of at least one invertebrate organism, Rangia cuneata, appear to be permeable to NADH (Chen & Awapara, 1969). If the D. latum mitochondria are likewise permeable to NADH, the electron transfer from NADH to CoQ and cytochrome c could be coupled to oxidative phosphorylation in a similar manner to that of vertebrate tissues. The considerably lower activity of plerocercoid NADH-CoQ oxidoreductase supports this view, since the adult enzyme would be better adapted to aerobic conditions. Moon & Schofield (1968) were able to demonstrate that a mitochondrial fraction of H. contortus showed NADH-cytochrome c reductase activity, which was completely inhibited by antimycin A, an inhibitor known to influence the NADH oxidation coupled to oxidative phosphorylation. The existence of nonphosphorylating NADH-cytochrome c reductase located in the outer mitochondrial membrane has only occasionally been considered in parasites. The mitochondria of the hookworm Ancylostoma caninum were shown to be unable to couple NADH oxidation to ATP synthesis (Warren, 1970). The role of enzymes like these would be meaningful under aerobic conditions, where the cytochrome c can be further oxidized by cytochrome oxidase. Consequently this pathway exists in adult D. latum, which lives in an environment containing enough oxygen to permit an aerobic metabolism. The role of the enzyme in plerocercoid is an open question. Perhaps the enzyme is of potential character, being able to adapt further as the larva undergoes the transition to the adult stage. REFERENCES AGOSIN M. & REPETTO Y. (1963) Studies on the metabolism of Echinococcus granulosus--VI I. Reactions of the tricarboxylic acid cycle in E. granulosus scolices. Comp. Biochem. Physiol. 8, 245-261.
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BERNTZEN A. K. (1962) In vitro cultivation of t a p e w o r m s - - I I . Growth and maintenance of Hymenolepis nana (Cestoda: Cyclophyllidae). J. ParasitoI. 48, 785-797. BUEDINC E. (1963) Comparative biochemistry of parasitic helminths. Proc. Fifth Int. Congr. Biochem. 3, 280-290. CHEAH K. S. (1967) T h e oxidase systems of Moniezia expansa (Cestoda). Comp. Biochem. Physiol. 23, 277-302. CHEAH K. S. & BRYANT C. (1966) Studies on the electron transport system of Moniezia expansa (Cestoda). Comp. Biochem. Physiol. 19, 197-223. CHEN C. • A'vVAPARAJ. (1969) Intracellular distribution of enzymes catalyzing succinate production from glucose in Rangia mantle. Comp. Biochem. Physiol. 30, 727-737. DAVEY R. A. & BRYANT C. (1969) The tricarboxylic acid cycle and associated reactions in Moniezia expansa (Cestoda). Comp. Biochem. Physiol. 31,503-511. ERNSTER L., LEE I.-Y., NORDLING B. & PERSSON B. (1969) Studies with ubiquinonedepleted submitochondrial particles. Eur..7. Biochem. 9, 299-310. GOLDBERG E. (1957) Studies on the intermediary metabolism of TrichinelIa spiralis. Expl Parasit. 6, 367-382. ttOBSON A. D. (1948) T h e physiology and cultivation in artificial media of nematodes parasitic in the alimentary tract of animals. Parasit. 38, 183-277. KMETEC E. & BUEDING E. (1961) Succinic and reduced diphosphopyridine nucleotide oxidase systems of Ascaris muscle..7, biol. Chem. 236, 584-591. LASER H. (1944) The oxidative metabolism of .4scaris suis. Biochem. J. 38, 333-338. LowRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALLR. J. (1951) Protein measurements with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MACHINIST J. M. & SINGER T. P. (1965) Studies on the respiratory chain-linked reduced nicotinamide adenine dinucleotide dehydrogenase---IX. Reactions with coenzyme Q. J. biol. Chem. 240, 3182-3190. MASSEY V. & ROGERS W. P. (1950) The intermediary metabolism of nematode parasites--I. T h e general reactions of the tricarboxylic acid cycle. Aust. J. Sci. Res. B. 3, 251-264. MINAKAMI S., RINGLER R. L. & SINGER T. P. (1962) Studies on the respiratory chain-linked diphosphopyridine nucleotide dehydrogenase. J. biol. Chem. 237, 569-576. MOON K. E. & SCHOFIELDP. J. (1968) Reduction of cytochrome c by Haemonchus contortus larvae. Comp. Biochem. Physiol. 26, 745-748. OYA H., KIKUCHI G., BANDO T. & HAYASHI H. (1965) Muscle tricarboxylic acid cycle in Ascaris lumbricoides vat. suis. Expl Parasit. 17, 229-240. PRESCOTT L. & CAMPBELL J. W. (1965) Phosphoenolpyruvate carboxylase activity and glycogenesis in the flatworm, Hymenolepis diminuta. Comp. Biochem. Physiol. 14, 491-511. PRICHARD R. K. & SCHOFIELD P. J. (1968) A comparative study of the tricarboxylic acid cycle enzymes in Fasciola hepatica and rat liver. Comp. Biochem. Physiol. 25, 1005-1019. SEIDMAN I. & ENTNER N. (1961) Oxidative enzymes and their role in phosphorylation in sarcosomes of adult .4scaris lumbricoides. J. biol. Chem. 236, 915-919. SIMPSON J. W. & AWAPARAJ. (1966) The pathway of glucose degradation in some invertebrates. Comp. Biochem. Physiol. 18, 537-548. SINGER T. P. & GUTMAN M. (1971) T h e D P N H dehydrogenase of the mitochondrial respiratory chain. Adv. Enzymol. 34, 79-153. SOTTOCASA G. L., KUYLENSTIERNAB., ERNSTER L. ~; BERGSTRANDA. (1967) An electrontransport system associated with the outer membrane of liver mitochondria, d~. Cell Biol. 32, 415-438. THORSELL W. (1963) Some acids belonging to the citric acid cycle in the liver fluke Fasciola hepatica L. Acta chem. Scand. 17, 2129-2131. WARD C. W. & SCHOFIELD P. J. (1967) Comparative activity and intracellular distribution of tricarboxylic acid cycle enzymes in Haemonchus contortus larvae and rat liver. Comp. Biochem. Physiol. 23, 335-359.
O X I D A T I O N OF EXTERNAL
NADH
BY D I P H Y L L O B O T H R I U M L A T U M
289
WAm~N L. G. (1970) Biochemistry of the dog hookworm--III. Oxidative phosphorylation. Expl Parasitol. 27, 417-423.
Key Word Index---Cestode; tapeworm; DiphyUobothrium laturn; NADH, NADH dehydrogenase; parasite development; parasite metabolism.
10
THE OXIDATION OF EXTERNAL NADH BY A D U L T AND PLEROCERCOID D I P H Y L L O B O T H R I U M LATUM KALEVI SALMINEN Department of Food Hygiene, College of Veterinary Medicine, H/imeentie 57, 00550 Helsinki 55, Finland
(Received 16 May 1972) Abstract--1. The apparent Km's and relative rates of mitochondrial NADH dehydrogenase and specific activity of NADH-CoQ e oxidoreductase of adult and plerocercoid Diphyllobothrium latura were determined. 2. As electron accepters for NADH dehydrogenase K-ferricyanide, 2,6dichlorphenolindophenol and cytochrome c were used. 3. The g,, ferrocrtoohromoo of the adult enzyme is the same as that observed for the plerocercoid, whereas the Km NADa of the adult enzyme is only onetwenty-fifth of that of the plerocercoid enzyme. 4. The activity of the adult NADH-CoQ8 oxidoreductase was over twenty times greater than that of the plerocercoid enzyme. INTRODUCTION THE aEACTIONS of the tricarboxylic acid cycle occur in vertebrate tissues in the soluble cytoplasm of the cells in which N A D H is produced. N A D H is unable to diffuse from this compartment into the mitochondria. The diffusion takes place through the glycerol phosphate shuttle and N A D H is further oxidized by the mitochondrial electron transport chain. This respiratory chain-linked N A D H dehydrogenase is amytal and rotenone sensitive (Singer & Gutman, 1971). Studies with mammalian liver mitochondria have shown that there also exists an enzyme system capable of oxidizing external NADH. This enzyme system is located in the outer mitochondrial membrane (Sottocasa et al., 1957). The oxidation of external N A D H by cytochrome c seems to be antimycin A, amytal and rotenone insensitive and it is not coupled to oxidative phosphorylation. The role of these mitochondrial NADH-oxidizing enzymes in helminths is not known. In an aerobic environment N A D H can be oxidized by the mitochondrial electron transport chain, whereas under anaerobic conditions various pathways have been suggested for the dehydrogenation of NADH. Since the various developmental stages of Diphyllobothrium latum inhabit greatly different hosts, this helminth was selected for a comparative study of the NADH-dehydrogenating enzyme systems. The plerocercoid lives in water organisms under virtually anaerobic conditions; the adult worm, on the other hand, inhabits the intestinal tract of 283
284
KALEVI SALMINEN
several mammalianspecies,includingman, wherethere is enoughmolecularoxygen present to permit an aerobic metabolism (Laser, 1944; Hobson, 1948; Berntzen, 1966). MATERIALS AND METHODS
Preparation of the particulate fraction The plerocercoid material used in the experiments was recovered from naturally infested pikes. The adult worms were obtained from experimentally infested golden hamsters, which were sacrificed by decapitation and autopsied about 2 weeks after infestation. Immediately after the plerocercoids had been isolated from the pikes and the adult worms removed from the golden hamsters, they were washed several times with ice-cold 0"4 M sucrose containing 0"001 M E D T A , 0"2% heparin, pH 6"9 to remove the remains of fish tissue and intestinal contents. Homogenization was carried out in a tenfold amount of the sucrose solution. The material to be homogenized was at first cut into small pieces with an Ultra-Turrax homogenizer operated for 10 sec at a low cutter rotation speed. The prehomogenized slurry was further homogenized with a Potter-Elvehjem type tissue grinder equipped with a tightfitting Teflon pestle (Arthur A. Thomas Co., Philadelphia, U.S.A.) at 200 rev/min for 1 min. The vessel was kept immersed in ice-water. The homogenate was centrifuged for 10 min at 1800 g to remove the unbroken cells and nuclei. T h e supernatant was further spun for 20 min at 25,000 g to sediment the mitochondrial particulate fraction. The pellet was washed once. Finally, the pellet was suspended in an 0"05 M triethanol amine buffer, pH 7"6 and stored for enzyme assays at 45 °C. Protein determination was carried out according to the Folin method of Lowry et al. (1951), with bovine serum albumin as the protein standard. -
N A D H dehydrogenase assay T h e enzyme assays were carried out with a Perkin Elmer UV-VI S 139 spectrophotometer with cuvettes of 300 ktl capacity. The spectrophotometer was equipped with a Digital Concentration Read-out unit, which allowed a numerical recording of the absorbance at 2-sec intervals. The enzyme activity was measured by the methods of Minakami et al. (1962). The following were used as electron acceptors (1) ferricyanide, (2) 2,6-dichlorphenolindophenol (DCPIP), (3) cytochrome c and (4) coenzyme Q6- The decrease or increase of absorbance was monitored at the following wavelengths: (1) 420 nm, (2) 600 nm, (3) 550 n m and (4) 275 nm. The assay mixtures contained, in addition to the protein, in a total volume of 300 ~1: (1) 0"02-0"3/~moles K-ferricyanide, 0'018-0"13/xmoles N A D H and 100/L1 0"12M triethanolamine buffer, pH 7"8; (2) 5-25 n-moles DCPIP, 9-90 n-moles N A D H and 100/xl 0"12 M triethanolamine buffer, pH 8"5 ; (3) 5-40 n-moles cytochrome c, 0.9/xmoles KCN, 5"427 n-moles N A D H and 100/.d 0"12 M triethanolamine buffer, pH 8"5. All reactions were initiated by the addition of the substrate. (4) The reaction medium, containing 0"5 M Tris sulphate at pH 8"0 30/xl, 30 m M K C N 10/xl, 6 m M CoQe in methanol 5/xl and protein 5 or 10/xl was preincubated for 2"5 min at 30°C. The reaction was initiated by the addition of 10/xl of 4 m M N A D H . All the chemicals were commercial products.
Calculation of the values of Km and V T h e values of K,~ and V were calculated according to the double reciprocal method. From the successive readings of the individual assays a regression equation characterizing the actual reaction rate was calculated. When calculating the initial reaction rate the readings recorded during the first 20 sec were in most cases used. Occasionally, when the
OXIDATION OF EXTERNAL BY
NADH
DIPHYLLOBOTHRIUM L A T U M
285
course of the reaction was linear during a shorter time than 20 sec, the readings of the linear part of the reaction were chosen for the calculations. The linearity of the regression equations was checked by calculating the regression coefficient, which varied within a range of 0.995-0"980. From reciprocals of the reaction rate and substrate concentration a regression equation was developed giving the values of Km and V. RESULTS T h e Michaelis constants for N A D H dehydrogenase of adult and plerocercoid D. latum are shown in Table 1. T h e artificial electron acceptors ferricyanide and D C P I P can act as electron acceptors in the dehydrogenation of N A D H , as can T A B L E 1 - - T H E MEASURED MICHAELIS CONSTANTS OF THE NADH DEHYDROGENASE OF ADULT AND PLEROCERCOID D . l a t u m : K m VALUES DETERMINED AT FOUR TO FIVE CONCENTRATIONS OVER A FIVE- TO FIFTEEN-FOLD RANGE BY DOUBLE RECIPROCAL PLOTS
Adult
K., acceptor Oxidant Ferricyanide DCPIP Cytochrome c
(M) 8"7 x 10 -5 3"3 x 10 -s 1-1 x 10 -4
Plerocercoid KmNADH
(M) 8-8 x 10 -4 2-2 x 10 -5 1"2 x 10 -s
K~ acceptor (M) 4"6 x 10 -5 1"8 x 10 -4 5.9 x 10 -5
K m NADH
(M) 9"5 x 10 -5 1"3 x 10 -5 3"2 x 10 -4
also the natural electron acceptor cytochrome c. A comparison of the numerical values of K m acc,ptor for adult and plerocercoid enzymes shows that the Km values for ferricyanide and cytochrome c with the adult enzyme are about two orders of magnitude higher than with the plerocercoid enzyme, while for D C P I P the Km with the adult enzyme is only about one-fifth of that with the plerocercoid enzyme. T h e values of Km NADH with adult and plerocercoid enzymes vary considerably; for ferricyanide the K m with the adult enzyme is about nine orders of magnitude and for D C P I P about two orders of magnitude higher but for cytochrome c only one-twenty-fifth of the Km of the plerocercoid enzyme. T h e relative rates (V acc,ptorin terms of oxidant-reduced and NADH-oxidized) of dehydrogenation of N A D H by the previously mentioned electron acceptors relative to ferricyanide are shown in Table 2. In general the rates of cytochrome c reduction and N A D H dehydrogenation by D C P I P and cytochrome c are higher with the adult enzyme, whereas the relative rate of D C P I P reduction with the plerocercoid enzyme is about three times higher than with the adult enzyme. This finding is consistent with the situation regarding Kin, which for D C P I P with the adult enzyme is only about one-fifth of that with the plerocercoid enzyme. T h e activity of N A D H - C o Q 6 oxidoreductase was found to be 8.7 and 0.37 n-moles NADH-oxidized/min per mg protein for the adult and plerocercoid enzyme, respectively. CoQ6 can act as an electron acceptor both for the adult and plerocercoid N A D H dehydrogenase, the activity of the former being over twenty times higher than that of the latter.
286
KALEVI SALMINEN
TABLE 2 - - N A D H DEHYDROGENASE OF ADULT AND PLEROCERCOID D. RATE ( V aeeept°r IN TERMS OF OXIDANT-REDUCED AND N A D H - o x I D I Z E D )
latum: THE RELATIVE OF OXIDATION OF
N A D H BY SOME ELECTRON ACCEPTORS RELATIVE TO FERRICYANIDE (ARBITRARILY SET AT 1 0 0 )
Adult Oxidant
Ferricyanide DCPIP Cytochrome c
V aecept°r
100 15"5 2"9
Plerocercoid V NADH
100 5"2 3"2
V accept°r
V NADtt
100 45"0 1"8
100 3"7 1-1
DISCUSSION The presence of all the enzymes or acids of the tricarboxylic acid cycle has been demonstrated in several parasites, such as Echinococcusgranulosus (Agosin & Repetto, 1963), Ascaridia galli and Nematodirus spp. (Massey & Rogers, 1950), Fasciola hepatica (Thorsell, 1963; Prichard & Schofield, 1968), Trichinella spiralis larvae (Goldberg, 1957), Ascaris lumbricoides muscle (Kmetec & Bueding, 1961, Oya et al., 1965), Moniezia expansa (Davey & Bryant, 1969) and Haemonchus contortus (Ward & Schofield, 1967). The operating TCA cycle includes the transfer of four pairs of electrons. Three pairs of electrons are removed by forming in the cytosol NADH, which must be dehydrogenated in some way in order for the cycle to continue to function. If the analogy between vertebrates and invertebrates holds true, NADH cannot penetrate the mitochondrion and be oxidized in this way. It is therefore necessary to postulate that (a) a shuttle mechanism exists, (b) the mitochondria of invertebrate tissue are permeable to N A D H and (c) a mitochondrial enzyme system capable of transferring electrons to the cytochrome system without a phosphorylating effect exists. There seems to be as yet no data reported for helminths concerning the existence of a shuttle mechanism. A NADH-oxidase system, whether phosphorylating or nonphosphorylating, has been shown for helminths (Cheah, 1967). Kmetec & Bueding (1961) have demonstrated the coupling of NADH oxidation to fumarate reduction under anaerobic condition in a particulate preparation from A. lumbricoides muscle, and have suggested that this transfer is mediated by electron transport enzymes. This finding was further supported by Cheah & Bryant (1966) by showing that in M. expansa the coupling of NADH oxidation to the reduction of fumarate to succinate was mediated by an electron transport system involving a cytochrome of the b type and flavoprotein carriers. Similarly, since many helminths contain very low lactate dehydrogenase activity, the production of succinate has been suggested as a means of dehydrogenating the NADH formed during glycolysis (Bueding, 1963). This mechanism has even been shown in A. lumbricoides to provide the energy-rich bonds in the form of ATP (Seidman & Entner, 1961). On the other hand, an active phosphoenolpyruvate carboxykinase has been demonstrated in H. diminuta (Prescott & Campbell, 1965). The oxalacetate so formed was
OXIDATION OF EXTERNALNADH BY DIPHYLLOBOTHRIUM LATUM
287
then reduced by an active malate dehydrogenase, this reoxidizing the NADH produced during glycolysis (Simpson & Awapara, 1966). In conclusion we can state that the particulate fraction of several parasites is able to dehydrogenate NADH, and that of A. lumbricoides, in addition, is able to couple ATP production to both aerobic and anaerobic dehydrogenation of NADH. The problem of NADH penetration into mitochondria has not been considered. The present studies have shown that both adult and plerocercoid D. latum mitochondria are able to transfer electrons from NADH to CoQ and cytochrome c. In mammals the evidence strongly supports the hypothesis that CoQ mediates the electron transport between NADH dehydrogenase and cytochrome system (Ernster et al., 1969). In the present study the nonphysiological reduction of exogenous CoQ was assayed. However, in all preparations which reduce external CoQ, endogenous CoQ and phospholipids are present. In fact it seems very likely that in such systems external CoQ accepts electrons from endogenous CoQ, rather than from the dehydrogenase directly (Machinist & Singer, 1965). The question of NADH penetration remains open, but a shuttle mechanism function does not seem to be involved, since only the particulate fraction without any cytosol components was utilized. The possibility that D. latum mitochondria might be permeable to NADH, is however, not excluded. The mitochondria of at least one invertebrate organism, Rangia cuneata, appear to be permeable to NADH (Chen & Awapara, 1969). If the D. latum mitochondria are likewise permeable to NADH, the electron transfer from NADH to CoQ and cytochrome c could be coupled to oxidative phosphorylation in a similar manner to that of vertebrate tissues. The considerably lower activity of plerocercoid NADH-CoQ oxidoreductase supports this view, since the adult enzyme would be better adapted to aerobic conditions. Moon & Schofield (1968) were able to demonstrate that a mitochondrial fraction of H. contortus showed NADH-cytochrome c reductase activity, which was completely inhibited by antimycin A, an inhibitor known to influence the NADH oxidation coupled to oxidative phosphorylation. The existence of nonphosphorylating NADH-cytochrome c reductase located in the outer mitochondrial membrane has only occasionally been considered in parasites. The mitochondria of the hookworm Ancylostoma caninum were shown to be unable to couple NADH oxidation to ATP synthesis (Warren, 1970). The role of enzymes like these would be meaningful under aerobic conditions, where the cytochrome c can be further oxidized by cytochrome oxidase. Consequently this pathway exists in adult D. latum, which lives in an environment containing enough oxygen to permit an aerobic metabolism. The role of the enzyme in plerocercoid is an open question. Perhaps the enzyme is of potential character, being able to adapt further as the larva undergoes the transition to the adult stage. REFERENCES AGOSIN M. & REPETTO Y. (1963) Studies on the metabolism of Echinococcus granulosus--VI I. Reactions of the tricarboxylic acid cycle in E. granulosus scolices. Comp. Biochem. Physiol. 8, 245-261.
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BERNTZEN A. K. (1962) In vitro cultivation of t a p e w o r m s - - I I . Growth and maintenance of Hymenolepis nana (Cestoda: Cyclophyllidae). J. ParasitoI. 48, 785-797. BUEDINC E. (1963) Comparative biochemistry of parasitic helminths. Proc. Fifth Int. Congr. Biochem. 3, 280-290. CHEAH K. S. (1967) T h e oxidase systems of Moniezia expansa (Cestoda). Comp. Biochem. Physiol. 23, 277-302. CHEAH K. S. & BRYANT C. (1966) Studies on the electron transport system of Moniezia expansa (Cestoda). Comp. Biochem. Physiol. 19, 197-223. CHEN C. • A'vVAPARAJ. (1969) Intracellular distribution of enzymes catalyzing succinate production from glucose in Rangia mantle. Comp. Biochem. Physiol. 30, 727-737. DAVEY R. A. & BRYANT C. (1969) The tricarboxylic acid cycle and associated reactions in Moniezia expansa (Cestoda). Comp. Biochem. Physiol. 31,503-511. ERNSTER L., LEE I.-Y., NORDLING B. & PERSSON B. (1969) Studies with ubiquinonedepleted submitochondrial particles. Eur..7. Biochem. 9, 299-310. GOLDBERG E. (1957) Studies on the intermediary metabolism of TrichinelIa spiralis. Expl Parasit. 6, 367-382. ttOBSON A. D. (1948) T h e physiology and cultivation in artificial media of nematodes parasitic in the alimentary tract of animals. Parasit. 38, 183-277. KMETEC E. & BUEDING E. (1961) Succinic and reduced diphosphopyridine nucleotide oxidase systems of Ascaris muscle..7, biol. Chem. 236, 584-591. LASER H. (1944) The oxidative metabolism of .4scaris suis. Biochem. J. 38, 333-338. LowRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALLR. J. (1951) Protein measurements with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MACHINIST J. M. & SINGER T. P. (1965) Studies on the respiratory chain-linked reduced nicotinamide adenine dinucleotide dehydrogenase---IX. Reactions with coenzyme Q. J. biol. Chem. 240, 3182-3190. MASSEY V. & ROGERS W. P. (1950) The intermediary metabolism of nematode parasites--I. T h e general reactions of the tricarboxylic acid cycle. Aust. J. Sci. Res. B. 3, 251-264. MINAKAMI S., RINGLER R. L. & SINGER T. P. (1962) Studies on the respiratory chain-linked diphosphopyridine nucleotide dehydrogenase. J. biol. Chem. 237, 569-576. MOON K. E. & SCHOFIELDP. J. (1968) Reduction of cytochrome c by Haemonchus contortus larvae. Comp. Biochem. Physiol. 26, 745-748. OYA H., KIKUCHI G., BANDO T. & HAYASHI H. (1965) Muscle tricarboxylic acid cycle in Ascaris lumbricoides vat. suis. Expl Parasit. 17, 229-240. PRESCOTT L. & CAMPBELL J. W. (1965) Phosphoenolpyruvate carboxylase activity and glycogenesis in the flatworm, Hymenolepis diminuta. Comp. Biochem. Physiol. 14, 491-511. PRICHARD R. K. & SCHOFIELD P. J. (1968) A comparative study of the tricarboxylic acid cycle enzymes in Fasciola hepatica and rat liver. Comp. Biochem. Physiol. 25, 1005-1019. SEIDMAN I. & ENTNER N. (1961) Oxidative enzymes and their role in phosphorylation in sarcosomes of adult .4scaris lumbricoides. J. biol. Chem. 236, 915-919. SIMPSON J. W. & AWAPARAJ. (1966) The pathway of glucose degradation in some invertebrates. Comp. Biochem. Physiol. 18, 537-548. SINGER T. P. & GUTMAN M. (1971) T h e D P N H dehydrogenase of the mitochondrial respiratory chain. Adv. Enzymol. 34, 79-153. SOTTOCASA G. L., KUYLENSTIERNAB., ERNSTER L. ~; BERGSTRANDA. (1967) An electrontransport system associated with the outer membrane of liver mitochondria, d~. Cell Biol. 32, 415-438. THORSELL W. (1963) Some acids belonging to the citric acid cycle in the liver fluke Fasciola hepatica L. Acta chem. Scand. 17, 2129-2131. WARD C. W. & SCHOFIELD P. J. (1967) Comparative activity and intracellular distribution of tricarboxylic acid cycle enzymes in Haemonchus contortus larvae and rat liver. Comp. Biochem. Physiol. 23, 335-359.
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WAm~N L. G. (1970) Biochemistry of the dog hookworm--III. Oxidative phosphorylation. Expl Parasitol. 27, 417-423.
Key Word Index---Cestode; tapeworm; DiphyUobothrium laturn; NADH, NADH dehydrogenase; parasite development; parasite metabolism.
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