- Email: [email protected]
Succinate and lactate oxidoreductases of bivalve mollusks
Comp. Biochem. Physiol., 1975, Vol. 50B, pp. 407 to 412. PergamonPress. Printed in Great Britain
SUCCINATE A N D LACTATE OXIDOREDUCTASES OF BIVALVE MOLLUSKS C. S. HAM_MEN Department of Zoology, University of Rhode Island, Kingston, R.I. 02881, U.S.A. (Received19February1973) Abstract--1. Succinic acid is a major end product of anaerobic glycolysis in mollusks, and lactic acid is a minor product. On the hypothesis that production of these acids is partially controlled by properties of the enzymes catalyzing the final step in each pathway, extracts of the adductor muscle were prepared and used in oxidoreductase assays. 2. The relations between substrate concentration and velocity of reaction were determined for pyruvate reduction (PR), lactate oxidation (LO), fumarate reduction (FR) and succinate oxidation
(so). 3. The animals were Mercenaria mercenaria, Mytilus edulis and Crassostrea virginica, and the assay mixtures included nicotinamide adenine dinucleotide (NAD) or dichlorophenolindophenol (DCIP). 4. The data were summarized as apparent Michaelis constants (Kin) and maximal velocities (V). Reductas¢ properties varied little between species, Km of PR 0.54--0.61 mM, Km of FR 25-30 mM. Oxidas¢ properties were more varied, Km of LO from the clam Mereenaria twice as great as the other two species, 250 and 120 mM; Km of SO from the oyster Cra~sostrea three to four times as great as the others, 120 vs. 30 and 40 raM. The smallest species, Mytilus, had the greatest Vfor both reductases, while the species varied little in V of the oxidases. 5. The SO of Mytilus was inactive with NAD until ATP was added, and its activity with DCIP was stimulated fourfold by 2 mM ATP; the FR of Mytilus was stimulated threefold by 8 mM ADP.
INTRODUCTION IN BIVALVEmollusks, succinic acid is a major end product of anaerobic giycolysis, and lactic acid is a minor product. This has been noticed in the oyster Crassostrea virginica (Hammen & Osborne, 1959; Simpson & Awapara, 1966), the hard clam Mercenaria mercenaria (Crenshaw & Neff, 1969) and the blue mussel Mytilus edulis (de Zwaan & Zandee, 1972). One simple hypothesis to account for the partition between acid end products is control by the enzymes catalyzing the final step in each pathway. Among micro-organisms, succinate dehydrogenase from anaerobes catalyzes reduction of fumarate more rapidly and oxidation o f succinate more slowly than the same enzyme from aerobes (Singer, 1971). Bivalve mollusks exhibit varying degrees of facultative anaerobiosis, which are reflected in varying ratios of forward and reverse rates of oxidoreductases. The ratio of fumarate reduction to succinate oxidation (FR/SO) varied from 0.23 in Mercenaria to 6"2 in Crassostrea, a greater range than found in any other group of marine invertebrates (Hammen, 1969). Ratios of pyruvate reduction to lactate oxidation were more uniform, and indicated a bias against lactate formation, compared to more active invertebrates. Experiments with a single concentration of substrate, however, did not reveal whether differences in rates were due to differences in tissue concentra-
tion of the enzyme or to differences in affinity for substrate. This new work was performed to find maximal rates per unit weight of tissue, and Michaelis constants for two oxidoreductases in both directions. The enzymes concerned are commonly known as succinate dehydrogenase and lactate dehydrogenase. The former enzyme is a flavoprotein with a quinone as the physiological hydrogen acceptor; a useful artificial acceptor is the blue dye dichlorophenolindopbenol (DCIP), giving the systematic name succinate : DCIP oxidoreductase (E.C. 1.3.99.1). The latter enzyme ordinarily requires nicotinamide adenine dinucleotide (NAD) as hydrogen acceptor, and in mollusks is specific for D-lactate, giving the name D-lactate : N A D oxidoreductase (E.C.
1.1.1.28). MATERIALS AND METHODS Three species of bivalve mollusks were collected in Narragansett Bay or its estuaries between 10 June and 27 August 1973 and were used in experiments within 24 hr. The ranges of shell length and total weight were: Mytilus edulis, 4.6-5.0cm and 13.1-15.9 g; Mercenaria mercenaria, 5"1-8"5cm and 36-3-152"0g; and Crassostrea virginica, 6.5-9"0 cm and 37.0-74.0 g. Freshly dissected adductor muscles were homogenized with nine times their weight of 0.250 M Tris-HCl buffer, pH 7.4, then centrifuged for 10 min at 800 g and 0°C
407
408
C.S. ~
in a Model B-20 (International Equipment Co.) centrifuge. Enzyme assays were performed with a Zeiss PMQII spectrophotometer, using the supematant fraction as the enzyme source. The oxidase activities were measured by a decrease in absorbance at 600 nm of mixtures containing 0-05 mM DCIP and 1-0mM phenazine methosulfate (PMS), essentially the method of Arrigoni & Singer (1962). The reductase activities were measured by a decrease in absorbance at 340 nm of mixtures containing 0.16 mM NADH, a procedure similar to the method of Sanadi & Fluharty (1963). Some experiments were also performed with 4"8 mM NAD + as the hydrogen acceptor for both lactate and succinate oxidation with Mytilus preparations. Reaction rates were calculated by means of the molar absorptivity, 6.22 x 10a for NADH, and 3-90-5.40 x 108 for DCIP in the presence of PMS. Temperatures of reaction mixtures were 19.0-22.0°C, and the final volume was 3.0 ml in each case.
003
f
~A
jra
NAD* 3 /
o, )
RESULTS Fumarate reductase and succinate oxidase activities are summarized as apparent Michaelis constants (Km) and maximum velocities (V) derived from double-reciprocal graphs of rate vs. substrate concentration (Table 1). Fumarate was readily reduced by N A D H over the range of 4-40 m M fumarate, and K m values fell in a narrow range, Table 1. Michaelis constants (mM) and maximum velocities (pmoles/min per g) of succinate oxidoreductases. Fumarate reductase Km
Mercenaria Mytilus Crassostrea
27 30 25
Succinate oxidase
V
Km
0.227 0-286 0"141
30 40 120
Km (SUC) V Km (FUM) 0"577 1"11 0-279 1"33 0 " 4 5 5 4.80
25-30 raM. In each experiment, there was substantial oxidation of N A D H in the absence of added fumarate, and these blank rates were subtracted. V was lowest in Crassostrea, and highest in Mytilus, 0.141 and 0.286 pmole/min per g tissue, respectively. Succinate oxidase properties varied more between species, with the lowest Km and highest V found in Mercenaria, and an extraordinarily high Km of 120 mM found in Crassostrea. The ratio Km(SUC)/ Km(FUM), indicating reductive tendency or probability of producing succinate, was nearly the same for Mercenaria and Mytilus, but fourfold greater for Crassostrea. As expected, N A D + was not reduced by succinate alone in the presence of Mytilus homogenate, but rapid reduction (succinate oxidation) occurred after the addition of 2 m M ATP. I n addition, succinate oxidation and concomitant DCIP reduction were accelerated more than fourfold by ATP (Fig. 1).
-0-04 0
,
I
I
2I
)
• 3
I
I
4 rain
5
I
6
7
8
Fig. 1. Stimulation of NAD+-succinate oxidase and DCIP--succinate oxidase reactions by added ATP, M. edulis adductor muscle. Change in absorbance at 340 nm and at 600 nm vs. time. Assay mixture represented by upper curve contained buffer, extract of 150 mg tissue and NAD+, 4.8 raM. That represented by lower curve contained 0.05 mM DCIP and 1.0mM PMS, instead of NAD +. Both reactions were started with the addition of succinate; final concentration 100 mM. Both mixtures received 2.0 mM ATP at points marked by
arrows.
The reverse reaction, fumarate reduction by NADH, was similarly accelerated by A D P at concentrations of 4 m M and above (Fig. 2). Pyruvate reductase and lactate oxidase are also expressed as Michaelis constants and maximum velocities (Table 2). The K m for pyruvate varied Table 2. Michaelis constants (mM) and maximum velocities (pmoles/min per g) of lactate oxidoreductases. Pyruvate reductase Km
Mercenaria 0"61 Mytilus 0.54 Crassostrea 0.54
Lactate oxidase
V
K,,
V
0.445 1.250 0.392
250 118 120
0.483 0"389 0"526
Km(LAC) Km(PYR) 410 218 222
little among species, only 0.54-0-61 mM. The V of this reaction was three times greater for Mytilus than the others. The K m for lactate was nearly the same for Mytilus and Crassostrea, and twice as great, 250 mM, for Mercenaria. The Vof lactate oxidation varied little among species. The same Km(LAC)
o
Oxidoreductases of mollusks
409
4C--
°//
I/v AA
_
k
20
0.05
-
-
I
I
I
2
l
5
E
4
!
5
I
6
rain
PiE. 2. Stimulation of NADH-fumarate reductase reaction by added ADP, M. edulis adductor muscle. Change in abserbance at 340 nm vs. time. Blank contained only buffer, extract of 100 mg tissue and 0"16 mM NADH. All reactions were started with the addition of NADH. The lowest curve represents rapid reaction with 40 mM fumarate and 8 mM ADP in mixture. The middle curves depict reactions with only fumarate and only ADP added.
was obtained for Mytilus when N A D + served as hydrogen acceptor and when D C I P was used (Fig. 3). The maximum velocity was quite different. The ratio Km(LAC)/Km(PYR), indicating the likelihood of lactate production, was twice as great for Mercenaria as for the other two species. DISCUSSION The reduction of fumarate by N A D H was catalyzed readily by extracts of these bivalve mollusks. This reaction has been studied in the parasitic worms Ascaris lumbricoides (Kmetec & Bueding, 1961; Seidman & Entner, 1961) and Fasciola hepatica (Prichard & Schofield, 1968; de Zoeten & Tipker, 1969). The general conclusion was that fumarate reduction occurred more rapidly in these helminths than in mammalian tissues. However, fragmented mitochondria from mammals also catalyzed the N A D H - f u m a r a t e reductase reaction (Sanadi & Fluharty, 1963; Wilson & Cascarano, 1970). Mitochondria from heart muscle were more effective than those from other tissues. The fumarate reductase of nineteen species of marine invertebrates was assayed by F M N H , oxidation (Hammen, 1969). Before the present work, only one marine invertebrate, the crab, Pachygrapsus crassipes,
-I0
0
I0
20
I/s Fig. 3. Velocity of lactate oxidation in relation to substrate concentration, M. edulis adductor muscle, double reciprocal plot. The upper curve based on reaction with 4.8 mM NAD +, lower curve on reaction with DCIP-PMS mixture as described in the text. Mean total weight of animals used was 24.2 and 14.1 g respectively. had been shown to reduce fumarate with N A D H (Vatsis & Schatzlein, 1972). Comparison of the apparent / ~ ( F U M ) of the mollusks with the literature is difficult, because no K m values are found in the studies cited. The mollusk values are 40-100 times greater than Km(FUM) for purified enzymes from bacteria and yeasts (Singer, 1971). Most authors have been content to measure fumarate reductase activity at a single substrate concentration, and compare it with activity in the reverse direction. The oxidation of succinate to fumarate, by means of the dye mixture PMS--DCIP, gave Michaelis constants of 30-120 mM. These values are ten to twenty times Km(SUC) for other organisms (Singer, 1971). In fact, the value for M. edulis, 40 mM, is twenty times greater than the K m of 2 m M determined for a soluble form of succinate dehydrogenase from the same species (Ryan & King, 1962). The higher K m values, however, probably represent more closely the properties of succinate oxidase within mollusk cells, because purification processes eliminate several thousand compounds normally present in the intracellular environment, and destroy fine structure more completely than simple homogenization. Since succinate oxidation and fumarate reduction are two sides of the same coin, ratios of activities and ratios of Michaelis constants can provide more information. The ratio Km(SUC)/Km(FUM)
410
C.S. ~ N
indicates the reductive tendency or probability of forming succinate, while the reciprocal indicates the tendency towards oxidizing succinate. Among microorganisms, Km(SUC)/Km(FUM) was 0.58 for an aerobe, 17.6 for an anaerobe and 3.14 for a facultative anaerobe (Singer, 1971). The ratio for these mollusks is 1.1 (Mytillus), 1.3 (Mercenaria) and 4.8 (Crassostrea). This suggests that they are all facultative anaerobes, with the best enzymatic adaptation shown by the oyster. The ratio of activity in both directions at a fixed concentration of each substrate, FR/SD, has proved useful for identifying the relative ability of various species to cope with temporary anaerobiosis by producing succinate (Hammen, 1969). The ratio of Michaelis constants, Km(SUC)/Km(FUM), accomplishes the same purpose with more precision. The ratio of maximum velocities, VFR/VSO, has also been obtained by varying concentrations of FMNH2 and ferricyanide, the H donor and acceptor, at fixed concentration of substrates, and gives similar information (de Zoeten et al., 1969). Much more information can be presented, and predictions about the direction of metabolism can be made, if results are presented as equations containing both K m and V. The Lineweaver Burk form of the Michaelis-Menten equation is
1/v = (Km/V) (1/S)+ 1/V. This has the same form as the simple linear equation:
y = ax+b, where y is the reciprocal of velocity, the slope a is Km/V and the intercept b is 1/V. After finding Km and V by experiment, an equation can be written for each direction of a reaction, an equation that permits calculation of the velocity at any specified substrate concentration. For example, the fumarate reductase activity of Mercenaria, according to these results is expressed by y = 119x+4-40. The fumarate content of Mercenaria tissues is unknown, but at 1.2 mM fumarate, a concentration within the range found in oyster tissues, the rate is y = 119(1/1.2)+4.40 = 103-5 = 1/v, v = 0.0097/~mole/min per g = 0.582/~mole/hr per g. This rate of succinate production can be obtained only by calculation, because the concentration of fumarate is too low to permit actual measurement. The succinic acid content of tissue and fluid of Mercenaria increased from 2-0 to 5.5 mM after 6 hr of anaerobiosis (Crenshaw & Neff, 1969). Assuming a 1-g tissue equivalent to 1 ml, this was an increase of 3.5 ymoles/g in 6 hr, or a rate of succinate
production of 0.583 pmole/hr per g, a rate identical to that calculated above from K m and V. This rate could be sustained only if other reactions were continuously supplying fumarate to maintain the assumed 1.2 mM concentration. The rate equation of succinate oxidation in Mercenaria adductor extract is y = 52.0x+ 1.73. This gives 0.0361/~mole/min per g or 2.16 ~moles/hr per g for succinate at 2-0 mM. Since each mole of succinate yields two g-atoms of hydrogen, which combine with one g-atom of oxygen, the equivalent rate of oxygen consumption is 1.08/~moles Os/hr per g. At 20°C the adductor muscle of large Mercenaria consumed oxygen at the rate of 5.36/~moles/hr per g dry or, assuming 80~o water, 1.08/~moles/hr per g fresh (Hopkins, 1946), exactly the rate expected ff oxygen comsumption were solely a consequence of succinate oxidation. Further, if the same equation is valid for mantle tissue, an increase in succinate concentration from 2.0 to 5.5 mM yields an increase in oxygen consumption of 1.60/zmoles/hr per g. Isolated mantle consumed oxygen at 7.91/,moles/hr per g. Thus, after 6 hr of valve closure, mantle tissue is predicted to show a 20 per cent increase in Os consumption, due to the increased succinate concentration. Higher respiration rates, immediately after anaerobic periods (oxygen debt) are well known among bivalve mollusks. For example, in the first 30 min open, after closure during handling, oysters consumed 16 per cent more rapidly than during 5-8 hr continuously open (Galtsoff, 1964). The acceleration of fumarate reduction by ADP, and the absolute dependence on ATP of the reverse reaction, NAD+-succinate oxidation, provides strong evidence for a coupled phosphorylation accompanying fumarate reduction in M. edulis. This is the first demonstration of this phosphorylation in an invertebrate other than,4scaris (Seidman & Entner, 1961), and Fasciola (de Zoeten & Tipker, 1969), and the first in a free-living marine invertebrate. Previously, we had attempted to demonstrate phosphorylation associated with fumarate reduction by F M N H I in homogenate of C. virginica, but the results were equivocal (Wegener et al., 1969). The present result indicates that this type of phosphorylation is readily demonstrable, once appropriate methods are found. All the fumarate reductions reported here occurred without any steps taken to exclude oxygen, so the coupled phosphorylation probably can occur during aerobic as well as anaerobic phases of metabolism, although at a lower rate during the aerobic phase, because the reaction shifts toward succinate oxidation. Pyruvate reductase activities have been measured in at least ten species of bivalve mollusks (Hammen,
Oxidoreduetases of mollusks
411
1969; G~tde & Zebe, 1973), and the values were F r o m our determination of K m and V, the usually about 10 per cent of activities for more active, appropriate equations for pyruvate reduction are invertebrates, and 1 per cent of those for vertebrate y = 1.37x+2.25 and y = 0.432x+0.80. tissues. The only molluscan K m (PYR) in the literature seems to be 0.64 m M for squid axoplasm These predict the pyruvate concentrations (Roberts et aL, 1958). This work gave values very necessary to support the observed rates of lactate near this, 0.54-0.61 mM. Recently, the K m of pyruvate reduction catalyzed by purified v-lactate production as 0.014 m M for Mercenaria and 0.003 dehydrogenases from the horseshoe crab Limulus m M for Mytilus. Pyruvate concentrations in these polyphemus and the polychaete Nereis virens have animals have not been determined. In C. commerbeen reported as 0.07 and 0.13mM (Long & cialis adductor, however, the pyruvate concentration Kaplan, 1973). The lower K m values for these was below the limit of detection (Humphrey, 1950), species may be related to greater rates of lactate and our unpublished results suggested that pyruvate formation. In experiments on uptake of 14C- did not exceed 0.001 m M in the mantle of C. bicarbonate, more radioactivity appeared in lactic virginica. This suggests that bivalve mollusks do not acid in Limulus than in any other of the fourteen accumulate pyruvate, do not produce much lactate and, therefore, have no need for development of species of marine invertebrates examined (Hammen lactate oxidase activity. Rates calculated from these & Osborne, 1959). The oxidation of lactate to pyruvate, with the equations and reported acid concentrations indicate P M S - D C I P mixture as hydrogen acceptor, gave that after anaerobiosis succinate oxidation proceeds Michaelis constants of 118-250mM. These are from 90 to 150 times more rapidly than lactate consistent with values previously reported for oxidation. The conclusion is that lactate metabolism simply Mytilus and Crassostrea (Hammen & Lum, 1972). is not very important to marine bivalve mollusks. They are thirty to eighty times greater than Km (LAC) for Limulus and Nereis (Long & Kaplan, F r o m the similarity between species, I suggest that 1973). The possibility that use of N A D + in the natural selection has never required bivalves to assay, rather than DCIP, might give much lower develop a highly active lactate dehydrogenase system. On the other hand, succinate production K m (LAC) was considered, and proved unfounded (Fig. 3); with Mytilus adductor homogenate, the via the fumarate reductase reaction probably was same Km was obtained with both acceptors. The needed to deal with anaerobiosis since the earliest difference in V is interpreted as a consequence of stages of bivalve evolution, and some species have size. This phenomenon was observed in the lactate become better adapted to aerobic life by developing oxidase of a marine gastropod; a decrease in weight- increased capacity to oxidize succinate. specific enzyme activity with increase in size, and REFERENCES presumably age, of the animal (Hammen & Lum, 1972). Such changes in enzyme content probably lie ARRIGONI O. • SINGERT. P. (1962) Limitations of the at the basis of the well-known decrease in overall phenazine methosulphate assay for succinic and tissue metabolic rate with increased size within a related dehydrogenases. Nature, Lond. 193, 1256-1258. species. Cm~NSI-IAWM. A. & NEFF J. M. (1969) Decalcification at Ratios of activity P R / L D suggested that mollusks the mantle-sheU interface in molluscs. Am. ZooL have a lower probability of producing lactate than 9, 881-885. certain marine invertebrates such as a crab and GgvE G. & ZEnE E. (1973) Ober den Anaerobiosestoffwechsel yon Molluskenmuskeln. J. Comp. Physiol. Limulus, but not the lowest of all (Hammen, 1969). 85, 291-301. Ratios of Michaelis constants Km(LAC)/Km(PYR) were in the range 218 to 410, while the same ratios GALTSOFFP. S. (1964) The American oyster, Crassostrea virginica. Fishery Bulletin 64, U.S. Govt. Printing for Nereis and Limulus were 23.8 and 54.3 (Long & Office, Washington, D.C. Kaplan, 1973). This suggests that the ability of HAMMEN C. S. (1969) Lactate and succinate oxidorebivalves to oxidize lactate is both absolutely and ductases in marine invertebrates. Mar. Biol. 4, relatively poor, and that lactic acid might be 233-238. expected to accumulate. In fact, however, succinic HAMM~NC. S. & LLrMS. C. (1972) D-Lactate oxidation in a marine gastropod and a sea urchin. Comp. Biochem. acid was present in Mercenaria fluid at fifteen times Physiol. 4211, 123-129. the concentration of lactic acid, and increased elevenfold in 24 hr of anaerobic conditions, while HAMPtoN C. S. & OSnORNE P. J. (1959) Carbon dioxide fixation in marine invertebrates: a survey of major lactic only increased threefold (Crenshaw & Neff, phyla. Science, Wash. 130, 1409-1410. 1969). Similarly, succinate in M. edulis, initially HOPKINSH. S. (1946) The influence of season, concentraabout the same as lactate, increased twelvefold in tion of sea water and environmental temperature upon 48 hr while lactate only doubled in concentration the oxygen consumption of tissues in Venus mercenaria. (de Zwaan & Zandee, 1972). These data suggest J. Exp. Zool. 102, 143-158. in vivo rates of lactate production of 0.010 and 0-007 Htnorn~Y G. F. (1950) Glycolysis in oyster muscle. Austral. J. exp. Biol. Med. Sci. 28, 151-160. t~mole/hr per g.
412
C . S . Hmq~N
I ~ T e C E. & BU'~n~O E. (1961) Succinic and reduced diphosphopyridine nucleotide oxidase systems of Ascaris muscle. J. biol. Chem. 236, 584-591. LONG G. L. & K~t.AN N. O. (1973) Diphosphopyridine nucleotide-linked D-lactate dehydrogenascs from the horseshoe crab,/.,/mu/us polyphemus, and the seaworm, Nereis virens---II. Catalytic properties. Archs Bioehem. Biophys. 154, 711-725. Peacl~,nD R. K. & SCHOFn~r~P. J. (1968) A comparative study of the tricarboxylic acid cycle enzymes in Fasciola hepatica and rat liver. Comp. Biochem. Physiol. 25, 1005-1019. ROBERTS N. R., COI~J~O R. R., LOWRY O. H. & CRAW~m3 E. J. (1958) Enzyme activities of giant squid axoplasm and axon sheath. J. Neurochem. 3, 109-115. RYAN C. A. & KING T. E. (1962) Succinate dehydrogenase from the bay mussel, Mytilus edulis. Biochem. biophys. Acta 62, 269-278. SANADID. R. & FLUHAR'rgA. L. (1963) On the mechanism of oxidative phosphorylation--VII. The energyrequiring reduction of pyridine nucleotide by succinate and the energy-yielding oxidation of reduced pyridine nucleotide by fumarate. Biochemistry 2, 523-528. SEn3~J,~ I. & E ~ n ' ~ N. (1961) Oxidative enzymes and their role in phosphorylation in sarcosomes of adult Ascaris 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. (1971) Evolution of the respiratory chain and of its flavo-proteins. In Biochemical Evolution and the Origin of Life. (Edited by SctxorremeLS E.) pp. 203-223. North-Holland, Amsterdam.
VATSIS K. P. & SCHATZ[.~ F. C. (1972) Tricarboxylic acid cycle enzymes of the striped shore crab, Pachygrapsus crassipes. Comp. Biochem. Physiol. 42, 591610. W~OENERB. A., B,~q_~uTrA. E. & H ~ C. S. (1969) Reduction of fumarate and oxidation of succinate in Craasostrea virginica (Gmdin). Life Sei. 8, 335-343. W~.soN M. A. & C ~ A N O J. (1970) The energyyielding oxidation of NADH by fumarate in submitochondrial particles of rat tissue. Bioehem. biophys. Acta 216, 54-62. DE Zo~r~4 L. W., POSrHUMA D. & TIPKER J. (1969) Intermediary metabolism of the liver fluke Fasciola hepatica--I. Biosynthesis of propionic acid. Hoppe-
Seyler's Z. physiol. Chem. 350, 683-690. DE ZoFr~N L. W. & Tn,rant J. (1969) Intermediary metabolism of the liver fluke Fasciola hepatica--II. Hydrogen transport and phosphorylation. HoppeSeyler's Z. physiol. Chem. 350, 691-695. DE ZWAANA. & Z A ~ E D. I. (1972) The utilization of glycogen and accumulation of some intermediates during anaerobiosis in Mytilus edulis L. Comp. Biochem. Physiol. 43B, 47-54.
Key Word lndex---Succinate oxidase; fumarate reductase, phosphorylation; mollusk muscle; Mercenaria; Mytilus; Crassostrea; pyruvate; lactate; oxidoreductase.
SUCCINATE A N D LACTATE OXIDOREDUCTASES OF BIVALVE MOLLUSKS C. S. HAM_MEN Department of Zoology, University of Rhode Island, Kingston, R.I. 02881, U.S.A. (Received19February1973) Abstract--1. Succinic acid is a major end product of anaerobic glycolysis in mollusks, and lactic acid is a minor product. On the hypothesis that production of these acids is partially controlled by properties of the enzymes catalyzing the final step in each pathway, extracts of the adductor muscle were prepared and used in oxidoreductase assays. 2. The relations between substrate concentration and velocity of reaction were determined for pyruvate reduction (PR), lactate oxidation (LO), fumarate reduction (FR) and succinate oxidation
(so). 3. The animals were Mercenaria mercenaria, Mytilus edulis and Crassostrea virginica, and the assay mixtures included nicotinamide adenine dinucleotide (NAD) or dichlorophenolindophenol (DCIP). 4. The data were summarized as apparent Michaelis constants (Kin) and maximal velocities (V). Reductas¢ properties varied little between species, Km of PR 0.54--0.61 mM, Km of FR 25-30 mM. Oxidas¢ properties were more varied, Km of LO from the clam Mereenaria twice as great as the other two species, 250 and 120 mM; Km of SO from the oyster Cra~sostrea three to four times as great as the others, 120 vs. 30 and 40 raM. The smallest species, Mytilus, had the greatest Vfor both reductases, while the species varied little in V of the oxidases. 5. The SO of Mytilus was inactive with NAD until ATP was added, and its activity with DCIP was stimulated fourfold by 2 mM ATP; the FR of Mytilus was stimulated threefold by 8 mM ADP.
INTRODUCTION IN BIVALVEmollusks, succinic acid is a major end product of anaerobic giycolysis, and lactic acid is a minor product. This has been noticed in the oyster Crassostrea virginica (Hammen & Osborne, 1959; Simpson & Awapara, 1966), the hard clam Mercenaria mercenaria (Crenshaw & Neff, 1969) and the blue mussel Mytilus edulis (de Zwaan & Zandee, 1972). One simple hypothesis to account for the partition between acid end products is control by the enzymes catalyzing the final step in each pathway. Among micro-organisms, succinate dehydrogenase from anaerobes catalyzes reduction of fumarate more rapidly and oxidation o f succinate more slowly than the same enzyme from aerobes (Singer, 1971). Bivalve mollusks exhibit varying degrees of facultative anaerobiosis, which are reflected in varying ratios of forward and reverse rates of oxidoreductases. The ratio of fumarate reduction to succinate oxidation (FR/SO) varied from 0.23 in Mercenaria to 6"2 in Crassostrea, a greater range than found in any other group of marine invertebrates (Hammen, 1969). Ratios of pyruvate reduction to lactate oxidation were more uniform, and indicated a bias against lactate formation, compared to more active invertebrates. Experiments with a single concentration of substrate, however, did not reveal whether differences in rates were due to differences in tissue concentra-
tion of the enzyme or to differences in affinity for substrate. This new work was performed to find maximal rates per unit weight of tissue, and Michaelis constants for two oxidoreductases in both directions. The enzymes concerned are commonly known as succinate dehydrogenase and lactate dehydrogenase. The former enzyme is a flavoprotein with a quinone as the physiological hydrogen acceptor; a useful artificial acceptor is the blue dye dichlorophenolindopbenol (DCIP), giving the systematic name succinate : DCIP oxidoreductase (E.C. 1.3.99.1). The latter enzyme ordinarily requires nicotinamide adenine dinucleotide (NAD) as hydrogen acceptor, and in mollusks is specific for D-lactate, giving the name D-lactate : N A D oxidoreductase (E.C.
1.1.1.28). MATERIALS AND METHODS Three species of bivalve mollusks were collected in Narragansett Bay or its estuaries between 10 June and 27 August 1973 and were used in experiments within 24 hr. The ranges of shell length and total weight were: Mytilus edulis, 4.6-5.0cm and 13.1-15.9 g; Mercenaria mercenaria, 5"1-8"5cm and 36-3-152"0g; and Crassostrea virginica, 6.5-9"0 cm and 37.0-74.0 g. Freshly dissected adductor muscles were homogenized with nine times their weight of 0.250 M Tris-HCl buffer, pH 7.4, then centrifuged for 10 min at 800 g and 0°C
407
408
C.S. ~
in a Model B-20 (International Equipment Co.) centrifuge. Enzyme assays were performed with a Zeiss PMQII spectrophotometer, using the supematant fraction as the enzyme source. The oxidase activities were measured by a decrease in absorbance at 600 nm of mixtures containing 0-05 mM DCIP and 1-0mM phenazine methosulfate (PMS), essentially the method of Arrigoni & Singer (1962). The reductase activities were measured by a decrease in absorbance at 340 nm of mixtures containing 0.16 mM NADH, a procedure similar to the method of Sanadi & Fluharty (1963). Some experiments were also performed with 4"8 mM NAD + as the hydrogen acceptor for both lactate and succinate oxidation with Mytilus preparations. Reaction rates were calculated by means of the molar absorptivity, 6.22 x 10a for NADH, and 3-90-5.40 x 108 for DCIP in the presence of PMS. Temperatures of reaction mixtures were 19.0-22.0°C, and the final volume was 3.0 ml in each case.
003
f
~A
jra
NAD* 3 /
o, )
RESULTS Fumarate reductase and succinate oxidase activities are summarized as apparent Michaelis constants (Km) and maximum velocities (V) derived from double-reciprocal graphs of rate vs. substrate concentration (Table 1). Fumarate was readily reduced by N A D H over the range of 4-40 m M fumarate, and K m values fell in a narrow range, Table 1. Michaelis constants (mM) and maximum velocities (pmoles/min per g) of succinate oxidoreductases. Fumarate reductase Km
Mercenaria Mytilus Crassostrea
27 30 25
Succinate oxidase
V
Km
0.227 0-286 0"141
30 40 120
Km (SUC) V Km (FUM) 0"577 1"11 0-279 1"33 0 " 4 5 5 4.80
25-30 raM. In each experiment, there was substantial oxidation of N A D H in the absence of added fumarate, and these blank rates were subtracted. V was lowest in Crassostrea, and highest in Mytilus, 0.141 and 0.286 pmole/min per g tissue, respectively. Succinate oxidase properties varied more between species, with the lowest Km and highest V found in Mercenaria, and an extraordinarily high Km of 120 mM found in Crassostrea. The ratio Km(SUC)/ Km(FUM), indicating reductive tendency or probability of producing succinate, was nearly the same for Mercenaria and Mytilus, but fourfold greater for Crassostrea. As expected, N A D + was not reduced by succinate alone in the presence of Mytilus homogenate, but rapid reduction (succinate oxidation) occurred after the addition of 2 m M ATP. I n addition, succinate oxidation and concomitant DCIP reduction were accelerated more than fourfold by ATP (Fig. 1).
-0-04 0
,
I
I
2I
)
• 3
I
I
4 rain
5
I
6
7
8
Fig. 1. Stimulation of NAD+-succinate oxidase and DCIP--succinate oxidase reactions by added ATP, M. edulis adductor muscle. Change in absorbance at 340 nm and at 600 nm vs. time. Assay mixture represented by upper curve contained buffer, extract of 150 mg tissue and NAD+, 4.8 raM. That represented by lower curve contained 0.05 mM DCIP and 1.0mM PMS, instead of NAD +. Both reactions were started with the addition of succinate; final concentration 100 mM. Both mixtures received 2.0 mM ATP at points marked by
arrows.
The reverse reaction, fumarate reduction by NADH, was similarly accelerated by A D P at concentrations of 4 m M and above (Fig. 2). Pyruvate reductase and lactate oxidase are also expressed as Michaelis constants and maximum velocities (Table 2). The K m for pyruvate varied Table 2. Michaelis constants (mM) and maximum velocities (pmoles/min per g) of lactate oxidoreductases. Pyruvate reductase Km
Mercenaria 0"61 Mytilus 0.54 Crassostrea 0.54
Lactate oxidase
V
K,,
V
0.445 1.250 0.392
250 118 120
0.483 0"389 0"526
Km(LAC) Km(PYR) 410 218 222
little among species, only 0.54-0-61 mM. The V of this reaction was three times greater for Mytilus than the others. The K m for lactate was nearly the same for Mytilus and Crassostrea, and twice as great, 250 mM, for Mercenaria. The Vof lactate oxidation varied little among species. The same Km(LAC)
o
Oxidoreductases of mollusks
409
4C--
°//
I/v AA
_
k
20
0.05
-
-
I
I
I
2
l
5
E
4
!
5
I
6
rain
PiE. 2. Stimulation of NADH-fumarate reductase reaction by added ADP, M. edulis adductor muscle. Change in abserbance at 340 nm vs. time. Blank contained only buffer, extract of 100 mg tissue and 0"16 mM NADH. All reactions were started with the addition of NADH. The lowest curve represents rapid reaction with 40 mM fumarate and 8 mM ADP in mixture. The middle curves depict reactions with only fumarate and only ADP added.
was obtained for Mytilus when N A D + served as hydrogen acceptor and when D C I P was used (Fig. 3). The maximum velocity was quite different. The ratio Km(LAC)/Km(PYR), indicating the likelihood of lactate production, was twice as great for Mercenaria as for the other two species. DISCUSSION The reduction of fumarate by N A D H was catalyzed readily by extracts of these bivalve mollusks. This reaction has been studied in the parasitic worms Ascaris lumbricoides (Kmetec & Bueding, 1961; Seidman & Entner, 1961) and Fasciola hepatica (Prichard & Schofield, 1968; de Zoeten & Tipker, 1969). The general conclusion was that fumarate reduction occurred more rapidly in these helminths than in mammalian tissues. However, fragmented mitochondria from mammals also catalyzed the N A D H - f u m a r a t e reductase reaction (Sanadi & Fluharty, 1963; Wilson & Cascarano, 1970). Mitochondria from heart muscle were more effective than those from other tissues. The fumarate reductase of nineteen species of marine invertebrates was assayed by F M N H , oxidation (Hammen, 1969). Before the present work, only one marine invertebrate, the crab, Pachygrapsus crassipes,
-I0
0
I0
20
I/s Fig. 3. Velocity of lactate oxidation in relation to substrate concentration, M. edulis adductor muscle, double reciprocal plot. The upper curve based on reaction with 4.8 mM NAD +, lower curve on reaction with DCIP-PMS mixture as described in the text. Mean total weight of animals used was 24.2 and 14.1 g respectively. had been shown to reduce fumarate with N A D H (Vatsis & Schatzlein, 1972). Comparison of the apparent / ~ ( F U M ) of the mollusks with the literature is difficult, because no K m values are found in the studies cited. The mollusk values are 40-100 times greater than Km(FUM) for purified enzymes from bacteria and yeasts (Singer, 1971). Most authors have been content to measure fumarate reductase activity at a single substrate concentration, and compare it with activity in the reverse direction. The oxidation of succinate to fumarate, by means of the dye mixture PMS--DCIP, gave Michaelis constants of 30-120 mM. These values are ten to twenty times Km(SUC) for other organisms (Singer, 1971). In fact, the value for M. edulis, 40 mM, is twenty times greater than the K m of 2 m M determined for a soluble form of succinate dehydrogenase from the same species (Ryan & King, 1962). The higher K m values, however, probably represent more closely the properties of succinate oxidase within mollusk cells, because purification processes eliminate several thousand compounds normally present in the intracellular environment, and destroy fine structure more completely than simple homogenization. Since succinate oxidation and fumarate reduction are two sides of the same coin, ratios of activities and ratios of Michaelis constants can provide more information. The ratio Km(SUC)/Km(FUM)
410
C.S. ~ N
indicates the reductive tendency or probability of forming succinate, while the reciprocal indicates the tendency towards oxidizing succinate. Among microorganisms, Km(SUC)/Km(FUM) was 0.58 for an aerobe, 17.6 for an anaerobe and 3.14 for a facultative anaerobe (Singer, 1971). The ratio for these mollusks is 1.1 (Mytillus), 1.3 (Mercenaria) and 4.8 (Crassostrea). This suggests that they are all facultative anaerobes, with the best enzymatic adaptation shown by the oyster. The ratio of activity in both directions at a fixed concentration of each substrate, FR/SD, has proved useful for identifying the relative ability of various species to cope with temporary anaerobiosis by producing succinate (Hammen, 1969). The ratio of Michaelis constants, Km(SUC)/Km(FUM), accomplishes the same purpose with more precision. The ratio of maximum velocities, VFR/VSO, has also been obtained by varying concentrations of FMNH2 and ferricyanide, the H donor and acceptor, at fixed concentration of substrates, and gives similar information (de Zoeten et al., 1969). Much more information can be presented, and predictions about the direction of metabolism can be made, if results are presented as equations containing both K m and V. The Lineweaver Burk form of the Michaelis-Menten equation is
1/v = (Km/V) (1/S)+ 1/V. This has the same form as the simple linear equation:
y = ax+b, where y is the reciprocal of velocity, the slope a is Km/V and the intercept b is 1/V. After finding Km and V by experiment, an equation can be written for each direction of a reaction, an equation that permits calculation of the velocity at any specified substrate concentration. For example, the fumarate reductase activity of Mercenaria, according to these results is expressed by y = 119x+4-40. The fumarate content of Mercenaria tissues is unknown, but at 1.2 mM fumarate, a concentration within the range found in oyster tissues, the rate is y = 119(1/1.2)+4.40 = 103-5 = 1/v, v = 0.0097/~mole/min per g = 0.582/~mole/hr per g. This rate of succinate production can be obtained only by calculation, because the concentration of fumarate is too low to permit actual measurement. The succinic acid content of tissue and fluid of Mercenaria increased from 2-0 to 5.5 mM after 6 hr of anaerobiosis (Crenshaw & Neff, 1969). Assuming a 1-g tissue equivalent to 1 ml, this was an increase of 3.5 ymoles/g in 6 hr, or a rate of succinate
production of 0.583 pmole/hr per g, a rate identical to that calculated above from K m and V. This rate could be sustained only if other reactions were continuously supplying fumarate to maintain the assumed 1.2 mM concentration. The rate equation of succinate oxidation in Mercenaria adductor extract is y = 52.0x+ 1.73. This gives 0.0361/~mole/min per g or 2.16 ~moles/hr per g for succinate at 2-0 mM. Since each mole of succinate yields two g-atoms of hydrogen, which combine with one g-atom of oxygen, the equivalent rate of oxygen consumption is 1.08/~moles Os/hr per g. At 20°C the adductor muscle of large Mercenaria consumed oxygen at the rate of 5.36/~moles/hr per g dry or, assuming 80~o water, 1.08/~moles/hr per g fresh (Hopkins, 1946), exactly the rate expected ff oxygen comsumption were solely a consequence of succinate oxidation. Further, if the same equation is valid for mantle tissue, an increase in succinate concentration from 2.0 to 5.5 mM yields an increase in oxygen consumption of 1.60/zmoles/hr per g. Isolated mantle consumed oxygen at 7.91/,moles/hr per g. Thus, after 6 hr of valve closure, mantle tissue is predicted to show a 20 per cent increase in Os consumption, due to the increased succinate concentration. Higher respiration rates, immediately after anaerobic periods (oxygen debt) are well known among bivalve mollusks. For example, in the first 30 min open, after closure during handling, oysters consumed 16 per cent more rapidly than during 5-8 hr continuously open (Galtsoff, 1964). The acceleration of fumarate reduction by ADP, and the absolute dependence on ATP of the reverse reaction, NAD+-succinate oxidation, provides strong evidence for a coupled phosphorylation accompanying fumarate reduction in M. edulis. This is the first demonstration of this phosphorylation in an invertebrate other than,4scaris (Seidman & Entner, 1961), and Fasciola (de Zoeten & Tipker, 1969), and the first in a free-living marine invertebrate. Previously, we had attempted to demonstrate phosphorylation associated with fumarate reduction by F M N H I in homogenate of C. virginica, but the results were equivocal (Wegener et al., 1969). The present result indicates that this type of phosphorylation is readily demonstrable, once appropriate methods are found. All the fumarate reductions reported here occurred without any steps taken to exclude oxygen, so the coupled phosphorylation probably can occur during aerobic as well as anaerobic phases of metabolism, although at a lower rate during the aerobic phase, because the reaction shifts toward succinate oxidation. Pyruvate reductase activities have been measured in at least ten species of bivalve mollusks (Hammen,
Oxidoreduetases of mollusks
411
1969; G~tde & Zebe, 1973), and the values were F r o m our determination of K m and V, the usually about 10 per cent of activities for more active, appropriate equations for pyruvate reduction are invertebrates, and 1 per cent of those for vertebrate y = 1.37x+2.25 and y = 0.432x+0.80. tissues. The only molluscan K m (PYR) in the literature seems to be 0.64 m M for squid axoplasm These predict the pyruvate concentrations (Roberts et aL, 1958). This work gave values very necessary to support the observed rates of lactate near this, 0.54-0.61 mM. Recently, the K m of pyruvate reduction catalyzed by purified v-lactate production as 0.014 m M for Mercenaria and 0.003 dehydrogenases from the horseshoe crab Limulus m M for Mytilus. Pyruvate concentrations in these polyphemus and the polychaete Nereis virens have animals have not been determined. In C. commerbeen reported as 0.07 and 0.13mM (Long & cialis adductor, however, the pyruvate concentration Kaplan, 1973). The lower K m values for these was below the limit of detection (Humphrey, 1950), species may be related to greater rates of lactate and our unpublished results suggested that pyruvate formation. In experiments on uptake of 14C- did not exceed 0.001 m M in the mantle of C. bicarbonate, more radioactivity appeared in lactic virginica. This suggests that bivalve mollusks do not acid in Limulus than in any other of the fourteen accumulate pyruvate, do not produce much lactate and, therefore, have no need for development of species of marine invertebrates examined (Hammen lactate oxidase activity. Rates calculated from these & Osborne, 1959). The oxidation of lactate to pyruvate, with the equations and reported acid concentrations indicate P M S - D C I P mixture as hydrogen acceptor, gave that after anaerobiosis succinate oxidation proceeds Michaelis constants of 118-250mM. These are from 90 to 150 times more rapidly than lactate consistent with values previously reported for oxidation. The conclusion is that lactate metabolism simply Mytilus and Crassostrea (Hammen & Lum, 1972). is not very important to marine bivalve mollusks. They are thirty to eighty times greater than Km (LAC) for Limulus and Nereis (Long & Kaplan, F r o m the similarity between species, I suggest that 1973). The possibility that use of N A D + in the natural selection has never required bivalves to assay, rather than DCIP, might give much lower develop a highly active lactate dehydrogenase system. On the other hand, succinate production K m (LAC) was considered, and proved unfounded (Fig. 3); with Mytilus adductor homogenate, the via the fumarate reductase reaction probably was same Km was obtained with both acceptors. The needed to deal with anaerobiosis since the earliest difference in V is interpreted as a consequence of stages of bivalve evolution, and some species have size. This phenomenon was observed in the lactate become better adapted to aerobic life by developing oxidase of a marine gastropod; a decrease in weight- increased capacity to oxidize succinate. specific enzyme activity with increase in size, and REFERENCES presumably age, of the animal (Hammen & Lum, 1972). Such changes in enzyme content probably lie ARRIGONI O. • SINGERT. P. (1962) Limitations of the at the basis of the well-known decrease in overall phenazine methosulphate assay for succinic and tissue metabolic rate with increased size within a related dehydrogenases. Nature, Lond. 193, 1256-1258. species. Cm~NSI-IAWM. A. & NEFF J. M. (1969) Decalcification at Ratios of activity P R / L D suggested that mollusks the mantle-sheU interface in molluscs. Am. ZooL have a lower probability of producing lactate than 9, 881-885. certain marine invertebrates such as a crab and GgvE G. & ZEnE E. (1973) Ober den Anaerobiosestoffwechsel yon Molluskenmuskeln. J. Comp. Physiol. Limulus, but not the lowest of all (Hammen, 1969). 85, 291-301. Ratios of Michaelis constants Km(LAC)/Km(PYR) were in the range 218 to 410, while the same ratios GALTSOFFP. S. (1964) The American oyster, Crassostrea virginica. Fishery Bulletin 64, U.S. Govt. Printing for Nereis and Limulus were 23.8 and 54.3 (Long & Office, Washington, D.C. Kaplan, 1973). This suggests that the ability of HAMMEN C. S. (1969) Lactate and succinate oxidorebivalves to oxidize lactate is both absolutely and ductases in marine invertebrates. Mar. Biol. 4, relatively poor, and that lactic acid might be 233-238. expected to accumulate. In fact, however, succinic HAMM~NC. S. & LLrMS. C. (1972) D-Lactate oxidation in a marine gastropod and a sea urchin. Comp. Biochem. acid was present in Mercenaria fluid at fifteen times Physiol. 4211, 123-129. the concentration of lactic acid, and increased elevenfold in 24 hr of anaerobic conditions, while HAMPtoN C. S. & OSnORNE P. J. (1959) Carbon dioxide fixation in marine invertebrates: a survey of major lactic only increased threefold (Crenshaw & Neff, phyla. Science, Wash. 130, 1409-1410. 1969). Similarly, succinate in M. edulis, initially HOPKINSH. S. (1946) The influence of season, concentraabout the same as lactate, increased twelvefold in tion of sea water and environmental temperature upon 48 hr while lactate only doubled in concentration the oxygen consumption of tissues in Venus mercenaria. (de Zwaan & Zandee, 1972). These data suggest J. Exp. Zool. 102, 143-158. in vivo rates of lactate production of 0.010 and 0-007 Htnorn~Y G. F. (1950) Glycolysis in oyster muscle. Austral. J. exp. Biol. Med. Sci. 28, 151-160. t~mole/hr per g.
412
C . S . Hmq~N
I ~ T e C E. & BU'~n~O E. (1961) Succinic and reduced diphosphopyridine nucleotide oxidase systems of Ascaris muscle. J. biol. Chem. 236, 584-591. LONG G. L. & K~t.AN N. O. (1973) Diphosphopyridine nucleotide-linked D-lactate dehydrogenascs from the horseshoe crab,/.,/mu/us polyphemus, and the seaworm, Nereis virens---II. Catalytic properties. Archs Bioehem. Biophys. 154, 711-725. Peacl~,nD R. K. & SCHOFn~r~P. J. (1968) A comparative study of the tricarboxylic acid cycle enzymes in Fasciola hepatica and rat liver. Comp. Biochem. Physiol. 25, 1005-1019. ROBERTS N. R., COI~J~O R. R., LOWRY O. H. & CRAW~m3 E. J. (1958) Enzyme activities of giant squid axoplasm and axon sheath. J. Neurochem. 3, 109-115. RYAN C. A. & KING T. E. (1962) Succinate dehydrogenase from the bay mussel, Mytilus edulis. Biochem. biophys. Acta 62, 269-278. SANADID. R. & FLUHAR'rgA. L. (1963) On the mechanism of oxidative phosphorylation--VII. The energyrequiring reduction of pyridine nucleotide by succinate and the energy-yielding oxidation of reduced pyridine nucleotide by fumarate. Biochemistry 2, 523-528. SEn3~J,~ I. & E ~ n ' ~ N. (1961) Oxidative enzymes and their role in phosphorylation in sarcosomes of adult Ascaris 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. (1971) Evolution of the respiratory chain and of its flavo-proteins. In Biochemical Evolution and the Origin of Life. (Edited by SctxorremeLS E.) pp. 203-223. North-Holland, Amsterdam.
VATSIS K. P. & SCHATZ[.~ F. C. (1972) Tricarboxylic acid cycle enzymes of the striped shore crab, Pachygrapsus crassipes. Comp. Biochem. Physiol. 42, 591610. W~OENERB. A., B,~q_~uTrA. E. & H ~ C. S. (1969) Reduction of fumarate and oxidation of succinate in Craasostrea virginica (Gmdin). Life Sei. 8, 335-343. W~.soN M. A. & C ~ A N O J. (1970) The energyyielding oxidation of NADH by fumarate in submitochondrial particles of rat tissue. Bioehem. biophys. Acta 216, 54-62. DE Zo~r~4 L. W., POSrHUMA D. & TIPKER J. (1969) Intermediary metabolism of the liver fluke Fasciola hepatica--I. Biosynthesis of propionic acid. Hoppe-
Seyler's Z. physiol. Chem. 350, 683-690. DE ZoFr~N L. W. & Tn,rant J. (1969) Intermediary metabolism of the liver fluke Fasciola hepatica--II. Hydrogen transport and phosphorylation. HoppeSeyler's Z. physiol. Chem. 350, 691-695. DE ZWAANA. & Z A ~ E D. I. (1972) The utilization of glycogen and accumulation of some intermediates during anaerobiosis in Mytilus edulis L. Comp. Biochem. Physiol. 43B, 47-54.
Key Word lndex---Succinate oxidase; fumarate reductase, phosphorylation; mollusk muscle; Mercenaria; Mytilus; Crassostrea; pyruvate; lactate; oxidoreductase.