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Anaerobic metabolism in the oyster heart Collicutt, Janet Margaret 1975

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ANAEROBIC METABOLISM IN THE OYSTER HEART by JANET MARGARET COLLICUTT B.Sc. (Hons.)* University of Manitoba, 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the department of ZOOLOGY We accept t h i s thesis as conforming to the required stanldard THE UNIVERSITY OF BRITISH COLUMBIA In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 D e p a r t m e n t i ABSTRACT Previous studies indicate that the i n t e r t i d a l oyster, Crassostrea gigas, i s a f a c u l t a t i v e anaerobe. The end-products of anaerobiosis i n the oyster and i n other i n t e r t i d a l bivalves-are succinate and alanine. The production of these compounds i s energetically advantageous over lactate production i n that ATP-producing steps, other than those i n g l y c o l y s i s , appear to be coupled to succinate production. In t h i s study, anoxia adaptations of the v e n t r i c l e , a tissue with a high aerobic capacity, were examined. TWJ sources of energy areravailable to the anoxic v e n t r i c l e : glycogen stores and a large free amino acid pool. 14 C-labelled glucose, aspartate, and glutamate were tested as substrates of anaerobic metabolism i n the isolated v e n t r i c l e . Glucose and aspartate are readily metabolized by the anoxic v e n t r i c l e . The major end-product of glucose metabolism i s alanine while aspartate i s metabolized mostly to succinate. Glutama&e i s a poor substrate of anaerobic metabolism. Correspondingly, the i n t r a c e l l u l a r pool of aspartate decreases i n anoxia while the glutamate pool i s not depletedd; It i s proposed that the simultaneous mobilization of one mole of glucose and one mole of aspartate to two moles of alanine and one mole of succinate respectively, can maintainethe system i n redox balance, account for the production of 2-3 times more alanine than succinate i n the anoxic v e n t r i c l e , and provide the v e n t r i c l e with an ATP-yielding step at the fumarate reductase reaction. 'Amino groups are transfered from aspartate to alanine through the combined functioning of aspartate and alanine aminotransferases. However, the t o t a l number of amino groups needed for alanine synthesis cannot be accounted for by anoxic changes i n the pools of aspartate, other amino acids, or the adenylates. i i 14 14 A minimal accumulation of succinate- C from glutamate- C as the precursor shows that forward functioning of the Krebs cycle from tf-ketoglutarate i s of minor importance i n anoxia and the potential for generating GTP at the succinyl thiokinase reaction cannot be u t i l i z e d . 14 A t h i r d anaerobic end-product i s produced from both glucose- C and 14 aspartate- C. The compound appears to be metabolically closely linked to alanine or pyruvate but i t could not be i d e n t i f i e d . Aerobic ATP concentrations are not maintained i n anoxia. The concentration of .ATP decreases while ADP and AMP concentrations increase. These changes would activate g l y c o l y t i c enzymes i n anoxia and reactivate the Krebs cycle immediately upon return to an aerobic environment. i i i TABLE OE CONTENTS Page Abstract i Table of Contents i i i L i s t of Tables i v L i s t of Figures v i Chapter I. Introduction 1 Chapter I I . Materials and Methods 11 Chapter I I I . Results 19 Chapter IV. Discussion 45 Chapter V. Literature Cited 59 Appendix L i s t of Abbreviations 63 i v LIST OF TABLES Table I. Table I I . Table I I I . Table IV. Table V. Table VI. Table VII. Table VIII. Table IX. Table X. Anaerobic incubation of isolated v e n t r i c l e s 14 with glucose- C(U) for one hour. Anaerobic incubation of isolated v e n t r i c l e s 14 with aspartate- C(U) fornone hour. Anaerobic incubation of isolated v e n t r i c l e s 14 with glutamate- C(U) for one hour. Anaerobic incubation of isolated Ventricles with alanine- 1-^C for one hour. Anaerobic incubation of isolated v e n t r i c l e s 14 with glucose- C(U) for three hours. Anaerobic incubation of isolated v e n t r i c l e s 14 with aspartate- C(U) for three hours. Anaerobic incubation of isolated v e n t r i c l e s 14 with glutamate- C(U) for three hours. Anaerobic incubation of isolated v e n t r i c l e s 14 with glucose- C(U) for one hour i n sea water with pH adjusted to 7.0. Anaerobic incubation of isolated v e n t r i c l e s 14 with aspartate- C(U) for one hour i n sea water with pH adjusted to 7.0. Anaerobic incubation of isolated v e n t r i c l e s 14 with glutamate- C(U) for one hour i n sea water with pH adjusted to 7.0. Page 21 22 23 24 28 29 30 32 33 34 V Table XI. Table XII. Table X I I I . Table XIV. Table XV. Metabolite concentrations i n v e n t r i c l e s before and after anaerobiosis _in v i t r o . Metabolite concentrations i n the v e n t r i c l e i n vivo before and after anaerobiosis. I n t r a c e l l u l a r free amino acids i n the oyster v e n t r i c l e before and after anaerobiosis. The concentrations of the adenylates i n the v e n t r i c l e before and after anaerobiosis i n  v i t r o and i n vivo. The a c t i v i t i e s of some enzymes i n the adductor muscle and v e n t r i c l e of the oyster, €. gigas. Page 37 39 41 43 51 v i LIST OF FIGURES Page Figure 1. Pathway of anaerobic glucose metabolism i n the 3 oyster adductor muscle. Inset shows the known control interactions at the PEP branchpoint. Figure 2. Proposed coupling of amino acid and 7 carbohydrate metabolism i n anoxia i n f a c u l t a t i v e l y anaerobic bivalves. Figure 3. Representative chromatogram showing the 20 14 d i s t r i b u t i o n of C-label i n the amino acid and carboxylic acid fractions from a v e n t r i c l e 14 incubated with glucose- C. Figure 4. Proposed scheme for anaerobic metabolism i n 53 the v e n t r i c l e of the oyster. CHAPTER I INTRODUCTION 1 I n t e r t i d a l bivalve molluscs function as f a c u l t a t i v e anaerobes to survive the periods of anoxia that may be imposed on them by the t i d a l cycle. In nature i t would be unli k e l y f o r i n t e r t i d a l bivalves to be exposed to a i r for more than 24 hours but under laboratory conditions, Mytilus g a l l o p r o v i n c i a l i s can survive at least three days of anoxia (Zs-Nagy and Ermini,1972) and the oyster, Crassostrea gigas, can l i v e as long as 22 days out of water with no measurable oxygen uptake (Pedlow,1974). The oxygen storage capacity (hemoglobin, myoglobin, hemocyanin) of bivalves i s very limited (Zs-Nagy,1974) such that within a few minutes of sh e l l closure, a l l oxygen stores are used up. Pedlow (1974) showed that immediately upon s h e l l closure the P^^ of the p a l l i a l f l u i d dropped from 160 mmHg to 40-50 mmHg and remained at t h i s l e v e l for several days. Shell closure i s accompanied by an immediate drop i n the pH of the p a l l i a l f l u i d (from 7.5 aerobically to 6.7-7.0 within a few minutes) and, i t i s assumedrj of the tissues as w e l l . By decreasing the pH of sea water, Galtsoff (1964) inh i b i t e d oxygen uptake by the oysterm Crassostrea virg'inica. In sea water at pH 5.8, the oxygen consumption was only 10% of i t s normal l e v e l . As internal oxygen i s depleted within a few minutes of s h e l l closure, the switch from aerobic to anaerobic metabolism must be quite rapid and pH may be an important control i n the t r a n s i t i o n . When oxygen-based metabolism can no longer function, the bivalve must modify i t s metabolism i n such a way that the energy requirements of the tissues can s t i l l be met. During anoxia the tissue demand for energy decreases. For example, the rate of the heartbeat decreases (Bayne,1971) and the l a t e r a l c i l i a of the g i l l , which are responsible for maintaining the water current during f i l t e r feeding, cease beating (Malanga and A i e l l o , 1972). During anoxia i n vertebrates, g l y c o l y s i s i s the chief mechanism for 2 energy production and l a c t i c acid accumulates as the major end-product. In bivalves capable of surviving anoxia, lactate dehydrogenase i s either absent or i n very low a c t i v i t i e s (Simpson and Awapara,1966) and lactate i s a minor end-product of anaerobiosis (de Zwaan and Zandee,1972; Stokes and Awapara, 1968). Many studies have shown that the major end-products of anaerobiosis are succinate and alanine (Stokes and Awapara,1968; de Zwaan and Zandee,1972; de Zwaan and van Marrewijk,1973a;Malanga and Aiello,1972). The mantle tissue 14 14 of Rangia cuneata produces succinate- C and alanine- C i n a 1:1 r a t i o when 14 given glucose- C under anoxia (Stokes and Awapara,1968). Succinate and alanine accumulate i n equimolar amounts during anaerobiosis i n Mytilus 14 edulis (de Zwaan and Zandee,1972) but when glucose- C i s the substrate, the 14 14 r a t i o of alanine- C : succinate- C i s 2:1 (de Zwaan and van Marrewijk,1973a). The metabolic scheme i n Figure 1 was proposed to explain the anaerobic production of equimolar amounts of succinate and alanine by the tissues of bivalve molluscs (Hochachka and Somero,1973). The energy store for anaerobic metabolism i s glycogen and bivalve tissues are r i c h i n glycogen. tie Zwaan and Zandee (1972) report that 10-35% of the dry weight of Mytilus edulis i s glycogen. Fat cannot be oxidized anaerobically and, correspondingly, f a t stores i n i n t e r t i d a l bivalves are very low (1-2.5% of wet wt.)(Hammen,1969). Metabolism to the l e v e l of phosphoenolpyruvate (PEP) occurs by the c l a s s i c a l g l y c o l y t i c pathway (Hochachka ejt a l . , 1973) . Aerobically, pyruvate kinase converts PEP to pyruvate, which i s then oxidized by the Krebs cycle. The pyruvate kinases from the mantle and adductor of the oyster, Crassostrea  gigas, (Mustafa and Hochachka,1971) and from the mantle (Livingstone and Bayne,1974) and adductor (Holwerda and de Zwaan,1973) of Mytilus edulis have been described. They a l l have alkalin e pH optima such that the drop i n pH of the tissues during anaerobiosis would decrease the a c t i v i t y of the enzyme. Pathway of anaerobic metabolism i n the oyster adductor muscle. Inset shows the knowm control interactions at the PEP branchpoint. 3b "A NAD NADH G6P T F6P Y FDP T triose-P f" s^ATP 3-PG /TP I IOXAI | N A D H ^ i j N A D - * - ! V_. ATP malate -«^ -|pyruvate|. NADP NADPH Itumarafe] • aninei glutamate a- ketoglutarate NADH NAD Cyfop/asm M/fochonc/r/on FDP 4 Alanine and ATP i n h i b i t pyruvate kinase but fructose-1,6-diphosphate (FDP) can override t h i s i n h i b i t i o n . Hochachka e_t a l . (1973) propose that the combined effects of the drop i n pH and the increase i n alanine concentrations that accompany anaerobiosis serve to inactivate pyruvate kinase and at the same time activate PEP carboxykinase (Figure 1, in s e t ) . To produce succinate v i a the pathway i n Figure 1, carboxylation of a trio s e must take place. The enzymes PEP carboxykinase, malic enzyme, and pyruvate 'carboxylase can a l l accomplish t h i s function. However, pyruvate carboxylase which catalyses the reaction pyruvate + C0 2 + ATP • a c e t y 1 ~ C o A » M§ oxaloacetate + ADP + P i s i n low a c t i v i t i e s i n bivalve tissues (de Zwaan and van Marrewijk,1973b) and malic enzyme i s strongly poised for anaerobic function i n the malate ^ pyruvate dir e c t i o n (Mustafa and Hochachka,1973a). In mammalian tissues, PEP carboxykinase functions i n gluconeogenesis (Scrutton and Utter,1968) but i n f a c u l t a t i v e anaerobes such as Ascaris (Saz,1971) and i n t e r t i d a l bivalves (de Zwaan and van Marrewijk,1973b; Mustafa and Hochachka,1973b,c), i t i s k i n e t i c a l l y poised i n the d i r e c t i o n of oxaloacetate production. PEP carboxykinase i s activated by decreasing pH and by increasing alanine concentrations and so would be activated during anaerobiosis (Hochachka and Mustafa, 1972H Figure 1 i n s e t ) . The conversion of oxaloacetate to malate regenerates the NADH produced i n g l y c o l y s i s . Malate dehydrogenase, therefore, plays a role analogous to that of lactate dehydrogenase i n vertebrate tissues. Malate can be metabolized by two alternate routes and to explain a 1:1 r a t i o of succinate-14 14 C : alanine- C (Stokes and Awapara,1968), the two routes must compete equally for cytoplasmic malate. Fumarase and fumarate reductase (the reverse dir e c t i o n of succinate 5 dehydrogenase) convert malate to succinate. Fumarate reductase i s the only enzyme of those involved i n succinate production that i s located solely i n the mitochondria (Chen and Awapara,1969). The capacity for fumarate reduction vs. succinate oxidation increases i n animals capable of withstanding anoxia. In ^ / v i r g i n i c a the succinate dehydrogenase : fumarate reductase r a t i o i s 0.08 while i n the subtidal mussel, Mercenaria mercenaria, the r a t i o i s 2.5 (Hammen and Lum,1966). In anoxia-tolerating p a r a s i t i c worms, ATP production i s coupled to fumarate reduction (Seidman and Entner,1961; de Zoeten and Tipker,1969), the energetic advantage of succinate vs. lactate production i n the anoxic environment i s therefore obvious. Recently, Hammen, (1975) demonstrated a three-fold stimulation of. fumarate reductase by ADP and an absolute requirement of succinate dehydrogenase for ATP i n the bivalve, Mytilus edulis, a resu l t taken as goo& evidence for a coupled phosphorylation of ADP accompanying fumarate reduction. As Figure 1 shows, the production of one mole of succinate uses the reducing equivalents formed i n generating two moles of PEP. The disposal of half of the trioses produced i n gly c o l y s i s must therefore occur with no net oxido-reduction. Alanine i s the other known end-product of anaerobiosis. If pyruvate kinase i s absent, as i n adult Ascaris (Saz,1971), or i s non-functional during anaerobiosis, then pyruvate must be formed from malate v i a the malic enzyme reaction. The enzyme i s found i n i n t e r t i d a l bivalves (de Zwaan and van Marrewijk,1973b) and has properties that suit i t to anaerobic production of pyruvate'(Mustafa and Hochachka,1973a). Alanine i s formed v i a the alanine aminotransferase reaction: pyruvate + glutamate ) alanine + o(-ketoglutarate . The enzyme occurs i n high a c t i v i t i e s i n the tissues of bivalves (Read,1962; Dupaul and Webb,1974; Hammen,1969). Thus the anaerobic production of succinate 6 and alanine accounts for the trioses produced i n g l y c o l y s i s , keeps the system i n redox, and i s energetically advantageous. The amino groups for alanine production are donated by glutamate, and yet, Dupaul and Webb, (1971'J) have shown that i n t r a c e l l u l a r glutamate i s not depleted during anoxia. Glutamate can be replenished i n two ways : a) by the glutamate dehydrogenase reaction which catalyses <^-ketoglutarate + NH*—' ^ glutamate b) by transamination with another amino acid. In molluscs, the a c t i v i t y of glutamate dehydrogenase i s very low (Campbell and Bishop,1970). Dupaul and Webb (1971) showed a correlation between aspartate depletion and alanine accumulation i n the isolated g i l l of Mya arenaria. I t appears, therefore, that glutamate i s regenerated through the asparfiate aminotransferase reaction : aspartate + o(-ketoglutarate ^ oxaloacetate + glutamate. Like alanine aminotransferase, t h i s enzyme i s i n high a c t i v i t i e s i n bivalve tissues (Dupaul and Webb,1974? Hammen,1969). Figure 2 shows one proposed scheme for the coupling of amino acids into anaerobic metabolism..After deamination, the carbon skeleton of aspartate can be metabolized to alanine add succinate by the same pathways used i n glucose metabolism. Mobilization of two moles of aspartate to one mole of succinate plus one mole of alanine re s u l t s i n a net gain of one mole of NAD+. To balance redox, Hochachka e_t a l . (1973) propose the simultaneous mobilization of two moles of glutamate. By metabolizing the carbon skeleton of glutamate to succinate, the organism gains a mole of GTP (produced at the succinyl thiokinase step) per mole of glutamate. However, i f glutamate l e v e l s do not decrease i n anoxia (Dupaul and Webb,1971), then a net synthesis of glutamate must occur during anoxia i n order for t h i s scheme to be functional. Proposed coupling of amino acidsand carbohydrate metabolism i n anoxia i n f a c u l t a t i v e l y anaerobic bivalves. 7 b G6P - N A D — - G 3 P N A D H -) * - ^ NADH -NAD 1,3 DPG PEP I OX A*-3 amino acid (glycine) NADH -• - O X A ; N A D - . — \ N A D P ~ - M A L A T E N A D P H - — malate "-pyruvate : GLUTAMATE ADP+Pi ATP CoASH NADH *-succinylCoA 8 The coupling of amino acid and carbohydrate metabolism during anoxia has been proposed. The i n t r a c e l l u l a r free amino acid pool of marine bivalves i s very large (Schoffeniels and Gilles,1972; Campbell and Bishop,1970) and i t i s well demonstrated that the osmotic pressure due to i n t r a c e l l u l a r free amino acids constitutes a large part of the t o t a l i n t r a c e l l u l a r osmotic pressure (Lynch and Wood,1966). In C. V i r g i n i c a , the i n t r a c e l l u l a r free amino acid concentration ranges from 0.035 to 0.164 M depending on s a l i n i t y and makes up 14-21% of the i n t r a c e l l u l a r osmotic pressure (Hammen,1969). The free amino acid pool,responds to changes i n the e x t r a c e l l u l a r osmotic pressure. Pierce and Greenberg (1972) have shown that when the external s a l i n i t y i s reduced, the isolated v e n t r i c l e of Modiolus demissus releases amino, acids into the incubation medium, thereby decreasing the i n t r a c e l l u l a r osmotic pressure. When external s a l i n i t y i s increased, the tissue responds by increasing the synthesis of i n t r a c e l l u l a r amino acids. In Mya arenaria (Dupaul and Webb,1971) and i n Modiolus demissus (Baginski and Pierce,1975) the immediate response i s an increase i n the concentration of alanine. Accompanying t h i s , Dupaul and Webb (1971) noticed a decrease i n aspartate concentrations. This implicates anaerobic g l y c o l y s i s as the pathway for rapid production of amino acids during s a l i n i t y stress. Dupaul and Webb (1974) discuss a " f a s t " and a "slow" component i n the adjustment to high s a l i n i t y . The " f a s t " component i s characterized by a rapid increase i n alanine concentrations. In Modiolus demissus, the SsiBw" component i s a gradual increase i n the concentration of glycine (Baginski and Pierce,1975). Glycine replaces alanine and alanine concentrations return to normal. Thus carbohydrate and amino acid metabolism seem to be closely linked i n the responses to two of the most important stresses placed upon i n t e r t i d a l bivalves: anaerobiosis and s a l i n i t y stress. Although aspartate and glutamate 9 have been implicated i n the anaerobic metabolism of these organisms, their roles have not been tested. In t h i s study the u t i l i z a t i o n of the i n t r a c e l l u l a r free amino acid pool as an energy source during anoxia was assessed. As the most thorough studies of the properties of enzymes involved i n anaerobic metabolism have been done i n C_. gigas, metabolite studies i n t h i s species would be meaningful. In the present work, the u t i l i z a t i o n of 14 14 14 glucose= C, aspartate-, C, and glutamate- C as substrates of anaerobic metabolism was studied and compared with measurements of the changes i n concentrations of intermediates and end-products. The isolated v e n t r i c l e of C^. gigas was chosen as the system to be used. The v e n t r i c l e of the oyster i s composed of unitary smooth muscle (Irisawa et al.,1969). Glycogen granules are abundant. Electron micrographs show a p l e n t i f u l supply of mitochondria (Irisawa et a l . ,1969) unlike the adductor muscle which has very few mitochondria (Mustafa and Hochachka,1973). In t h i s sense the v e n t r i c l e resembles the vertebrate heart which i s highly aerobic and dependant on the Krebs cycle and oxidative phosphorylation for the generation of ATP while skeletal muscle has a much higher capacity for g l y c o l y t i c generation of ATP (Drummond,1966; Scrutton and Utter,1968). The bivalve heart i s very sensitive to oxygen tension. In declining oxygen tensions, the hearts of Mytilus edulis (Bayne,1971) and Pecten maximus (Brand and Roberts,1973) maintain a constant beat down to approximately 50 mmHg pO^ after which heartbeat rate rapidly declines u n t i l cardiac arrest occurs at approximately 20 mmHg. Oxygen consumption by the heart i s constant down to approximately 75 mmHg after which i t declines u n t i l there i s no oxygen consumption by 20 mmHg. Upon s h e l l closure, therefore, the v e n t r i c l e makes a true aerobic to anaerobic t r a n s i t i o n . The drop i n oxygen tension found i n the oyster body f l u i d s immediately upon s h e l l closure (Pedlow,1974), combined with the effect of pH on oxygen consumption (Galtsoff ,1964) would 10 e f f e c t i v e l y prevent further oxygen consumption by the heart and i n i t i a t e anaerobic metabolism. The v e n t r i c l e i s well suited for studying anaerobic metabolism for several reasons. The v e n t r i c l e continues to beat i n anoxia (although at a slower rate)(Bayne,1971), i t i s composed of a single tissue type, and the metabolism i s geared to a single purpose — that of generating ATP to be used for muscle contraction. The v e n t r i c l e i s thi n enough to be used as a tissue s l i c e i n i t s e l f once cut open, and i t can be removed from the animal without any great physical damage to the tissue. The isolated molluscan v e n t r i c l e has long been used as a simple and hardy preparationein which to study the e l e c t r i c a l , i o n i c , and neurochemical events associated with the heartbeat (for example, Greenberg,1969). From these studies, i t has been shown that the isolated v e n t r i c l e i n a pysiological saline w i l l r e t a i n a regular beat for several hours (Greenberg,1969) and t h i s i s good evidence that normal metabolism persists i n the isolated v e n t r i c l e . CHAPTER I I MATERIALS AND METHODS 11 Animals Oysters, Crassostrea gigas, were purchased from Bi l l i n g s g a t e Fish Co., Vancouver and held, starved, for one week i n running aerated sea water at o 15 C before use. Radiotracer experiments The v e n t r i c l e (avg.wt. = 72.5 ± 19.2 mg) was excised, cut open for maximum surface area, blotted, weighed, and put into the incubation f l a s k . The f l a s k was a 25 ml erlemyer which had been autoclaved and into which 5 ml m i l l i p o r e f i l t e r e d sea water had been added. After adding the v e n t r i c l e , the f l a s k was sealed with a serum stopper and flushed with nitrogen gas (99.5 % pure) for 15 minutes, to drive off oxygen. The labelled compound was then 14 injected ( o.l ml of solution containing approx. LuCi of C). The labelled 14 14 compounds were: D-glucose- C (U) (15mCi/mmih<he) , L-aspartic acid- C(U) (204mCi/ mmole), L-glutamic acid-'^C(U) (260m CL/mmole) , and L-alanine-l-^C(13mCi/mmole) , a l l purchased from New England Nuclear. The v e n t r i c l e s were incubated, shaking, for 1 or 3 hours at room temperature, then removed f.Eom the saline and frozen i n l i q u i d nitrogen. Each incubation was with a single v e n t r i c l e , with 3 or 4 v e n t r i c l e s per experimental group. Incubations to control for b a c t e r i a l contamination were done i n which streptomycin (0.5 mg/ml) and p e n i c i l l i n (500 U/ml)(Anderson,1974) were added to the incubation saline. Sea water at room temperature i s pH 7.8. In some experiments, the pH of the saline was lowered to simulate the drop i n internal pH known to occur during anaerobiosis. To do t h i s , lOmM imidazole was added to the saline and the pH adjusted to 6.8 with IN HCl. The pH of t h i s solution rose to 7.0 after flushing with nitrogen gas but remained constant during the rest of the incubation. 12 Each v e n t r i c l e was homogenized i n 5ml 80% ethanol i n a centrifuge tube using a V i r t i s t e f l o n homogenizer, and l e f t to stand one hour i n ic e . Precipitated protein was removed be centrifugation at 7000g for 10 minutes. The supernatent was saved and the precipitate resuspended i n 2ml 20% ethanol. Sfter centrifugation, the wash was added to the f i r s t supernatent. This ethanol extract was then shaken i n 3 volumes of chloroform, and the aqueous layer collected. The organic layer was re-extracted twice with 2ml d i s t i l l e d water each time, and the aqueous layers added to the f i r s t aqueous layer (Stokes and Awapara, 1968). Amino acids were separated from carboxylic acids, neutral compounds, and phosphorylated compounds using an Anberlite IR-120 H + column(3 cm x 1 cm). After passing the extract through the column, the column was washed with 10 ml d i s t i l l e d water and a l l eluants to t h i s point were collected as the carboxylic acid f r a c t i o n . Amino acids were eluted with 20 ml 2N NH^ OH. The two fractions were evaporated down to a small volume using a f l a s h evaporator and chromatographed. Descending paper chromatography on Watman #1 paper was employed using the solvent system of Crowley et al.(1963) consisting of: EDTA 1.2 gm 17N ammonia 100 ml water 950ml n-propanol 350 ml isopropanol 75 ml n-butanol / 75 ml isobutyric acid ' 2500 ml The chromatograms were developed for 14 hours. The dried chromatograms were cut i n one cm s t r i p s and counted using l i q u i d s c i n t i l l a t i o n ( s c i n t i l l a t i o n 13 c o c k t a i l consisting of toluene with 4% w/v PPO and 0.02% w/v POPOP). Radioactive compounds were i d e n t i f i e d by comparison to co-chromatographed standards. The data were corrected for quenching, standardized to dpm per 100 mg tissue weight, and background was subtracted. Other solvent systems were used to aid i n ide n t i f y i n g the radiolabelled compounds and to ensure that a single compound was responsible for each peak. These were: for amino acids a) phenol:water, 80:20 v/v (Stokes and Awapara, 1968), b) propanolrammonia:water, 73:20:7 v/v (Regnouf and van Thoai,1970), c) butanol:propionic acid:water, 46:23:31 v/v (Crowley et al.,1963); for carboxylic acids: a) ethanol: ammonia:water, 80:5:15 v/v (Stokes and Awapara, 1968). The incubation saline was extracted with 20 ml 80% ethanol, centrifuged down and the . supernatent saved. The precipitate was re-extracted with 5 ml 20% ethanol,centrifuged, and the supernatent added to the f i r s t . Total r a d i o a c t i v i t y remaining i n the saline was determined, and f r a c t i o n i z a t i o n and chromatography done to look for metabolized r a d i o a c t i v i t y i n the saline. Metabolite Experiments In these experiments only oysters whose shell s were open and were ac t i v e l y pumping water were used. This ensured that the animals were aerobic at the start of the experiment. In the isolated v e n t r i c l e experiments, the v e n t r i c l e s were excised and incubated as described i n the previous section except that radiotracers were not added. Each v e n t r i c l e was cut i n h a l f , one half frozen immediately i n l i q u i d nitrogen, and the other half incubated anaerobically for one hour and then frozen. Eight half hearts were pooled i n each aerobic and anaerobic group. To makertehe tissues anaerobic as quickly as possible, the saline was 0 14 flushed with nitrogen gas before the v e n t r i c l e was added and again afterwards. In the i n vivo experiments, ve n t r i c l e s were subjected to anaerobiosis while s t i l l i n the intact animal. Four oysters were opened immediately upon removal from the tank,their v e n t r i c l e s excised, blotted, and frozen i n l i q u i d nitrogen. These control aerobic v e n t r i c l e s were pooled for analysis. Experimental^ were l i f t e d out of the tank, blotted dry, and l e f t exposed to a i r for four hours, after which they were opened and the v e n t r i c l e s excised and frozen. The v e n t r i c l e s were pooled i n groups of four. The v e n t r i c l e s were homogenized i n 8% perchloric acid i n 40% ethanol (containing ImM EDTA) (Williamson and Corkey,1968) using a V i r t i s t e f l o n homogenizer. Solutions and homogenates were kept cold i n a dry i c e - ethanol bath. A volume:weight r a t i o of 3:1 perchloric acid solution : tissue was used. The precipitated protein was removed by centrifugation ot 25,000 g for 10 minutes and the supernatent collected. The p e l l e t was resuspended i n the same volume of perchloric acid, reeentEm£uged, and the second supernatent added to the f i r s t . The extract was neutralized with a solution of 3M K^CO^ a n <* 0.5M triethanolamine. The precipitated KCIO^ was removed by centrifugation at 25,000 g for 10 minutes. The t o t a l volumes of both therneutralized and unneutralized extracts were recorded. The neutralized extracts were stored at -20°C u n t i l use. Metabolites were measured i n enzyme assays coupled to the pyridiriee nucleotides and the change i n absorbance of these cofactors measured at 340nm i n a Unicam SP1800 spectrophotometer. Enzymes and chemicals were from Sigma. In each metabolite assay, the amount of pyridine nucleotide used up i s .directly proportional to. the amount of substrate used. If the assays are run to completion i n a system where only the metabolite of interest i s l i m i t i n g then the number of ^ /moles of pyridine nucleotide used up i s d i r e c t l y 15 comparible to the number of ^ moles of substrate used up. The amount of pyridine nucleotide used up i s calculated v i a the formula: JJL moles = AOD x v o l . / £ where:AOD i s the change i n absorbance of the pyridine nucleotide at 340nm, v o l . i s the t o t a l volume i n the cuvette, and £ i s the extinction c o e f f i c i e n t of the pyridine nucleotides at 340nm = 6.22 (Williamson and Corkey,1968). Knowing the following: a) the volume of neutralized extract added to the cuvette b) the t o t a l volume of the neutralized and unneutralized extracts c) the vol.:wt. r a t i o of perchlorate : .tissue used, and assuming that the tissue i s 80% water, the concentration of the metabolite of interest i n the c e l l can be determined. A l l assays were allowed to run to completion. Standard ImM solutions of each metabolite to be measured were made up. With the exception of oxaloacetate, each assay gave an accurate determination of the concentration of the standard. Blank cuvettes were run with every assay so that changes i n absorbance that were not due to u t i l i z a t i o n of the substrate could be corrected f o r . Alanine assay pyruvate + NADH + H — — > malate + NAD GPT alanine +«t-ketoglutarate > pyruvate + glutamate Lactate dehydrogenase(LDH) i s added to a cuvette containing buffer (50mM Tris,pH 8.1), NADH(100/uM) , o(-ketoglutarate(200/tfM), and neutralized extract. When endogenous pyruvate has been used up, glutamate-pyruvate transaminase (GPT) i s added and the change i n O D ^ Q recorded (Lowry and Passonneau,1972). Aspartate assay GOT aspartate + *-ketoglutarate ^ oxaloacetate + glutamate 16 _ l MDH + oxaloacetate + NADH + H - > malate + NAD Malate dehydrogenase(MDH) Is added to a cuvette containing buffer( 50mM imidazole,pH 7.0), NADH(100/(M) , <X-ketoglutarate(200;«M) , and neutralized extract. When endogenous oxaloacetate has been used upm glutamate-oxaloacetate transaminase(GOT) i s added and the change i n O D J ^ Q recorded(Lowry and Passpnneau,1972). * ATP assay H K ATP + glucose > ADP + glucose-6-phosphate 4" G6PDH -H glucose-6-phosphate + NADP ^ 6-P-gluconolactone + NADPH + H Glucose-6-phosphate dehydrogenase(G6PDH) i s added to a cuvette containing buffer(50mM Tris,pH 8.1), MgCl 2(lmM), dithiothreitol(0.5mM), NADP+ (0.5mM),glucose(lmM), and neutralized extract. When the reaction i s complete, hexokinase i s added and the change i n OD^^q i s recorded (Lowry and Passonneau,1972). Glutamate assay . *t~ G D H + + glutamate + NAD - ^ c*-ketoglutarate + NH^ + NADH + H Glycine-hydrazine buffer (purchased from Sigma) was pH'd to 8.4. Glutamate dehydrogenase(GDH) i s added to a cuvette containing buffer, NAD+ (ImM) , and neutralized extract. The change i n O D - ^ Q i s recorded. This i s a modification of Lowry and Passonneau(1972). "(-ketoglutarate assay + GDH + ef-ketoglutarate + NADH + H ) glutamate + NAD + H20 Glutamate dehydrogenase(GDH) i s added to a cuvette containing buffer (50mM triethanolamine,pH 7.0), MgS04(10mM), EDTA(5mM), NADH(IOO^M), (m^)?S0^ (2mM) , and neutralized extract. The change i n OD-J^Q i s recorded (Lowry and Passonneau,1972). Lactate assay G P T pyruvate + glutamate > alanine + «(. -ketoglutarate + LDH + lactate + NAD ^ pyruvate + NADH + H Lactate dehydrogenase(LDH) and glutamate-pyruvate transaminase(GPT) are added to a cuvette containing buffer(lOOmM methyl-aminopropanol,pH 9.9), NAD+(2mM) , glutamate(50mM), and neutralized extract. The change i n O D ^ Q i s recorded. GPT i s added to draw off the pyruvate as i t i s formed and speed the reaction (Lowry and Passonneau,1972). Malate assay GOT oxaloacetate + glutamate • ) aspartate + d-ketoglutarate •+• MDH + malate + NAD — — » oxaloacetate + NADH + H Malate dehydrogenase and glutamate-oxaloacetate transaminase are added to a cuvette containing buffer(100mM methyl-aminopropanol,pH 9.9), NAD+(2mM), glutamate(50mM), and neutralized extract. The change i n O D i s recorded. Glutamate-oxaloacetate transaminase i s added to draw off the oxaloacetate as i t i s formed and speed the reaction(Lowry and Passonneau,1972). Oxaloacetate and Citrate assay CL c i t r a t e •+ ) oxaloacetate + acetate oxaloacetate + NADH + H —> malate + NAD Malate dehydrogenase(MDH) i s added to a cuvette containing buffer(100mM Tris,pH 7.6), NADH(100/«M) , ZnCl2(SO^uM) , and neutralized extract. The change i n f° r oxaloacetate reduction i s recorded. Then c i t r a t e lyase (CL) i s added and the change i n O D ^ Q for c i t r a t e u t i l i z a t i o n i s determined(Lowry and Passonneau,1972). Pyruvate,ADP, Succinate, and AMP assay AMP + ATP.—— > 2 ADP S T K succinate + ATP + CoA i £ i—> succinyJ-CoA + P + ADP 18 P K ADP + PEP-n—- > pyruvate + ATP + L D H + pyruvate + NADH + H;- — — — ^ lactate + NAD Lactate dehydrogenase(LDH) i s added to a cuvette containing buffer(50mM triethanolamine,pH 7.4),with lOmM MgSO^ and 5mM EDTA), NADH(100/<M), and neutralized extract, and the change i n OD^^Q recorded. Then PEP(1.6mM) and pyruvate kinase(PK) are added and the OD^^ due to ADP u t i l i z a t i o n recorded. Then ATP(9.33mM), coenzyme-A(l.7mM), and succinyl thiokinase(STK) are added and the change i n O D ^ Q for succinate recorded. F i n a l l y myokinase(MK) i s added and the amount of AMP measured. Each of these four reactions i s allowed to go to completion before the reagents and enzymes for the next assay are added(Lowry and Passonneau,1972; Williamson and Corkey,1968). Amino acid analyses Amino acids other thahnthose measured i n the metabolite assays were measured i n the neutralized extract using a Beckman 120C amino acid analyser. Only neutral and acidic amino acids were determined. The amount of each amino acid i n the sample added to the column was determined by comparison to a previously run set of standard amino acids of known concentrations. A norleucine standard was run with a l l samples to correct for decay of the ninhydrin. The following calculation gave the number of nanomoles of amino acid i n the 10 lamdas of neutralized extract put on the column: A, ^ x nmoles, .. A x A, .. . (cal.IS) (cal.aa) (sample aa) ^(sample IS) X ^(cal.aa) where: ••^•(cai/];S) t* i e a r e a u n d e r the peak for the c a l i b r a t i o n run of the inter n a l standard norleucine, A, .. T M i s the area under the peak £>6r (sample IS) a sample run internal standard norleucine, A, N i s the area under the (sample aa) peak £Sr amino acid 'X' i n a sample, A, n , i s the area under the peak V.C3. i. • 3.3.) for 20 nmoles of amino acid 'X' on the c a l i b r a t i o n run, and n m o l e s ^ ^ a a ) ^ s 20 nmoles of norleucine put on column for the c a l i b r a t i o n run. CHAPTER III RESULTS 19 Radiotracer Experiments In control experiments, streptomycin and p e n i c i l l i n were added to the 14 saline i n which v e n t r i c l e s were incubating with glucose- C. The amount of 14 labelled glucose metabolized and the d i s t r i b u t i o n of the C-label i n intermediates and end-products i n these experiments was very similar to experiments without a n t i b i o t i c s (Table I ) . The recovery of labe l i n the various steps of the f r a c t i o n i z a t i o n was tested. The chloroform treatment of the ethanol extract was 97% e f f i c i e n t . The sum of r a d i o a c t i v i t y i n the amino acid and carboxylic acid fractions eluted from the Amberlite IR-120 H + column was equal to 99% of the ra d i o a c t i v i t y put onto the column. The carboxylic acid f r a c t i o n contained carboxylic acids, phosphorylated compounds, and neutral compounds. No further f r a c t i o n i z a t i o n was done. The phosphorylated compounds were e a s i l y separated from the carboxylic acids as they ran with very low R^'s i n the solvent system of Crowley et a l . (1963). (Figure 3). In preliminary experiments, neutral compounds (ex. glucose) were separated from carboxylic acids,and phosphorylated compounds on an Amberlite IR-400 CI column. No r a d i o a c t i v i t y was detected i n the neutral f r a c t i o n i f the v e n t r i c l e was removed from the saline (containing unmetabolized glucose-14 C) before homogenization. One Hour Incubations Tables I , I I , I I I , and IV show the re s u l t s of 1 hour incubations of oyster v e n t r i c l e s with glucose-*^(U) , aspartate-^C (U) , glutamate-'''^C(U) , 14 and alanine-1- C respectively. The uptake (assumed to be equal to 14 14 i n t r a c e l l u l a r C) of glucose- C from the saline was greater than the uptakes of any other radio-labelled compounds. In addition, no free 14 i n t r a c e l l u l a r glucose- C was detected. In to k a l , an average of 16.4% of the 20a 1 ^ Figure 3 Representative chromatogram showing the d i s t r i b u t i o n 14 of €€label i n the amino acid and carboxylie acid 14 f r a c t i o n s from a v e n t r i c l e incubated with glucose- C. The solvent system was that of Crowley et al.(1963). alanine 40,000 30,000 f DPM 20,000 10,000 4 10,000 -t DPM 5,000 unknown compound Amino Acid Fraction T 1 1 1 1 1 1 1 1 1 1 1 1 1 [— 2 4 6 18 10 1212 14 16 18 20 22 24 26 28 30 Strip # malate phosphorylated .\ cpds. ' Y Carboxylic Acid Fraction \ succinate pyruvate t-^*\ H 1 1 1 1 1 r — i 1 1 1 1 1 r -8 10 12 14 16 18 20 22 24 26 28 30 Strip # Table I 14 A n a e r o b i c i n c u b a t i o n o f i s o l a t e d v e n t r i c l e s w i t h g l u c o s e - C(U) f o r one hour V e n t r i c l e # C o n t r o l v e n t r i c l e s T o t a l dpm i n s a l i n e 1675500 + t i s s u e % u p t a k e o f l a b e l 2 3 . 5 i n t o t i s s u e % i n t r a c e l l u l a r l a b e l 0 . 0 i n g l u c o s e - 1 4 C % m e t a b o l i z e d i n t r a -c e l l u l a r l a b e l i n : unknown compound 3 7 . 2 a l a n i n e 4 6 . 9 p h o s p h o r y l a t e d c p d s . 4 . 8 m a l a t e 6 . 7 p y r u v a t e 0 . 4 s u c c i n a t e 4 . 0 2 1604100 1 5 . 5 0000 2 8 . 8 5 7 . 1 5 . 0 5 . 9 0 . 3 2 . 9 3 1613400 1 4 . 6 0 . 0 3 3 . 6 4 7 . 8 8 . 3 7 . 2 0 . 6 2 . 6 4 1611300 1 2 . 1 0 . 0 2 2 . 0 6 9 . 1 2 . 9 2 . 3 2 . 6 1 . 3 a v g . ± s . e . 3 0 . 4 ± 6 . 6 5 5 . 2 ± 1 0 . 3 5 . 3 ± 2 . 2 5 . 5 ± 2 . 2 1 . 0 ± 1 . 1 2 . 7 ± 1 . 2 1 1289500 1 5 . 0 0 . 0 2 0 . 7 5 0 . 5 8 . 7 8 . 5 7 . 1 4 . 4 1102300 2 1 . 1 0 . 0 20:9 5 4 . 3 1 4 . 2 4 . 2 4 . 8 1 . 6 T a b l e I I A n a e r o b i c i n c u b a t i o n o f I T o t a l dpm i n s a l i n e 1589000 + t i s s u e % u p t a k e o f l a b e l 1 3 . 2 i n t o t i s s u e % i n t r a c e l l u l a r l a b e l 2 7 . 6 i n a s p a r t a t e - 1 4 C % m e t a b o l i z e d i n t r a -c e l l u l a r l a b e l i n : g l u t a m a t e 1 .7 unknown compound 1 1 . 0 a l a n i n e 1 5 . 3 m a l a t e 2 6 . 4 p y r u v a t e 1 . 8 s u c c i n a t e 4 3 . 8 i s o l a t e d v e n t r i c l e s w i t h a s p a r t a t e - 1 4 c ( U ) f o r one h o u r , V e n t r i c l e # 2 3 4 a v g . ± s . e . 4 2257500 1421400 1974500 5 . 5 9 . 5 8 . 8 3 3 . 8 3 1 . 2 2 8 . 4 1 . 3 9 . 7 1 7 . 0 1 6 . 6 1 . 0 5 4 . 5 2 . 6 1 1 . 1 1 7 . 5 1 6 . 5 2 . 3 5 0 . 1 2 . 9 1 3 . 3 1 7 . 1 1 9 . 3 3 . 6 4 3 . 9 2 . 1 * 0 . 8 1 1 . 3 ± 1 . 5 1 6 . 7 ± 1 . 0 1 9 . 7 ± 4 . 7 2 . 2 ± 1 . 1 4 8 . 1 ± 5 . 2 IS3 ho Table I I I Anaerobic incubation of isolated ventricles with glutamate- C(U) for one hour. Ventricle # avg. ± s.e. Total dpm i n saline 1569400 1469800 1527400 1493300 + tissue % uptake of l a b e l 4.9 4.5 3.2 8.4 into tissue % i n t r a c e l l u l a r l a b e l 83.1 87.6 85411 86.7 i n glutamafee-14C ' % metabolized i n t r a -c e l l u l a r l a b e l i n : aspartate 14.1 6.4 8.8 11.2 other amino acids 19.7 3331 28.6 31.7 alanine 3.3 5.3 3.5 6.9 malate * <*-kga 12.1 13.2 7.0 10.5 succinate 50.8 42.1 52.1 39.7 10.1 ± 3.2" 28.3 ± 6.0 4.8 ± 1.7 10.7 ± 2.6 46.2 ± 6.2 Table IV Anaerobic incubation of isolated ventricles with alanine-1- C for one hour. Total dpm i n saline 1249400 + tissue % uptake of l a b e l 4.1 into tissue % i n t r a c e l l u l a r l a b e l 77.4 i n alanine-14C % metabolized i n t r a -c e l l u l a r l a b e l i n : unknown compound 66.8 pyruvate 33.2 2 1158200 6.5 77.5 72.9 27.1 Ventricle # 3 1161400 5.3 76.8 68.4 31.6 1156200 7.9 77.5 60.2 39.8 avg. ± s.e. -p-67.1 ± 5.3 32.9 ± 5.3 25 14 14 glucose- C was metabolized i n one hour. Unmetabolized C-labelled aspartate, glutamate, and alanine were found i n the tissues. Combining percentage uptake into the tissue with percentage of the i n t r a c e l l u l a r l a b e l that i s metabolized, i t can be seen that an average of 6.5%, 0.8%,and 1.4% of the 14 14 14 added aspartate- C, glutamate- C, and alanine- C are metabolized by the ve n t r i c l e after one hour of anaerobiosis. Figure 3 shows representative separations of the amino acid and 14 carboxylic acid fractions from a v e n t r i c l e incubated with glucose- C after 14 paper chromatography. Some C-label accumulates i n the phosphorylated g l y c o l y t i c intermediates (ex. glucose-6-P, fructose-6-P, and PEP). The phosphorylated compounds were not further separated. Table I shows that i n 14 14 the oyster v e n t r i c l e , glucose- C i s metabolized mainly to alanine- C. Only 14 14 small amounts of succinate- C are formed. The r a t i o of alanine- C to 14 succinate- C i s approximately 20:1. An unidentified compound i n the amino acid f r a c t i o n accounts for 30% of the metabolized r a d i o a c t i v i t y . This i s i n contrast to Stokes and Awapara (1968) who found equal amounts of succinate and alanine produced when the mantle tissue of Rangia cuneata was incubated anaerobically with labelled glucose. 14 14 As Table I I shows, aspartate- C i s metabolized mostly to succinate- C i n the oyster v e n t r i c l e . A large portion of the l a b e l also accumulates i n malate. Alanine and the unknown compound, although produced i n smaller 14 amounts from aspartate-, C are produced i n approximately the same r a t i o as 14 14 from glucose- C. If aspartate- C(U) i s used and i f aspartate i s 14 metabolized equally to alanine and succinate, then the r a t i o of alanine- C : 14 14 succinate- C should be 3:4 as there i s a one i n four chance that the C-labelled atom w i l l be l o s t i n the decarboxylation of malate to pyruvate. However, the r a t i o of alanine: succinate seen i n Table I I i s much less than 26 3:4. I t appears that i n the oyster v e n t r i c l e , aspartate i s p r e f e r e n t i a l l y metabolized to succinate during anoxia while glucose i s p r e f e r e n t i a l l y metabolized to alanine. As fumarate reductase i s mitochondrial (Chen and Awapara,1969), i t appears that the carbon skeleton from aspartate can enter the mitochondria and be converted to succinate while carbon from glucose does mot enter the mitochondria as r e a d i l y . In order to balance redox and amino groups and to explain a 1:1 r a t i o of succinate to alanine from glucose, Hochachka et a l . (1973) (Figure 2) propose the simultaneous mobilization of glucose, aspartate, and glutamate. 14 However, very l i t t l e glutamate- C i s used by the anaerobic v e n t r i c l e (Table 14 I I I ) . Not only i s uptake of glutamate- C into the tissue very low, but of the l a b e l taken up, 85% of i t remains as unreacted gluamate. Via transamination to o(-ketoglutarate, the carbon skeleton of glutamate i s spread into the Krebs cycle intermediates, succinate and malate, and into the amino 14 acids aspartate and alanine. Most of the metabolized l a b e l from glutamate- C accumulates i n succinate, indicating succinate as the end-product of both reverse and forward functioning of the Krebs cycle during anoxia. However, 14 as very l i t t l e glutamate- C i s metabolized, i t can be seen that the forward functioning of the Krebs cycle from °(-ketoglutarate is- of minor importance during anaerobiosis. 14 Uptake and u t i l i z a t i o n of alanine- C by the anaerobic v e n t r i c l e i s low and t h i s i s to be expected when alanine i s one of the end-products of anaerobiosis. I f the net flow of carbon i s into the alanine pool,,then i t i s 14 l i k e l y that l a b e l l i n g of compounds from alanine- C i s due to randomization of the la b e l amongst compounds, such as direct precursors, that are clo s e l y linked to alanine. Table IV shows that the d i r e c t precursor of alanine, pyruvate, i s indeed labelled. An unknown compound (which by chromatography i n several systems has been shown to be the same unknown as that produced 27 14 14 from glucose- C or, aspartate- C) i s the only other l a b e l l e d compound 14 produced from alanine- C. This leads to the supposition that the unknown i s metabolically closely linked to alanine or pyruvate. Three Hour Incubations The res u l t s of experiments i n which v e n t r i c l e s were incubated, for three 14 hours with C-labelled glucose, aspartate, and gluaamate are presented i n 14 14 Tables V,VI, and VII. The uptake of glucose- C and aspartate- C i s approximately double the uptake of the one hour incubations while the 14 uptake of glutamate- C i s s i x times that of the one hour incubations. An average of zero, 24.4, and 82.1 percent of the i n t r a c e l l u l a r l a b e l i s 14 14 14 unreacted glucose- C, aspartate- C, and glutamate- C respectively. These numbers are almost i d e n t i c a l to those obtained for the one hour incubations. The d i s t r i b u t i o n of the lab e l i s also almost the same for the one and three 14 hour incubations. From gluoose- C, the major end-products are alanine and the unknown compound (Table V). Small amounts of aspartate and glutamate are 14 14 also labelled. The major accumulation of C from aspartate- C i s i n succinate (Table VI). There i s es s e n t i a l l y no difference i n the d i s t r i b u t i o n 14 of la b e l from aspartate- C amongst intermediates and end-products after 14 one or three hours incubation. Of the small amount of glutamate- C that i s mebalbihMzed, succinate i s again the major end-product formed and the d i s t r i b u t i o n of l a b e l i n other compounds i s es s e n t i a l l y the same after one and three hour.incubations. Increasing the length of the incubations only increases the amount of r a d i o a c t i v i t y metabolized. There i s no evidence that end-products or r a t i o s of end-products change with time at least during the early stages (up to three hours) of anaerobiosis. The incubation salines from the one and three hour experiments with 14 14 14 glucose- C, aspartate- C,and glutamate- C were fractionated and analysed Table V Anaerobic incubation of isolated ventricles with glucose- C(U) for 3 hours. Ventricle # avg.± s.e. Total dpm i n saline 1340100 1483400 1486300 + tissue % uptake of la b e l 29.2 38.3 41.0 into tissue % i n t r a c e l l u l a r l a b e l 0.0 0.0 0.0 i n glucose-14C % metabolized i n t r a -c e l l u l a r l a b e l i n : aspartate 1.5 0022 00'1'Ll glutamate 2.8 0.9 0.7 unknown compound 24.5 34.7 28.3 alanine 52.9 44.4 34.2 phosphorylated cpds. 7.0 3.9 6.0 malate 7.2 4.6 5.8 pyruvate 0.4 7.3 16.4 succinate 3.7 4.0 8.5 0.6 ± 0.8 1.5 + 1.1 29.2 ± 5.2 43.8 ± 9.4 5.6 ± 1.6 5.9 ± 1.3 8.0 ± 8.0 5.4 ± 2.7 00 Table VI Anaerobic incubation of isolated ventricles with aspartate- C for 3 hours. Ventricle # Total dpm i n saline + tissue % uptake of l a b e l into tissue % i n t r a c e l l u l a r l a b e l i n aspartate-14C % metabolized i n t r a -c e l l u l a r l a b e l i n : glutamate unknown compound alanine malate pyruvate succinate 1 1589000 16.9 2?. 8 331 16.8 26.5 11.5 1.0 41.0 2257500 31.5 27.1 4.7 13.1 22.6 6.0 2.7 50.9 3 1421400 24.2 20.4 2.0 19.7 22.7 7.2 5.7 42.9 4 1974500 9977 23.5 avg. ± s.e. 2266 18.6 14.3 8.5 5.2 50.9 ho 3.1 ± 1.2 17.1 ± 2.9 21.5 ± 5.2 8.3 ± 2.4 3.7 ± 2.2 46.4 + 5.2 Table VII Anaerobic incubation of isolated ventricles with glutamate- C(U) for 3 hours. Ventricle # Total dpm i n saline 1084900 1042200 + tissue % uptake of l a b e l 27.8 33.1 into tissue % i n t r a c e l l u l a r l a b e l 83.4 74.2 i n glutamate-14C % metabolized i n t r a -c e l l u l a r l a b e l i n : aspartate 15.8 6.5 other amino acids 26.3 16.4 alanine 11.4 21.4 malate 15.8 10.3 succinate 30.9 45.4 3 1345600 39.8 84.2 5544 21.8 8.2 8.4 56.3 4 1357100 24.9 86.4 H 29.7 12.7 6.4 47.4 avg. ± s.e. o 7.9 ± 5.4 23.6 ± 5.8 13.4 ± 5.6 10.2 ± 4.1 45.0 ± 10.5 31 14 for metabolized r a d i o a c t i v i t y i n the saline. From glucose- C, small amounts 14 of alanine- C were found i n the saline but the amount would only increase 14 5 the t o t a l percentage of C i n alanine by 2-3% , so i t was not included when 14 14 determining the percentage of C i n alanine. Small amounts of succinate- C 14 were found i n the salines after incubations with aspartate- C of glutamate-14 C but againe these were not included i n t o t a l l i n g the percentage of the C label xn succxnate. pH 1_ Experiments In v i v a , p a l l i a l f l u i d pH drops to 6.7-7.0 early i n anoxia (Eedlow,1974). The experiments already described were done with sea water at pH 7.8 ( the pH of sea water at room temperature). If the i n t r a c e l l u l a r pH equilibrated with or was altered by the pH of the sa l i n e , then the radiotracer experiments could show the unnatural re s u l t s of anaerobiosis at a non-physiological pH. Incubations of isolated v e n t r i c l e s were rerun i n sea water containing 10 mM imidazole and with the pH adjusted to 6.8S7.0. Tables VIII,IX, and X show 14 14 14 the results of incubations with glucose- C, aspartate- C,and glutamate- C respectively. For each of the tfaEee labelled compounds , the uptake, percentage u t i l i z a t i o n of i n t r a c e l l u l a r l a b e l , and d i s t r i b u t i o n of the lab e l i n intermediates and end-products i s similar to the res u l t s obtained at pH 7.8. 14 Glucose- C i s s t i l l metabolized mainly to alanine with very l i t t l e succinate formed (Table V I I I ) . There i s less of the unknown compound and more pyruvate 14 14 formed. At pH 7.0 about twice as much malate- C i s formed from aspartate- C as at pH 7.8 (Table IX). There i s a corresponding decrease i n the amount of 14 succinate- C formed. This may indicate an affect of pH on the enzymes catalysing succinate formation from malate. The pH had no noticeable effect 14 on the metabolism of glutamate- C (Table X). Although the pH of the p a l l i a l f l u i d of the oyster can f a l l below 7.0 during anaerobiosis, these experiments Table VIII Anaerobic incubation of isolated ventricles with glucose- C(U) for one hour i n sea water with pH adjusted to 7.0 Ventricle # 1 2 3 avg. ± s.e. Total dpm i n saline 1987100 1731100 + tissue % uptake of l a b e l 14.6 22.4 into tissue % i n t r a c e l l u l a r l a b e l 0.0 0.0 0.0 i n glucose-14C N> % metabolized -i n t r a -c e l l u l a r l a b e l i n : aspartate 0.0 0.5 0.4 0.3 ±0.3 gikfcamateompound 1.6 1.1 0.0 0.9 ± 0.5 unknown compound 28.3 12.2 18.1 19.5 ± 8.1 alanine 47.6 47.8 54.9 . 50.1 ± 4.2 phosphorylated cpds. 8.9 7.9 8.9 8.6 ± 0.5 malate 6.3 10.4 13.0 9.6 ± 2.9 pyruvate 6.6 17.9 . 2.3 8.9 ± 8.1 succinate 0.7 2=?1 2.6 1.8 ± 1.0 Table IX Anaerobic incubation of isolated ventricles with aspartate- C(U) for one hour i n sea water with pH adjusted to 7.0. Total dpm i n saline + tissue % uptake of l a b e l into tissue % i n t r a c e l l u a i l a b e l i n aspartate-14G % metabolized i n t r a -c e l l u l a r l a b e l i n : glutamate unknown compound alanine malate pyruvate succinate 1703400 8.9 29.3 1.5 4.5 13.1 51.0 0.0 29.9 Ventricle # 2 1700400 10.5 37.0 1.3 5.8 14.0 34.2 1.5 42.2 1685100 9.1 40.3 3.8 3.8 21.3 31.0 3.3 33.8 avg. ± s.e. 1.9 ± 0.8 4.7 ± 1.0 16.4 ± 10.8 38.7 ± 10.8 1.6 ± 1.7 35.3 ± 6.3 ( 14 Table X Anaerobic incubation of isolated ventricles with glutamate- C(U) for one hour, i n sea water with pH adjusted to 7.0. Total dpm i n saline + tissue % uptake of la b e l into tissue % i n t r a c e l l u l a r l a b e l i n glutamate-14C % metabolized i n t r a -c e l l u l a r l a b e l i n : aspartate other amino acids malate,«C-kga succinate 1 1538000 3.7 85.6 21.5 32.2 18.7 27.6 Ventricle # 2 1246900 11.0 86.2 14.4 37.4 6.1 42.1 1689100 6.6 88.9 8.2 34.2 6.8-51.2 avg. ± s.e. 14.7 ± 6.6 34.6 ± 2.6 10.4 ± 7.2 40.3 ± 11.9 35 do not indicate alterations i n the r a t i o s of end-products produced from 14 C-precursors that could be further altered by.lowering the pH even more. Contrary to the situation i n Rangia cuneata (Stokes and Awapara,1968), there i s no evidence that the oyster v e n t r i c l e produces succinate and alanine i n a 1:1 r a t i o from glucose-^C. Unknown Compound An unknown compound accounts for a peak of r a d i o a c t i v i t y on the amino acid chromatograms from incubations with glucose-^C, aspartate-^C, and 14 alanine- C. Chromatography i n several systems has shown that i t i s a single 14 compound and that the same unknown i s produced from the three C-precursors. The compound has not been i d e n t i f i e d , but several tests have been done i n attempts to i d e n t i f y i t . The compound binds to the Amberlite IR-120 H + column along with the amino acids and therefore has a net positive charge. The net positive charge i s probably due to a group containing nitrogen,for example amino or guanidino. On paper chromatography, the unknown peak of r a d i o a c t i v i t y can be separated i n at least one solvent system from the following : aspartate,glutamate, glutamine, alanine («&y3 ) , serine, glycine, proline, asparagine, taurine, phenylalanine, cystine, cysteine, cysteic acid, tryptophan, threonine, v a l i n e , tyrosine, isoleucine, l y s i n e , leucine, h i s t i d i n e , methionine, arginine, c i t r u l l i n e , ornithine, and octopine. From 14 alanine- C, pyruvate arid the .unknown are the only products formed (Table IV). 14 From glucose- C, 55% of the counts are i n alanine, 30% i n the unknown 14 (Table I) and from aspartate- C 17% of the counts are i n alanine, 11% i n the unknown (Table I I ) . The unknown does not seem to be produced from glutamate-14 C. There seems to be a c&fase relationship between the unknown and alanine or pyruvate. The unknown, therefore, seems to be a product of the end of gl y c o l y s i s rather than of Krebs cycle functioning. The compound was stable 36 to acid hydrolysis (6N HCl for 22 hrs. at 110°C) and was therefore not a small peptide. A sample of the unknown was p u r i f i e d by sequential chromatography, elution,=and rechromatography of the radioactive peak i n three different solvent systems such that i t was separated from a l l of the compounds l i s t e d above/ The p u r i f i e d extract was ninhydrin p o s i t i v e , but subsequent analysis on an amino acid analyser showed contamination by alanine and taurine. No identifyable peak was found on the,amino acid analyser run although the column that separates basic amino acids was not run. The compound, therefore, probably does not have a free amino group. Metabolite Experiments In Vitr o Measurements 14 Having determined the fate of various C-labelled compounds i n the anaerobic v e n t r i c l e , i t was of interest bo correlate these findings with changes i n the concentrations of various intermediary metabolites during anaerobiosis. To ensure that aerobic and anaerobic concentrations of. metabolites were compatible, v e n t r i c l e s were cut i n half and one half frozen immediately i n l i q u i d nitrogen while the other half was incubated anaerobically (at pH 7.8) for one hour. Table XI shows the-results from measurements of the levels of metabolites i n four groups of aerobic vs. anaerobic h a l f - v e n t r i c l e s . The concentrations of oxaloacetate, pyruvate, and ot-ketoglutarate are low and do not change s i g n i f i c a n t l y during anaerobiosis. The concentration of oxaloacetate i n various animal tissues ranges between 2-10^M (Bergmeyer,1974). The high levels determined here are probably due to errors involved i n working at the lower l i m i t of s e n s i t i v i t y of the spectrophotometric assay. Although not s i g n i f i c a n t by the paired difference t e s t , L-lactate concentrations increased from 0.31-to 0.4+iflMM on the average. In comparisone. to the increases i n succinate and alanine, L-lactate i s a minor end-product of anaerobiosis i n oysters. De Zwaan and Zandee (1972) Table XI Metabolite concentrations i n ventricles before and after anaerobiosis i n v i t r o . Concentration (mM) 1 2 3 4 avg. aer. anaer. aer. anaer. aer. anaer. aer. anaer. aer. anaer. Oxaloacetate 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Citrate 2.1 0.6 2.4 0.2 1.6 1.8 2.7 1.5 2.3 1.2 *C-ket oglut ar at e 0.0 0.1 0.0 0.0 0.1 0.2 0.0 0.0 - -Pyruvate 0.2 0.1 0.2 0.1 0.1 0.1 0.1 0.2 0.2 0.1 L-lactate 0.2 0.4 0.4 0.4 0.1 0.3 0.5 0.6 0.3 0.4 Malate 0.3 0.6 0.2 0.6 0.2 0.8 0.3 0.6 0.3 0.6 Succinate 1.6 3.5 ©.9 3.-2 0.1 3.6 0.1 3.3 0.7 3.4 ** Aspartate 12.9 1.0 11.0 0.6 12.9 1.9 13.9 2.0 12.7 1.4 * * Glutamate 11.2 7.5 9.7 7.1 8.4 5.9 11.4 7.3 10.2 6.9 ** Alanine 7.4 12.7 3.2 10.0 8.6 12.8 7.8 12.3 6.7 11.98* * S t a t i s t i c a l l y s i g n i f i c a n t by the paired difference test ( P<0.05) (Snedecor and Cochran,1967) 38 found that the concentration of L-lactate i n the muscle (O.Z^mole/gm wet wt. or approx..0.25mM) of Mytilus edulis did not change during 48 hours of anoxia. D-lactate, however, increased from 0.11 to 0.44yumoles/gm wet wt.. D-lactate was not measured i n the oyster v e n t r i c l e but i t i s u n l i k e l y that D 6r L-14 lactate are major end-products of anaerobiosis as no l a c t a t e - C was formed 14 from any of the C-precursors used i n the radiotracer experiments. The concentration of c i t r a t e decreased i n anoxia. The c i t r a t e pool could be depleted to provide a source of o(-ketoglutarate for glutamate or succinate formation. Succinate and alanine are the major end-products of anaerobiosis i n the oyster as i n other i n t e r t i d a l bivalves (Stokes and Awapara,1968; de Zwaan and Zandee,1972). After one hour of anoxia, the concentration of succinate i n the isolated v e n t r i c l e increased by 2.7 mM while the concentration of alanine increased by 5.2 mM, double the increase i n succinate. Malate pools double i n size. As Figure 1 shows, malate i s a key branchpoint i n the route to either succinate or alanine (Hochachka et al.,1973). Aspartic acid i s a substrate of anaerobic metabolism i n the oyster. This i s now confirmed by two methods : the depletion of i n t r a c e l l u l a r aspartate pools (Table XI) and 14 the u t i l i z a t i o n of aspartate- C (Table I I ) . The isolated oyster v e n t r i c l e undergoes a large depletion of the aspartate pool (12.7 mM to 1.4 mM) while the concentration of glutamate decreases by only 3 mM during one hour of anaerobiosis. S i m i l a r i l y , Dupaul and Webb (1971) showed a decrease i n the i n t r a c e l l u l a r pools of aspartate, but not of glutamate, i n the isolated anaerobic g i l l of Mya arenaria. In Vivo Measurements The effecgs of anaerobiosis i n vivo on the metabolite concentrations i n the v e n t r i c l e are shown i n Table XII. The pattern of changes i s the same i n 39 e XII Metabolite concentrations i n the v e n t r i c l e i n vivo before and after anaerobiosis. Concentration (mM) Aerobic Anaerobic 1 2 Oxaloacetate 0.1 0.0 0.1 Citrate 1.3 2.2 1.6 c(-ketoglutarate 0.1 0.0 0.0 Malate 0.1 0.3 0.3 Pyruvate 0.1 0.1 0.2 Lactate (L) 0.0 0.1 .0.6 Succinate 0.2 3.5 2.8 Aspartate 10.5 4.6 6.2 Alanmme 8.8 16.8 19.5 Glutamate 10.1 12.9 12.7 40 these i n vivo experiments as i n the i n v i t r o experiments. The concentrations of oxaloacetate, pyruvate, oC-ketoglutarate, and c i t r a t e do not change over the four hours of anoxia. As i n the isolated system, malate and L-lactate levels increase somewhat. Aspartate i s depleted (a decrease of 5 mMj)) but glutamate levels do not decrease. Succinate and alanine accumulate as the end-products and as i n the isolated system, the increase i n alanine concentrations (+ 9.4 mM) i s greater than the increase i n succinate (+ 2.9 mM). Unlike the situation i n Rangia (Stokes and Awapara,1968) and i n Mytilus (de Zwaan and Zandee,1972), the oyster v e n t r i c l e does not produce succinate and alanine i n a one to one r a t i o during anoxia. The decrease i n aspartate concentrations i n the isolated v e n t r i c l e after one hour as much greater than the decrease i n aspartate concentrations i n the ve n t r i c l e i n vivo after 4 hours of anaerobiosis (Table XI vs. Table X I I ) . There are two possible explanations for t h i s . Small amounts of aspartate, glutamate, and alanine could be detected by enzyme assay i n the incubation saline. Some leakage of amino acids from the tissue could, therefore, have occured. This would also account for-, the decrease i n glutamate detected i n v i t r o but not i n vivo. Since aspartate i s nearly t o t a l l y depleted after one hour of anaerobiosis jin v i t r o , i t i s possibles that i n vivo aspartate of some other substrate could be supplied to the v e n t r i c l e i n the blood. When cut off from an exogenous supply of substrate, the v e n t r i c l e rapidly depletes i t s endogenous supply. Amino Acid Analyses Table XIII shows the concentrations of some neutral and acidic amino acids i n the v e n t r i c l e of the oyster under various conditions. Measureable amounts of cysteic acid, the precursor of taurine,. were found but th i s amino acid was not quantitated. The concentrations of taurine were high (greater Table X I I I 41 In t r a c e l l u l a r free amino acids i n the oyster v e n t r i c l e before and after anaerobiosis. Concentration (mM) i n v i t r o i n vivo before after before after Aspartate 14.8 1.7 11.7 5.2 Threonine 0.7 0.7 0.4 Serine 3.5 2.8 2.4 1.9 Glutamate 8.0 7.0 10.2 15.2 Proline 1.1 0.9 0.4 -Glycine 12.9 11.4 11.6 12.5 Alanine 9.0 15.9 9.5 21.1 Concentrations were determined using the amino acid analyser. The i n v i t r o measurements correspond to group 3 on Table XI. The i n vivo measumeasurements correspond to the aerobic and anaerobic #1 groups of Table XII. 42 than 20 mM). As i n JC. virginica-- (Lynch and Wood ,1966) and other marine bivalves (Schoffeniels and Gilles,1972), taurine i s the major, amino acid contributing to i n t r a c e l l u l a r osmotic pressure. The concentrations of aspartate, glutamate, and alanine are i n close agreement with those determined by enzymic methods. The concentrations of threonine, proline, and glycine did not change during anaerobiosis. The concentration of serine decreased s l i g h t l y . Other neutral and acidic amino acids were i n concentrations too low to be detected i n a 10 lamda sample. There i s no evidence for anaerobic u t i l i z a t i o n of amino acids other than aspartate. Adenylates Table XIV shows the aerobic and anaerobic concentrations of the adenylates (ATP,ADP,AMP) i n the v e n t r i c l e when anoxia i s carried out both i n vivo and i n v i t r o . The t o t a l adenylate pool size averages 1.85 mM. The size of the adenylate pool varies depending on the tissue type. In the r a t , the pool size i s approzimately 7.5 mM i n skeletal muscle, 4.7 mM i n l i v e r , and 1 mM i n mammary gland (Bergmeyer,1974). The adductor of the oyster, a str i a t e d muscle, had an adenylate pool size of approximately 6 mM ( C o l l i c u t t , unpublished). The t o t a l pool size remains constant during anoxia while the ra t i o s of the adenylates change. Aerobically, ATP makes up 47% of the t o t a l adenylates. In the isolated v e n t r i c l e , ATP drops to 20% of the t o t a l pool during anaerobiosis, while i n vivo 36% of the adenylates i s ATP after 4 hours. As ATP concentrations decrease, both ADP and AMP concentrations increase. The energy charge (ATP + SsADP)/(ATP+ ADP + AMP), a parameter which remains constant i n many c e l l types (Atkinson,1968) does not, therefore, remaine constant i n the oyster during anoxia. Similar changes i n the make-up of the adenylate pool occur during anaerobiosis i n Mytilus (Zs-Nagy and Ermini, 1972). ATP drops from 18% to 8% of the t o t a l pool during 24 hours of e XIV The concentrations of the adenylates i n the v e n t r i c l e before and after anaerobiosis jm v i t r o and i n vivo. Concentration (mM) i n v i t r o i n vivo 1 2 aerobic anaerobic aerobic anaerobic aerobic anaerobic ATP 0.60 (35) 0.44 (18) 0.85 (48) 0.37 (23) 0.89 (56) 0.69 (36) ADP 0.84 (50) 1.65 (66) 0.73 (41) 0.83 (52) 0.63 (39) 0.88 (46) AME 0.26 (15) 0.40 (16) 0.19 (11) 0.40 (25) 0.07 (5) 0.34 (18) t o t a l 1.70 adenylates 2.49 1.77 1.60 1.59 1.91 The i n v i t r o groups correspond to groups 3 and 4 on Table XI. The i n vivo group corresponds to Table XII i n which the values for the anaerobic groups have been averaged. The numbers i n brackets are the percentage of the t o t a l adenylate pool. 44 anaerobiosis. The fresh water clam, Anodonta, can,however, maintain a constant l e v e l of ATP during several days of anoxia (Zs-Nagy, 1973). CHAPTER IV DISCUSSION 45 The end-products of energy metabolism during anaerobiosis i n i n t e r t i d a l bivalves are succinate and alanine. This has now been demonstrated i n several species including.'the oyster, Crassostrea gigas (this study), the clam, Rangia cuneata (Stokes and Awapara,1968), the sea mussel, Mytilus  edulis (de Zwaan and Zandee,1972), Mya arenaria (Dupaul and Webb,1971), Mercenaria mercenaria (Crenshaw and Neff,1969), and Modiolus demissus (Malanga and Aiello,1972). However, although the end result of anaerobiosis i s the same i n a wide variety of bivalves, the pathways u t i l i z e d and the contributions made by various substrates may vary between animals and tissues. In Rangia mantle (Stokes and Awapara, 1968) the r a t i o of alanine-^C : 14 14 succinate- C produced from glucose- C i s 1:1 while i n Mytilus (de Zwaan and van Marrewijk,1973a) the r a t i o i s 2:1. In the oyster v e n t r i c l e the si t u a t i o n 14 i s even more extreme (Table I ) . Alanine- C i s the major end-product of 14 14 anaerobic u t i l i z a t i o n of glucose- C. The rafcio of alanine- C : succinate-14 C i s about 20:1. The pathway from glucose to alanine can be e n t i r e l y cytoplasmic. I t consists of gl y c o l y s i s wihh a terminal transamination b£ pyruvate to alanine (Figure 1) instead of a terminal reduction as i s the case i n lactate production. The l a s t enzyme i n the pathway to succinate i s located solely i n 14 the mitochondria (Chen and Awapara,1969). As very l i t t l e succinate- C i s 14 formed when glucose- C i s the precursor, i t can be postulated that i n the oyster v e n t r i c l e , the trioses foamied i n g l y c o l y s i s do not enter the mitochondria i n anoxia. Hochachka et al.(1973) propose that glucose carbon flows into alanine anaerobically v i a the malate pool (Figure 1). This explains how equimolar amounts of succinate and alanine can be formed from glucose. However, i f alanine i s the only product of glucose metabolism,itseems un l i k e l y that pyruvate should be formed v i a the circuitous route involving PEP 46 carboxykinase, malate dehydrogenase, and malic enzyme. I t i s probable, therefore, that pyruvate kinase i s s t i l l functional i n the v e n t r i c l e anaerobically. In the adductor, pH and alanine are important controls of pyruvate kinase and PEP carboxykinase and the drop i n pH and increase i n alanine concentrations that accompany anoxia are thought to "turn o f f " pyruvate kinase and "turn on" PEP carboxykinase (Hochachka et al.,1973).The production 14 14 14 of only alanine- C and not succinate- C from glucose- C during the incubations of ven t r i c l e s i n sea water at pH 7.8 could be due to the lack of pH i n h i b i t i o n of pyruvate kinase and of pH activation of PEP carboxykinase (assuming i n t r a c e l l u l a r and extsacellular pH's e q u i l i b r a t e ) . However, when incubations were carried out i n saline as pH 7.0, there was no difference i n 14 the pattern of end-products formed from glucose- C (Table I vs. Table V I I I ) . The scheme of Hochachka et_ a l . (1973) couples the u t i l i z a t i o n of the amino acids aspartate and glutamate to the oxidation of glucose. The u t i l i z a t i o n of aspartate as an energy source i n the anaerobic v e n t r i c l e has been demonstrated i n two ways : a) by the anaerobic u t i l i s a t i o n of aspartate-14 C and the production of the anaerobic end-produts succinate and alanine from i t TjSables II,VI, and IX) b) by demonstrating a decrease i n i n t r a c e l l u l a r aspartate concentrations after periods of anaerobiosis (Tables XI,XII, and X I I I ) . Figure 2 predicts that i f the r a t i o of succinate : alanine 14 produced from glucose i s 1:1, then the r a t i o w i l l be 1:1 from aspartate- C, due to mixing of carbon from both aspartate and glucose i n the common malate 14 pool. However, the oyster v e n t r i c l e p r e f e r e n t i a l l y makes alanine- C from 14 14 glucose- C and as Table I I shows, aspartate- C i s p r e f e r e n t i a l l y 14 14 14 metabolized to succinate- C. The r a t i o of succinate- C : alanine- C i s 3:1 instead of 4:3 as would be predicted i f equal amounts of succinate and alanine 47 were formed from aspartate. Aspartate can be ea s i l y introduced into intermediary metabolism by transamination to oxaloacetate. The metabolism of aspartate to the l e v e l of malate occurs i n the cytoplasm as oxaloacetate cannot traverse the mitochondrial membrane (Scrutton and Utter,1968).TEe conversion of malate to succinate i n the mitochondria most l i k e l y generates an ATP at the fumarate reductase step as i s the case i n other anoxia-tolerating invertebrates (Hammen,1975; Seidman and Entner,1961). The metabolism of one aspartate to one succinate generates one NAD+ and one oxidized flavoprotein ( F P o x ) • This i s equivalent to regenerating the two NADH formed i n the gly c o l y s i s of one glucose (Figure 2). As the fumarate reductase step i s located solely i n the mitochondria, i t seems possible that i n the oyster v e n t r i c l e anaerobic cytoplasmic production of NADH can be balanced by mitochondrial u t i l i z a t i o n of NADH. This requires that reducing equivalents be transferred across the mitochondria. The mitochondria are not permeable to NADH and therefore a shuttle system must carry reducing equivalents from the cytoplasm to the mitochondria. In mammals, the aspartate-malate shuttle i s used (LaNoue and Williamson,1971) while i n insect f l i g h t muscle the °<-glycero-phosphate— dihydroxyacetone-phosphate shuttle i s used.(Hochachka and Somero,1973). 14 14 Some alanine- C i s produced from aspartate- C as the precursor. This can be accomplished through the functioning of malic enzyme and alanine aminotransferase. Dupaul and Webb (1971) suggest the presence of an aspartate decarboxylase that would d i r e c t l y produce alanine from aspartate but no good evidence for t h i s enzyme has been demonstrated i n bivalves. Malic enzyme has been detected i n the oyster adductor (Mustafa and Hochachka,1973a) and i n the tissues of Mytilus edulis (deZwaan and van Marrewijk,1973b). The properties of the enzyme poise i t for anaerobic function i n theddirection of 48 pyruvate formation (Mustafa and Hochachka,1973a). Although i t i s u n l i k e l y that i n the oyster v e n t r i c l e malic enzyme plays a r o l e i n metabolizing carbon derived from glucose, i t does seem to be functional i n converting some of the carbon from aspartate ( and perhaps from other dicarboxylic acids) into pyruvate. The amino acid,glutamate, l i k e aspartate, i s metabolically closely linked to the Krebs cycle. After deamination, the carbon skeleton feeds d i r e c t l y into the Krebs cycle. Also l i k e aspartate, the i n t r a c e l l u l a r concentration of glutamate i s the oyster v e n t r i c l e i s high (Table XI) and glutamate would therefore be a good substrate for metabolism. Hochachka et^ a l . (1973) propose glutamate as an alternate energy source i n anoxia and show how the integrated metabolism of glucose, aspat£aee,and glutamate can produce a balanced metabolic scheme for anaerobiosis. However, i n comparison to aspartate, the anaerobic u t i l i z a t i o n of glutamate i n the v e n t r i c l e i s minimal (Tables III , V I I and X). The v e n t r i c l e i s capable of taking up 14 glutamate but the glutamate- C simply enters the glutamate pool. Of the glutamate that i s metabolized, the largest accumulation of l a b e l i s i n succinate. Measurements of i n t r a c e l l u l a r glutamate concentrations confirm the tracer studies. Tables XI,XII, and X I I I show that glutamate i s either not depleted or depleted to a small extent p a r t i c u l a r i l y i n comparison to aspartate. Forward functioning of the Krebs cycle from <X-ketoglutarate to succinate i s therefore minimal during anaerobiosis and the potential for productinn of GTP at the succinyl thiokinase step cannot be u t i l i z e d . 14 14 However, the formation of succinate-. C from glutamate- C, even i n small amounts, serves to show that succinate i s the sink for a l l carbon flow (whether forward of reverse) i n the Krebs cycle during anoxia. Although glutamate i s not an anaerobic energy source, the glutamate 49 pool does have an anaerobic function. The accumulation of alanine requires amino groups and the u t i l i z a t i o n of aspartate produces amino groups; Transfer of amino groups from aspartate to alanine occurs through the combined functioning of the two enzymes aspartate aminotranferase and alanine aminotransferase. Glutamate i s the intermediate amino c a r r i e r i n t h i s exchange. Both the aminotransferases are i n high concentrations i n the tissues of the oyster (Hammen,1969) and also i n other i n t e r t i d a l bivalves (Dupaul and Webb,1975). The major r o l e of the glutamate pool during anoxia, as envisioned by t h i s study, i s i n the transfer of amino groups. The radiotracer and metabolite data i n t h i s thesis do not f i t exactly with the model of anaerobic metabolism proposed by Hochachka and Mustafa (1972). The model i s based on the known enzymes and the properties of these enzymes i n the adductor muscle. A discussion of the properties of heart vs. muscle enzymes and, i n p a r t i c u l a r , what i s known about the properties of oyster v e n t r i c l e enzymes, w i l l aid i n interpreting the data and proposing a l t e r a t i o n ^ to the model of anaerobic metabolism to better suit i t to the metabolism of the v e n t r i c l e . Skeletal muscle i s characterized by i t s capacity for anaerobic g l y c o l y t i c work. This tissue has a much higher t i t r e of g l y c o l y t i c enzymes than heart. For example, the s p e c i f i c a c t i v i t y of pyruvate kinase i n rat skeletal muscle i s 780 units/gm wet wt. while i n heart i t i s 170 units/gm wet wt..(Scrutton and Utter,1968). The properties of the enzymes are such that the tissue can rapidly switch from low to maximal g l y c o l y t i c a c t i v i t y i n response to the energy demands for muscle work. This i s accomplished through Ca activation of phosphorylase kinase, release of ATP i n h i b i t i o n of phosphofructokinase (PFK) and pyruvate kinase, ADP and AMP activation of PFK, product activation of PFK, and feed-forward activation of pyruvate 50 kinase by fructose-l,6-diphosphate (FDP) (Hochachka and Somero,1973). The vertebrate heart has a much higher capacity for aerobic work. The tissue i s well supplied with mitochondria and has a high t i t r e of Krebs cycle enzymes. In p a t t i c u l a r , c i t r a t e synthase i s present i n much higher a c t i v i t i e s than i n skeletal muscle. Increased demand for energy generally results i n increased fl u x of substrate through the Krebs cycle but i f forced to do anaerobic work, the heart speeds up glycolysis by activating PFK i n a manner similar to that found i n muscle (Williamson,1966). The bivalve v e n t r i c l e i s a highly aerobic tissue and i s very sensitive to changes i n pO^ (Brand and Roberts, 1973; Bayne,1971). The tissue has a good supply of mitochondria (Irisawa et al.,1969) unlike the adductor which has few (Mustafa and Hochachka,1973a) and may depend on g l y c o l y t i c work for much of i t s energy even i n the presence of oxygen. The v e n t r i c l e must therefore make a true aerobic-anaerobmc t r a n s i t i o n upon s h e l l closure. The spe c i f i c a c t i v i t i e s of some of the g l y c o l y t i c and Krebs cycle enzymes have been determined for the v e n t r i c l e and adductor of the oyster (Fields, unpublished) (Table X¥). Ci t r a t e synthase levels are a good indication of the capacity for aerobic work. The oyster v e n t r i c l e has 10 times the amount of c i t r a t e sythase as the adductor. The c i t r a t e concentration i n the ve n t r i c l e (approx. 2 mM) (Tables XI,XII) i s also much higher than that found i n the adductor (approx. 0.4 mM)(Collicuttt,unpublished). The sp e c i f i c a c t i v i t y of malate dehydrogenase, another Krebs cycle enzyme, i s also higher i n the v e n t r i c l e . Pyruvate kinase and PFK a c t i v i t i e s are good indicators of the g l y c o l y t i c capacity of the tissue (Hochachka and Somero,1973). Pyruvate kinase a c t i v i t i e s are much higher i n the adductor, p a r t i c u l a r i l y at neutral (aerobic) pH. In agreement with the work of Mustafa and Hochachka (1971), pyruvate kinase a c t i v i t y i s decreased by decreasing'? the pH. Mustafa and 51 Table XV The a c t i v i t i e s of some enzymes i n the adductor muscle and v e n t r i c l e of the oyster, gigas. Specific A c t i v i t y (units/gm wet wt.) Aspartate aminotransferase Alanine aminotransfErase PEP carboxykinase Malic enzyme MalatMalate dehydrogenase Citrate synthase Pyruvate kinase pH 6.7 7.4 8.35 Adductor 10.0 14.9 0.7 0.9 125.0 1.2 6.7 (18.9) 15.4 (21.3) 8.1 (9.5) Ventricle 27.0 10.5 undetectable 0.9 311.0 11.6 2.3 (15.6) 4.9 (11.5) 3.4 (5.3) Numbers i n brackets represent s p e c i f i c a c t i v i t i e s i n the presence of 100//M fructose-l,6-diphosphate. Data from Fields (unpublished). 52 Hochachka (1971) found that FDP activated adductor pyruvate kinase, shifted i t s pH optimum to 7.0 from 8.5, and could override the i n h i b i t o r y effects of alanine and ATP. The percentage activation of pyruvate kinase by FDP i s not only much greater i n the v e n t r i c l e than i n the adductor, but FDP s h i f t s the maximal a c t i v i t y to pH 6.7 (andanaerobic pH). During the aerobic-anaerobic t r a n s i t i o n , FDP levels would l i k e l y r i s e and speed the PFK and pyruvate kinase reactions i n order to gear up gl y c o l y s i s . ATP, an i n h i b i t o r of pyruvate kinase, decreases i n concentration i n anoxia (Table XIV). Although the interacting effects of ATP, FDP, and alanine on v e n t r i c l e pyruvate kinase at low pH are not known, the interacting effects of pH and FDP indicate that v e n t r i c l e pyruvate kinase i s active anaerobically. Table XV shows that PEP carboxykinase, i f present, occurs i n low a c t i v i t i e s i n the v e n t r i c l e . Thus the t r a n s i t i o n from aerobic pyruvate kinase to anaerobic PEP carboxykinase u t i l i z a t i o n of PEP (Figure 1,inset) can take place i n the adductor but probably not i n the v e n t r i c l e . PEP can be metabolized only by pyruvate kinase i n the v e n t r i c l e . Malic enzyme i s present i n both tissues, as well as both aspartate and alanine aminotransferase. From these data, plus the radiotracer and metabolite data, a modified scheme representing anaerobic metabolism i n the v e n t r i c l e can be proposed. This scheme i s presented i n Figure 4. Carbon from glucose i s metabolized to alanine. The aerobic-anaerobic branchpoint for glucose carbon i s not at PEP but at pyruvate. Aerobically, pyruvate i s metabolized by the Krebs cycle, anaerobically, i t i s transaminated to alanine. The conversion of one mole of glucose to two moles of alanine generates two moles of NADH. Simultaneously one mole of aspartate / i s mobilized to one mole of succinate and i n the process generates one mole of NAD and one mole of FP . This balances the NADH produced i n g l y c o l y s i s . 53a Figure 4 Proposed scheme for anaerobic metabolism i n the v e n t r i c l e of the oyster. 53b glucose-6-P triose-P NAD + -NADH + H H 1,3-DPG 3-PG V PEP •ADP + P. i ATP ADP + P, ATP pyruvate aspartate glutamate • • ,.• o(-ketoglutarate h—> ot-ketoglutarate «*' .. glutamate alanine oxaloacetate NADH + H NAD +<—-Cytoplasm malate Mitochondrion NADH + H + NAD+ FP succinate ^ — FP red fumarate ATP ADP + P. 54 The transfer of amino groups from aspartate to alanine i s accomplished through the coupling of the alanine and aspartate aminotransferases, with the glutamate pool playing a central role i n t h i s transfer. For every mole of aspartate that i s mobilized to succinate, one mole of ATP i s produced at the fumarate reductase step. This i s the energetic advantage of the scheme over gl y c o l y s i s alone. If a l l glucose carbon flows into alanine and i f a l l aspartate carbon fshows into succinate, then the simultaneous mobilization of one mole of glucose and one mole of aspartate produces two moles of alanine and one mole of succinate and the system remains i n redox balance. Tables XI and XII do indeed show that the anoxic increase i n alanine i n the v e n t r i c l e i s 2-3 times greater than the increase i n succinate. In summary, the scheme proposed i n Figure 4 a) couples carbohydrate adn amino acid metabolism b) accounts for the anaerobic depletion of aspartate but not glutamate c) accounts for the end-products of glucose and aspartate metabolism as detected by radiotracers d) accounts for the r a t i o of alanine : succinate produced i n the ventricle.as determined i n the metabolite studies e) shows the energetic advantage over g l y c o l y s i s alone f) shows how redox can be balanced g) u t i l i z e s only enzymes known to exist i n the v e n t r i c l e . Two problems of t h i s scheme should be discussed : 1) If one aspartate i s mobilized per two alanine produced, then the transfer of amino groups from aspartate to alanine can account for only one-half of the alanine produced. 14 2) approximately 30% of the r a d i o a c t i v i t y from glucose- C appears as an unident i f ied compound. The _in vivo data '(Table XII) show that the depletion of aspartate (approx. 5 mM) i s greater than the increase i n succinate (approx. 3 mM) but less than the increase i n alanine (approx. 9 mM). The radiotracer studies 55 i 14 showed that aspartate- C was metabolized to malate, alanine, and the unkown compound as we l l as to succinate. The depletion of aspartate can account for more than half of the amino groups needed to synthesize alanine. Another source of amino groups i s s t i l l needed. The amino acid analyses (Table XIII) showed no change i n those neutral and a c i d i c amino acids present i n appreciable (>0.5 mM) quantities with the possible exception of serine. However, the decrease i n serine i s s t i l l not of the magnitude needed. Possibly a basic amino acid i s the source. The problem of a source of amino groups i s also encountered during adaptation to high s a l i n i t i e s when a net synthesis of amino acids occurs. In the isolated v e n t r i c l e 66 Modiolus demissus exposed to high s a l i n i t i e s , alanine and glycine increase over time u n t i l adaptation (as evidenced by the return of mechanical a c t i v i t y ) i s complete.(Baginski and Pierce,1975). Dupaul and Webb (1971) showed a small decrease i n aspartate p o l l s accompanying alanine accumulation during high s a l i n i t y stress i n the isolated g i l l of Mya arenaria. Baginski and Pierce (1975) found no decrease i n aspartate of any other amino acid i n their experiments. The glutamate dehydrogenase reaction can f i x ammonia into an amino group and t h i s can then be transfered to other amino acids. This enzyme functions i n some crabs during s a l i n i t y stress (Schoffeniels and Gilles,1970) but i t .cannon be detected i n i n t e r t i d a l bivalves (Campbell arid Bishop,1970). The degradation of special proteins, whose function i s simply to store amino acids, i s interesting but u n l i k e l y . Currently other ammonia f i x i n g reactions are being studied totdetermine i f they are present i n marine invertebrates and i f they can play a role i n generating amino acids i n s a l i n i t y and anaerobic stress (Bishop, pers. comm.). The AMP deaminase reaction i s .one that i s being studied. The v e n t r i c l e of the oyster makes a t h i r d anaerobic end-product. This i s 56 14 the unknown compound that i s labelled p r i n c i p a l l y from glucose- C. Due to i t s behaviour on the Amberlite H + column, the compound probably has some kind of nitrogen group i n i t , and the l a b e l l i n g pattern indicates that i t i s metabolically closely linked to alanine or pyruvate. Some molluscs produce the compound octopine during anaerobic stress (Regnouf and van Thoai,1970; Gade,1974; F l o r k i n and Bricteux-Gregoire,1972) p a r t i c u l a r i l y when the stress i s due to rapid muscle work and an i n a b i l i t y to provide the tissues with s u f f i c i e n t oxygen. Octopine replaces lactate as the end-product of rapid muscle gl y c o l y s i s i n the mantle of octopus and squid (Hochachka et^ a l . , unpublished), i n the mantle of Sepia (van Thoai and Robin, 196b.) , and i n the adductor of Pecten maximus (van Thoai et al.,1969). The enzyme octopine dehydrogenase catalyses the condensation : pyruvate + arginine + NADH + H + ^ ocjropine + NAD+ The synthesis of octopine serves a three-fold purpose : 1) disposal 66 pyruvate, the end-product of gl y c o l y s i s 2) drawing off the arginine a that accumulates as the phosphagen, arginine-phosphate, breaks down 3) regeneration of the NADH produced i n gl y c o l y s i s . The production of the unknown may l i k e l y regenerate NADH for the following reason..Between 2 and 3 times as much alanine i s produced i n the oyster v e n t r i c l e as succinate anaerobically. The reactions leading to alanine either from g l y c o l y s i s or from malate generate NADH or NADPH. The reactions producing succinate regenerate NAD+. Therefore with more alanine made than succinate, the system w i l l p i l e up NADH unless an alternate reaction i s present to regenerate i t . Although there are many reactions that could regenerate NADH without producing a compound that i s 14 also labelled by C from glucose, aspartate, or alanine, i t i s tempting to speculate that the presence of large amounts of an unknown compound during anaerobiosis (a situation i n which the tissue i s under energy stress) i s l i k e l y 57 to serve some useful purpose such as redox balance. The anoxic v e n t r i c l e i s not able to maintaine the aerobic concentrations of ATP (Table XIV). ATP concentrations decreases and ADP and AMP b u i l d up. A similar change takes place i n anoxia i n Mytilus (Zs-Nagy and Ermini,1972). The decrease i n ATP can probably be tolerated as the demand for energy also decreases i n anoxia (ex. heart rate decreases at least 50% (Bayne,1971)). The adenylates are effectors of several enzymes i n intermediary metabolism including PFK, pyruvate kinase, c i t r a t e synthase, arid NAD-1inked i s o c i t r a t e dehydrogenase.(Lehninger,1970). Williamson (1966) showed that the transient^ increases i n AMP and ADP and decrease i n ATP that accompany the i n i t i a t i o n of anoxia i n rat heart rapidly activated PFK thereby increasing g l y c o l y t i c f l u x . Oyster adductor PFK i s inhibited by ATP and arginine-phosphate. Arginine-phosphate i n h i b i t i o n i s greatly reduced as pH i s lowered (Storey, unpublished). ATP also i n h i b i t s adductor pyruvate kinase (Mustafa and Hochachka,1971). Decreasing ATP and arginine-phosphate levels that accompany the onset of anaerobiosis would therefore increase the a c t i v i t y of PFK and pyruvate kinase and speed up f l u x through g l y c o l y s i s . Onreeturn to an aerobic environment, the heart beat rapidly increases, sometimes overshooting the normal l e v e l s (Brand and Roberts,1973). The Krebs cycle can return to functioning i n the forward dire c t i o n and g l y c o l y t i c f l u x can be slowed down. NAD^linked i s o c i t r a t e dehydrogenase i s an important control s i t e i n the Krebs cycle. The enzyme i s regulated by ADP activation. ATP and NADH are i n h i b i t o r y (Lehninger,1970). The anoxic changes i n the adenylate pool w i l l activate the enzyme immediately upon return to aerobiosis. Anaerobic end-products can be e a s i l y remetabolized. Good aerobic tissues can e a s i l y metabolized both succinate and pyruvate. Malanga and A i e l l o (1972) showed that the g i l l s of Mytilus and Modiolus rapidly oxidize exogenous 58 succinate. After periods of anoxia, oxygen consumption i n isolated Modiolus g i l l s i s stimulated above that of aerobic controls and t h i s stimulation can be blocked by malonate ( a s p e c i f i c i n h i b i t o r of succinate dehydrogenase). Aerobic tissues such as the heart and g i l l s could oxidize the succinate and alanine produced by less aerobic tissues such as the adductor. As the Krebs cycle resumes i t s forward functioning, succinate can be completely oxidized, or reconverted to oxaloacetate and through transamination return to aspartate. By reversing the alanine aminotransferase - aspartate aminotransferase couple, amino groups are transferred from alanine to aspartate and the pyruvate can then be oxidized by the Krebs cycle. CHAPTER V LITERATURE CITED 59 Anderson,M.R. 1974. M.Sc. Thesis. Stanford University. Atkinson,D.E. 1968. 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APPENDIX LIST OF ABBREVIATIONS 63 Abbreviations used: (U) - uniformly labelled EDTA - ethylenediamine tetraacetic acid ATP, ADP, AMP - adenosine t r i - , d i - , and monophosphate GTP, GDP - guanosine t r i - , and diphosphate ITP, IDP - inosine t r i - , and diphosphate NAD, NADH - oxidized and reduced fomms of nicotinamide adenine ddnucleotide, respectively NADP, NADPH - oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate, respectively F P q x , F P r e cj ~ oxidized and reduced flavoprotein, respectively G6P - glucose-6-phosphate FDP - fructose-1,6,-diphosphate G3P - glyceraldehyde-3-phosphate 1,3-DPG - 1,3-diphosphoglycerate PEP - phosphoenolpyruvate OXA -oxaloacetate c(-KGA - A.-ketoglutarate PFK - phosphofructokinase 

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