Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Purification and properties of dolphin muscle glutamate-oxalacetate and glutamate-pyruvate transaminases… Owen, Terrance George 1974

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1974_A1 O94.pdf [ 3.59MB ]
Metadata
JSON: 831-1.0093494.json
JSON-LD: 831-1.0093494-ld.json
RDF/XML (Pretty): 831-1.0093494-rdf.xml
RDF/JSON: 831-1.0093494-rdf.json
Turtle: 831-1.0093494-turtle.txt
N-Triples: 831-1.0093494-rdf-ntriples.txt
Original Record: 831-1.0093494-source.json
Full Text
831-1.0093494-fulltext.txt
Citation
831-1.0093494.ris

Full Text

PURIFICATION AND PROPERTIES OF DOLPHIN MUSCLE GLUTAMATE-OXALACETATE AND GLUTAMATE-PYRUVATE TRANSAMINASES AND THEIR POSSIBLE ROLES IN THE ENERGY METABOLISM OF DIVING MAMMALS by TERRANCE GEORGE OWEN B.Sc. (Hons.), University of Victoria, 1968 M.Sc, University of New Brunswick, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of ZOOLOGY We accept this thesis as conforming to the requited standard THE UNIVERSITY OF BRITISH COLUMBIA April, 1974 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 advanced degree a t the 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 agree t h a t the L i b r a r y s h a l l make 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 and s t u d y . I f u r t h e r agree 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 urposes may be g r a n t e d by the Head o f my Department 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 ot 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 not 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 . Department o f The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada 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 the r e q u i r e m e n t s f o r an advanced degree a t the 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 agree t h a t the L i b r a r y s h a l l make 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 and s t u d y . I f u r t h e r agree 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 urposes may be g r a n t e d by the Head o f my Department 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 of 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 . The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada ABSTRACT Mitochondrial and supernatant glutamate-oxalacetate transaminases (EC 2.6.1.1) and supernatant glutamate-pyruvate transaminase (EC 2.6.1.2) were purified 89, 204 and 240-fold respectively, from dolphin muscle. Starch gel electrophoresis of crude and purified perparations revealed that a l l three enzymes exist as single forms. Km values of a-ketoglutarate, alanine, pyruvate and glutamate for the glutamate-pyruvate transaminase were 0.45, 8.2, 0.87 and 15 mM, respectively. For the glutamate-oxalacetate transaminases, the Km values of a-ketoglutarate, aspartate, oxalacetate and glutamate were 0.76, 0.50, 0.10 and 9.4 mM, respectively, for the mitochondrial form and 0.13, 2.4, 0.06 and 3.2 mM, respectively, for the supernatant form. In a l l cases, as the assay pH was decreased from pH 7.3, the Km values of the a-keto acids decreased while those of the amino acids increased. This caused the apparent equilibrium constants for the glutamate-oxalacetate transaminases to remain independent of pH. These values were 9.2 and 6.8 for the mitochondrial and supernatant forms, respectively where K'eo = t a sP a r t a t e 3[a-ketoglutarate] [glutamate][oxalacetate] Studies of the inhibition of the glutamate-oxalacetate transaminases by dicarboxylic acids indicated that these enzymes may be controlled by pools of metabolic intermediates. Three key roles are suggested for the transminases i n the energy metabolism of the diving mammal. F i r s t , i t is believed that a combined action of the transaminases could enhance energy production during hypoxia by providing (1) fumarate from aspartate for the ATP producing reversal of succinate dehydrogenase and (2) a-ketoglutarate from glutamate for the GTP producing succinyl thiokinase reaction. Next, diving mammals probably accumulate more NADH than other mammals during hypoxia. The glutamate-oxalacetate transaminases seem particularly well suited for restoring redox i i balance v i a the malate-aspartate cyc le a f t e r aerobic metabolism i s resumed. F i n a l l y , s ince migrat ing d ivers ox id i ze la rge amounts of s to red f a t s , the combined react ions of the transaminases could be instrumenta l i n p rov id ing increased suppl ies of oxalacetate to condense with the fat der ived a c e t y l CoA i n the c i t r a t e synthase r e a c t i o n . i i i TABLE OF CONTENTS Page Abstract i List of Tables v L i s t of Figures v i Acknowledgements i x Introduction 1 Materials and Methods 8 Reagents 8 Enzyme assays 8 Enzyme units 10 Enzyme kinetics 10 Specificity of glutamate-oxalacetate transaminase 10 Protein determinations 10 Electrophoresis 10 Results 12 Enzyme specific activities 12 Purification of glutamate-pyruvate transaminase 12 Purification of glutamate-oxalacetate transaminase 15 Electrophoresis 28 Molecular weight determinations 34 Effect of pH 34 Enzyme kinetics 44 Inhibition of glutamate-oxalacetate transaminase 68 Specificity of glutamate-oxalacetate transaminase 68 Discussion 83 The transaminase reaction 83 i v Page Comparative aspects of the transaminases 84 Role of the transaminases in the diving mammal 88 A) Energy production 88 B) Maintenance of redox balance 96 C) Augmentation of metabolites 99 Literature Cited 101 LIST OF TABLES Table I. Purification of dolphin muscle glutamate-pyruvate transaminase. II. Heat denaturation of dolphin muscle glutamate-oxalacetate transaminase. III. Purification of dolphin muscle glutamate-oxalacetate transaminase. IV. Summary of molecular weights obtained on Sephadex G-200. V. Summary of Km values for dolphin muscle glutamate-pyruvate transaminase. VI. Summary of Km values for dolphin muscle glutamate-oxalacetate trans aminas e. VII. Apparent equilibrium constants (K'eq) for dolphin muscle glutamate-oxalacetate transaminase. VIII. Summary of inhibition of dolphin muscle glutamate-oxalacetate transaminase by dicarboxylates. IX. Summary of inhibition of dolphin muscle glutamate-oxalacetate transaminase by the a-keto acid substrates. X. Apparent equilibrium constants (K'eq) for dolphin and rabbit muscle lactate dehydrogenase. v i LIST OF FIGURES Figure Page 1. Chromatography of dolphin muscle glutamate-pyruvate transaminase of Sephadex G-200. 13, 14 2. Chromatography of dolphin muscle glutamate-pyruvate transaminse of DEAE-cellulose. 17, 18 3. Chromatography of dolphin muscle glutamate-oxalacetate transaminse and malate dehydrogenase on Sephadex G-200. 21, 22 4. Chromatography of dolphin muscle mitochondrial (cationic) glutamate-oxalacetate transaminase on CM-cellulose. 24, 25 5. Chromatography of dolphin muscle supernatant (anionic) glutamate-oxalacetate transaminase and malate dehydrogenase on DEAE-cellulose. 26, 27 6. Isoelectric focusing of dolphin muscle supernatant (anionic) glutamate-oxalacetate transaminase and malate dehydrogenase. 29, 30 7. Starch gel electrophoresis of dolphin muscle glutamate-pyruvate and glutamate-oxalacetate transaminases. 32, 33 8. Spectrophotometric scan of disc gel containing a mixture of the two forms of dolphin muscle supernatant glutamate- 35, 36 oxalacetate separated by isoelectric focusing. 9. Calibration curve for Sephadex G-200 column used for molecular weight determinations. 37, 38 10. Effect of pH on the activity of dolphin muscle glutamate-pyruvate transminase. 40, 41 11. Effect of pH on the activity of dolphin muscle glutamate-oxalacetate transaminase. 42, 43 v i i F i g u r e Page 12. E f f e c t of amino a c i d s u b s t r a t e c o n c e n t r a t i o n on the a c t i v i t y of d o l p h i n muscle glutamate-pyruvate transaminase. ^5, 46 13. E f f e c t of a-keto a c i d s u b s t r a t e c o n c e n t r a t i o n on the a c t i v i t y of d o l p h i n muscle glutamate-pyruvate transaminase. 47, 48 14. Determination of the absolute Km of a - k e t o g l u t a r a t e f o r d o l p h i n muscle glutamate transaminase. 50, 51 15. Determination of the absolute Km of glutamate f o r d o l p h i n muscle glutamate-pyruvate transaminase. 52, 53 16. Determination of the absolute Km of pyruvate f o r d o l p h i n muscle glutamate-pyruvate transaminase. 54, 55 17. Determination of the absolute Km of a l a n i n e f o r d o l p h i n muscle glutamate-pyruvate transaminase. 56, 57 18. E f f e c t of a - k e t o g l u t a r a t e c o n c e n t r a t i o n on the a c t i v i t y of d o l p h i n muscle glutamate-oxalacetate transaminase. 58, 59 19. E f f e c t of glutamate c o n c e n t r a t i o n on the a c t i v i t y of d o l p h i n muscle glutamate-oxalacetate transaminase. 60, 61 20. E f f e c t of ox a l a c e t a t e c o n c e n t r a t i o n on the a c t i v i t y of d o l p h i n muscle glutamate-oxalacetate transaminase. 62, 63 21. E f f e c t of aspartate c o n c e n t r a t i o n on the a c t i v i t y of d o l p h i n muscle glutamate-oxalacetate transaminase. 64, 65 22. Succinate i n h i b i t i o n of d o l p h i n muscle glutamate-o x a l a c e t a t e transaminase. 69, 70 23. Glutamate i n h i b i t i o n of d o l p h i n muscle glutamate-o x a l a c e t a t e transaminase. 71, 72 24. Malate i n h i b i t i o n of d o l p h i n muscle glutamate-oxalacetate transaminase. 73, 74 v i i i Figure Page 25. a-ketoglutarate inhibition of dolphin muscle glutamate-oxalacetate transaminse. 75, 76 26. Oxalacetate substrate inhibition of dolphin muscle glutamate-oxalacetate transaminse. 78, 79 27. a-ketoglutarate substrate inhibition of dolphin muscle glutamate-oxalacetate transaminase. 80, 81 28. A metabolic map indicating the postulated role of glutamate-pyruvate and glutamate-oxalacetate transaminases during anaerobic excursions in the diving mammal. 89, 90 29. The malate-aspartate cycle. 97, 98 i x ACKNOWLEDGEMENTS I wish to extend my thanks to Dr . P. W. Hochachka fo r h i s c o n s i s t e n t l y wise counsel throughout the course of th is i n v e s t i g a t i o n and to the members of my committee fo r t h e i r comments and c r i t i c i s m . I am a l s o g r a t e f u l to the F i s h e r i e s Research Board of Canada and the U n i v e r s i t y of B r i t i s h Columbia fo r t h e i r generous f i n a n c i a l a s s i s t a n c e . INTRODUCTION The diving capacity of the marine mammals, when compared to that of terrestrial mammals, is remarkable. For example, trained human divers can remain submerged for about 2 minutes (Andersen, 1966) compared to 20 minutes for dolphins (Ewer, 1947). The best diver of comparable size, however, is the Weddell seal, Leptonychotes weddelli, which is capable of dives lasting for one hour (Kooyman, 1966; Eisner et a l . , 1970a). The diving times of other mammals are summarized by Kooyman.(1972). Such performance is permitted by impressive physiological adaptations for oxygen conservation such as bradycardia, a slowing of the heart rate during a dive. This reduction i s probably related to circulatory shunts which occlude the blood supply to the body (Prosser and Brown, 1961) so that only the heart and the brain are perfused (Eisner et a l . , 1970b). These adaptations, which are discussed in a number of reviews (Andersen, 1966; Eisner, 1969; Lenfant, 1969; Scholander, 1940, 1962) probably allow aerobic metabolism to continue at least in the brain and possibly the heart for the duration of most dives (Packer et a l . , 1969). The remainder of the body organs and tissues are ischemic but the oxygen stored in the large quantities of muscle hemoglobin which are present in diving mammals (see previous reviews) support aerobic metabolism in these tissues for at least a portion of most dives. However, i t is now considered common knowledge that these oxygen supplies are often consumed long before the end of a dive (Andersen, 1966; Ewer, 1947; Scholander, 1940; Scholander et a l . 1942). Diving mammals, then, must rely on anaerobic metabolism to maintain muscular activity during the remainder of such excursions. Although a l l aspects of this anaerobiosis have not yet been investigated, the conversion of glycogen or glucose to lactate via glycolysis is considered to be the major i f not the only source of ATP during prolonged diving in mammals and other 2 vertebrates (George et a l . , 1971; Kerem et a l , , 1973; Reeves, 1963; Vallyathari et a l . , 1969). This conclusion is supported by the observation that large quantities of lactate accumulate in the muscles during a dive and are flushed into the blood after the animal resurfaces (Andersen, 1966; Pickwell, 1968; Christiansen and Penney, 1973; Scholander, 1940, 1962; Scholander et a l . , 1942). It seems immediately obvious however that glycoly-sis may not be the only source of ATP during hypoxia since other substrate level phosphorylations that do not require the presence of oxygen are known to exist. One example is the conversion of a-ketoglutarate to succinate by succinyl thiokinase. This is a GTP producing reaction which could be supplied with a-ketoglutarate from the endogenous pools of free glutamate. Although such speculation has not been previously considered with respect to diving mammals, many experiments have been conducted with other animals. The most extensive of these studies concern the invertebrates which also u t i l i z e glycolysis but, in many of these organisms, this pathway does not proceed as far as the production of lactate during hypoxia. For example, i n the flatworm Fasciola hepatica (DeZoeten and Tipker, 1969), the roundworm Ascaris  lumbricoides (Saz, 1971; Seidman and Entner, 1961), and the molluscs Mytilus  edulis (DeZwaan and VanMarrewijk, 1973) and Crassostrea gigas (Hochachka and Mustafa, 1972; Mustafa and Hochachka, 1971, 1973), most, i f not a l l of the phosphoenolpyruvate is carboxylated by phosphoenolpyruvate carboxykinase in the cytoplasm to form oxalacetate rather than being converted to pyruvate by pyruvate kinase which is i t s usual fate. The oxalacetate is then reduced to malate by cytoplasmic malate dehydrogenase. This reduction serves an analogous function to that of pyruvate to lactate (which occurs in the more conventional glycolysis pathway mentioned above) in that both of these reductions regenerate NAD+- from NADH for the glyceraldehyde-3-phosphate dehydrogenase step of glycolysis, which cannot continue without NAD^ . The sequence of events after this reduction of oxalacetate to malate is a moot point. It i s generally agreed that the malate i s converted to fumarate by fumarase (which is freely reversible and hence, can operate i n the opposite direction to i t s function i n the Krebs cycle). However, depending on the organism, i t is estimated that this conversion involves either 50% (Saz, 1971; Hochachka and Mustafa, 1972), 75% (DeZwaan and VanMarrwijk, 1973) or 100% (DeZoeten and Tipker, 1969) of the malate and occurs in either the cytoplasm (Hochachka and Mustafa, 1972; De-Zoeten and Tipker, 1969) or the mitochondria (Saz, 1971; DeZwaan and VanMarrewijk, 1973). Regardless, the malate derived fumarate is reduced to succinate in the mitochondria by a reversal of the succinate dehydrogenase reaction which acts as a "fumarate reductase". This reaction requires NADH, produces ATP and results in the accumulation of succinate as an anaerobic end product. The following sequence therefore prevails: glucose - g^- -> phosphoenolpyruvate _ M — ^ oxalacetate NAD NADH IDP succinate NAD fumarate malate NADH ATP ADP Also i n question is the source of the NADH required for the fumarate reduction. For example, i t has been suggested that this reaction is coupled to (1) malate oxidation to pyruvate and C0_ by an NAD+-dependent malic 4 enzyme (Saz, 1971), (2) a more complex sequence of reactions involving a l l of the enzymes of the Krebs cycle except succinyl thiokinase (DeZwaan and VanMarrewijk, 1973), or (3) the oxidation of a-ketoglutarate by succinyl thiokinase to succinate which, again, accumulates (Hochachka and Mustafa, 1972). The a-ketoglutarate for this third reaction is believed to arise from the transamination of glutamate by glutamate-pyruvate transaminase which uses pyruvate originating from the phosphoenolpyruvate that i s not carboxylated to form oxalacetate. In the transaminase reaction, the pyruvate and glutamate are converted to alanine and a-ketoglutarate, respectively. The alanine i s not metabolized further and accumulates as an anaerobic end product. Despite the uncertainties i n the scheme presented above, i t explains the accumulation of succinate and alanine observed during hypoxia in invertebrates in other laboratories (Arillo and DeGiuli, 1971; Chen and Apawara, 1969; Malanga and Aiello, 1972; Stokes and Apawara, 1968; DeZwaan and Zandee, 1972; DeZoeten e_t a l . , 1969) . I t is also agreed that two main advantages are derived from such a metabolic strategy. F i r s t , the accumulated end products, succinate and alanine, are more easily oxidized (via the Krebs cycle) than lactate once aerobic metabolism is resumed (DeZwaan and VanMarrewijk, 1973). Second, i t is possible to increase the energy yield from the 2 ATP/glucose obtained through glycolysis to 2.5 to 4 ATP/glucose depending on which of the above authors' schemes is being considered. For example, the potential sites of ATP production include phosphoglycerate kinase, pyruvate kinase, phospho-enolpyruvate carboxykinase, "fumarate reductase", succinyl thiokinase, and i f succinyl CoA, derived from a-ketoglutarate, is converted to propionate (through reactions 1-3 catalyzed by methylmalonyl CoA mutase, propionyl CoA carboxylase and acetic thiokinase, respectively) rather than succinate, then the two phosphorylations that occur as a result of this conversion must be included (Hochachka and Mustafa, 1972; DeZoeten and Tipker, 1969) succinyl CoA •> methylmalonyl CoA (1) methylmalonyl CoA + ADP > propionyl CoA + ATP + CC>2 (2) propionyl CoA + AMP > propionate + ATP (3) The variations i n the estimates of ATP/glucose are therefore dependent on the number of the potential phosphorylation sites included in a particular scheme. Although the metabolic strategies described above have been elucidated in invertebrates i t has been demonstrated that similar pathways function to a significant degree in a variety of isolated, perfused, hypoxic, mammalian tissues (Chance and Hollunger, 1960; Hunter, 1949; Penney and Cascarano, 1970; Sanadi and Fluharty, 1963; Wilson and Cascarano, 1970; Randall and Cohen, 1966). As i n the invertebrates, ATP i s produced by the reduction of oxalacetate-derived fumarate by "fumarate reductase" which is generally considered to be coupled by NAD+ and NADH to the oxidation of a-ketoglutarate by succinyl thiokinase, also an energy producing reaction, i n the following manner: GTP GDP succinate < a-ketoglutarate NADH NAD fumarate > succinate ADP ATP Further evidence that these two reactions form an integral part of the anaerobic metabolism of mammals comes from the observation that succinate accumulates during hypoxia in their tissues (Arillo and DeGiuli, 1970, 1971; Hoberman and Prosky, 1967; Hochachka et a l . , 1974; Kondrashova and Chahovets, 6 1971; Mensen deSilva and Cazorla, 1973). In the mammalian tissues, i t seems unlikely that oxalacetate and hence, fumarate, arise from phosphoenolpyruvate since an active pyruvate kinase is present and also, the kinetic properties of phosphoenolpyruvate kinase favour the production, not the carboxylation, of phosphoenolpyruvate (Garber and Salganicoff, 1973; Saz, 1971). We therefore reasoned that an alternate source of oxalacetate was required and could be supplied by the transamination of free aspartate by glutamate-oxalacetate transaminase which converts aspartate and a-ketoglutarate to oxalacetate and glutamate respectively. The transamina-tion of glutamate by glutamate-pyruvate transaminase could provide the necessary a-ketoglutarate for the transamination of aspartate and for the succinyl thiokinase reaction in the mitochondria as follows: pyruvate alanine to fumarate to succinyl reductase thiokinase This idea is consistent with recent data (Davis and Bremer, 1973; Davis et a l . , 1972; Lowenstein, 1972; Safer et a l . , 1971; Safer and Williamson, 1973) suggesting that augmentation of Krebs cycle intermediates can occur at the expense of free amino acids through the combined reactions of the glutamate-pyruvate and glutamate-oxalacetate transaminases. This augmentation results in an accumulation of alanine that is equal to the sum of the increase of Krebs cycle intermediates. In the present study, these two transaminases were purified from dolphin muscle (Lagenorhynchus obliquidens) and characterized kinetically to gain 7 insight into their role i n the energy metabolism of this diving mammal, with particular reference to the possibility that they may provide substrates for ATP-producing reactions during hypoxia. 8 MATERIALS AND METHODS Reagents A l l s u b s t r a t e s , c o f a c t o r s , commercial enzymes and metabolites used i n i n h i b i t i o n experiments were obtained from Sigma; s t a r c h , from Connaught; reagents f o r polyacrylamide e l e c t r o p h o r e s i s , from Eastman; CM-cellulose and DEAE-cellulose, from Whatman; and Sephadex G-200, from Pharmacia. A l l other chemicals were of reagent grade. The d o l p h i n muscle (Lagenorhynchus  ob l i q u i d e n s ) was generously donated by the Vancouver P u b l i c Aquarium, Vancouver, B. C. and s t o r e d at -20°C. Enzyme assays A l l assays, unless otherwise i n d i c a t e d , contained NADH at a c o n c e n t r a t i o n of 0.2 mM, were brought to a f i n a l volume of 2.5 ml w i t h 100 mM sodium phos-phate b u f f e r s and were s t a r t e d by the a d d i t i o n of 0.1 ml of enzyme p r e p a r a t i o n c o n t a i n i n g approximately 3 u n i t s / m l . The A^^/min was then determined at 37°C using a Unicam SP800 or Unican Pye SP1800 spectrophotometer. This i s r e f e r r e d to as the "standard method". A survey of transaminases was made using d u p l i c a t e non-coupled assays c o n t a i n i n g 15 mM a-keto a c i d , 30 mM glutamate and 0.1 ml of enzyme p r e p a r a t i o n (d o l p h i n muscle homogenized i n 4 volumes of assay b u f f e r , pH 7.3, and c e n t r i -fuged at 600 X g f o r 15 min) i n a f i n a l volume of 2.0 ml adjusted to pH 7.3. A c o n t r o l assay c o n t a i n i n g no glutamate was prepared f o r each a-keto a c i d . Two a d d i t i o n a l c o n t r o l assays contained no a-keto a c i d . A l i q u o t s (0.6 ml) of the r e a c t i o n mixtures were added to 0.6 ml of 10% t r i c h l o r o a c e t i c a c i d at timed i n t e r v a l s . The p r e c i p i t a t e was sedimented and a 0.5 ml a l i q u o t of the supernatant was t r a n s f e r r e d to a c u v e t t e , n e u t r a l i z e d w i t h 1 N NaOH and assayed f o r a-ketoglutarate content by the standard method w i t h 0.02 ml of glutamate dehydrogenase (200 U/ml i n 2.0 M ammonium s u l p h a t e ) . 9 Glutamate dehydrogenase was assayed by the standard method at pH 7.3 with 2 mM a-ketoglutarate and 2 mM ammonium chloride. A l l GPT"*" and GOT assays, unless otherwise indicated, were by the standard method. GPT assays in the direction of glutamate production contained vary-ing amounts of a-ketoglutarate and alanine and 0.02 ml of lactate dehydrogenase (750 U/ml in 0.21 M ammonium sulphate). GOT assays i n the direction of glutamate production contained varying amounts of a-ketoglutarate and aspar-tate and 0.02 ml of malate dehydrogenase (500 u/ml i n 0.28 M ammonium sulphate). GPT and GOT assays in the direction of a-ketoglutarate production contained varying amounts of pyruvate and oxalacetate, respectively, varying amounts of glutamate and 0.02 ml of glutamate dehydrogenase (300 U/ml in 2.0 M ammonium sulphate). No activity was observed with any of the transaminase preparations used for kinetic studies when either the a-keto acid or amino acid was omitted from any of the assay mixtures. The presence of excess coupling enzyme was confirmed by adding either oxalacetate, pyruvate or a-ketoglutarate to representative assays after the assay had progressed for 60 to 90 seconds. Appropriate corrections were made to account for the a-ketoglutarate intro-duced into the GOT assays via the GOT preparations which contained 2 mM a-ketoglutarate for stabilization of the enzyme. A l l substrate and inhibitor stock solutions were adjusted to the pH of the assays. A l l assays were conducted at pH 7.3 ("normal") since this was the optimum pH for both trans-aminases and at pH 6.8 or 6.3 ("hypoxic") since pH decreases as hypoxia progresses (Chance et a l . , 1964). Abbreviations - GPT, glutamate-pyruvate transaminase; mGOT and sGOT, mitochondrial (cationic) and supernatant (anionic) glutamate-oxalacetate transaminase, respectively. 10 Lactate and malate dehydrogenases were assayed by the standard method at pH 7.3 and 1 mM pyruvate and oxalacetate, respectively. Enzyme units - One unit is defined as the amount of enzyme required to convert 1 micromole of substrate/min at pH 7.3 and 37°C. GOT and GPT specific activities were measured at 10 mM aspartate and alanine respectively and 5 mM a-ketoglutarate. Enzyme kinetics - A l l Km values were determined from Lineweaver-Burk plots using the formula: Km - Slope x V ^. A l l inhibitor data were expressed as the concentration of inhibitor required for 50% inhibition. These values were determined from Dixon plots of 1/Relative activity vs. [I] using the formula: [I] at 50% inhibition = (1/V)/Slope, where V » activity in the absence of inhibitor. GOT specificity - The a-keto acids of tyrosine, phenylalanine and leucine were substituted for oxalacetate to a fin a l concentration of 1 mM i n the standard GOT assay i n the direction of a-ketoglutarate production at pH 7.3. The rates of reduction of the a-keto acids by NADH were determined in the absence of glutamate and subtracted from the activities obtained after the transaminase reactions were started by the addition of glutamate to a concentration of 20 mM. To ascertain that the a-keto acids were not inhibiting the coupling enzyme, a-ketoglutarate was added after the assays had progressed for 3 to 5 min. Protein determinations - Protein concentrations were calculated using the formula (A^^ - A225^ X.0..154 = m § protein/ml. Electrophoresis - Horizontal starch gels (13%) were run at pH 7.0 and 250 volts for 18 to 24 hours using a 135 mM Tris-43 mM citrate buffer (Shaw and Prasad, 1970) i n the tanks and a 1:14 dilution of the same buffer for the gels. The gels were sliced in half lengthwise before staining. 11 Polyacrylamide electrophoresis was conducted by Davis 1 (1964) method using 7% separating gels, a Buchler apparatus and 1% Amido Schwartz in 7% glacial acetic acid to stain for protein. The GOT stain consisted of aspartate (500 mg), a-ketoglutarate (75 mg), pyridoxal-5-phosphate (50 mg) and Fast Violet B salt (200 mg) i n 100 ml of 50 mM Tris-Cl buffer adjusted to a f i n a l pH of 7.6. The GPT stain consisted of alanine (250 mg), NADH (15 mg) and a-ketoglu-tarate (37.5 mg) adjusted to pH 7.3 in 50 ml of 50 mM Tris-Cl buffer (Solution A). One g of Bactoagar (Difco) was boiled in another 50 ml of the same buffer u t n i l i t dissolved (Solution B). Both solutions were brought to 42°C and combined after 750 U of lactate dehydrogenase had been added to Solution A. This mixture was poured over the surface of the sliced starch gel. After approximately 30 minutes at room temperature the s o l i d i f i e d agar gel was peeled from the surface of the starch gel and the GPT could be localized by viewing the starch gel under a UV lamp equipped with a 340 nm f i l t e r . 12 RESULTS Enzyme Specific Activities The ot-keto acids of aspartate, alanine and tyrosine were transaminated at relative rates of 100, 12 and 0.3 per g of tissue, respectively, by crude dolphin muscle preparations in the non-coupled transaminase assay. There was no measurable activity with the a-keto acids of cysteine, glycine, isoleucine, leucine, phenylalanine or valine nor was there measurable glutamate dehydro-genase activity i n agreement with the findings of Copenhaver et a l . (1950). Purification of GPT Step 1 - 300 g of dolphin muscle were homogenized i n 900 ml of Buffer A (50 mM Tris-Cl, 10 mM EDTA, 10 mM 3-mercaptoethanol, pH 7.0 at 25°C) in a Sorvall Omnimixer at f u l l speed for 60 s and centrifuged at 10,000 x g for 40 min. The supernatant (775 ml) was placed i n a 60°C water bath, brought to 52°C and immediately placed in an ice bath unt i l i t s temperature was 4°C. Step 2 - The heat treated sample (775 ml) was brought to 30% saturation with 136 g of ammonium sulphate, stirred for 30 min and centrifuged at 10,000 x g for 30 min. The supernatant (790 ml) was brought to 55% saturation with 128 g of ammonium sulphate, stirred for 30 min and centrifuged at 10,000 x g for 30 min. The pellet was redissolved i n a minimal amount of Buffer B (15 mM Tris-Cl, 2 mM EDTA, 10 mM 3-mercaptoethanol, pH 7.25 at 25°C) and centrifuged at 39,000 x g for 30 min. Step 3 - The centrifuged sample was placed on a G-200 column (4 x 60 cm) equilibrated to Buffer B. The column was eluted at 17 ml/hr and 4 ml fractions were collected (Fig. 1). The peak fractions were pooled and adjusted to the same pH and conductivity as Buffer B. Step 4 - The sample was applied to a DEAE-cellulose column (1.9 x 25 cm) equilibrated to Buffer B. The column was flushed with 600 ml of Buffer B to 13 Figure 1: Chromatography of dolphin muscle glutamate-pyruvate transaminase on Sephadex G-200. A l l conditions are described in Results. Fraction 15 free the column of a l l lactate dehydrogenase activity and the GPT was then removed by applying a linear gradient of 0 to 100 mM NaCl in Buffer B (300 ml reservoirs). The column was eluted at 30 ml/hr and 3 ml fractions were collected (Fig. 2). The peak fractions were pooled, taken to 20 mM EDTA for stabilization (Saier and Jenkins, 1967) and stored at 4°C. Unlike the GPT purified from pig heart by these workers, dolphin muscle GPT was totally inactivated after one freeze-thaw cycle. However, at 4°C the purified preparations were stable and could be stored for periods of at least 2 months. A typical purification procedure is summarized in Table I. The highest specific activity obtained was 36.8 U/mg protein and a l l preparations were totally free of lactate dehydrogenase activity. Purification of GOT I n i t i a l attempts at purification of GOT failed due to in s t a b i l i t y of the enzyme. Pyridoxal-5-phosphate and a-ketoglutarate were found to be the most effective stabilizers (Table II) and were included in a l l buffers used for purification and storage of the enzyme. A l l steps of the purification were conducted at 4°C. Step 1 - 136 g of dolphin muscle were homogenized in 408 ml of Buffer C (100 mM sodium phosphate, 2 mM a-ketoglutarate, 0.25 mM pyridoxal-5-phosphate, pH 7.3) in a Sorvall Omnimixer at f u l l speed for 60 s and centrifuged at 10,000 x g for 30 min. The dolphin muscle pellets took up large quantities of buffer and always weighed more than the original portion of tissue. Consequent-ly, the pellet was rehomogenized in one volume of buffer and recentrifuged. This process was repeated again and the supernatants from each step were pooled. This rehomogenization procedure significantly increased the yield of GOT to 135 U/g of tissue. GPT activity was also measured and was found to be 16 U/g of tissue. 16 TABLE I Purification of dolphin muscle glutamate pyruvate transaminase Fraction Total Total Specific Fold- Yield Activity Protein Activity Purification units mg units/mg % Crude 1,750 11,700 0.15 1.0 100 Heat treated 1,700 9,750 0.18 1.2 98 30-55% ammonium sulphate 1,400 1,180 1.2 8.0 83 G-200 1,000 390 3.9 26 57 DEAE-cellulose 606 17 35.5 237 35 17 Figure 2: Chromatography of dolphin muscle glutamate-pyruvate transaminase on DEAE-cellulose. A l l conditions are described in Results. 4 0 Fraction 19 TABLE II Heat denaturation of dolphin muscle glutamate oxalacetate transaminase. Dolphin muscle was homogenized i n 0.1 M sodium phosphate buffer pH 7.3 (3 ml/g of tissue) and centrifuged at 30,000 x g for 30 min. Aliquots of 1.0 ml were taken to the indicated concentrations of reagents in a fi n a l volume of 2.0 ml, heated at 75° for 30 min, cooled, centrifuged and assayed at pH 7.3 for GOT activity by the standard method described i n "Materials and Methods" using 5 mM a-ketoglutarate and 10 mM aspartate. Additions Loss of Activity % None 99.5 EDTA, 5 mM 99.5 8-mercaptoethanol, 10 mM 99.5 Dithiothreitol, 2 mM 99.5 Glutamate, 25 mM 99.5 Aspartate, 25 mM 100 Pyridoxal-5-phosphate, 0.1 mM 96.5 " " , 0.5 mM 93.9 a-ketoglutarate, 0.5 mM 94.0 , 1.0 mM 90.0 Pyridoxal-5-phosphate, 0.1 mM + a-ketoglutarate, 1.0 mM 71.0 20 Step 2 - The pooled supernatants (630 ml) were placed i n a 60°C water bath for 40 min with frequent mixing and then centrifuged at 10,000 x g for 30 min. Step 3 - The supernatant (570 ml) was taken to 50% saturation with 178 g of ammonium sulphate, stirred for 30 min and centrifuged at 12,000 x g for 30 min. The pellet was discarded and the supernatant (640 ml) was taken to 75% saturation with 113 g of ammonium sulphate, stirred for 30 min and centrifuged at 12,000 x g for 40 min. The pellet was redissolved in Buffer C to a fina l volume of 24 ml and dialyzed against 4 l i t r e s of Buffer D (15 mM sodium phosphate, 2 mM a-ketoglutarate, 0.25 mM pyridoxal-5-phosphate, pH 7.0) for 8 hours. The contents of the dialysis bag were concentrated by covering the bag with crystalline sucrose for 11 hr. The contents were then forced down to one end of the dialysis bag and the bag was tied off and dialyzed against 4 l i t r e s of Buffer D for 4 hr. Step 4 - The contents of the dialysis bag (16 ml) were centrifuged at 30,000 x g for 30 min and then applied to a G-200 column (5 x 100 cm) equilibrated to Buffer D plus 0.02% sodium azide (to prevent bacterial growth). The column was eluted at 20 ml/hr and 5 ml fractions were collected after 900 ml buffer had passed through the column (Fig. 3). The peak fractions were pooled and adjusted to the same pH and conductivity as Buffer E (10 mM citrate-sodium phosphate, 2 mM a-ketoglutarate, 0.25 mM pyridoxal-5-phosphate, 0.02% sodium azide, pH 6.5). Step 5 - The sample (197 ml) was applied to a CM-cellulose column (1.6 x 10 cm) equilibrated to Buffer E. The column was washed with 50 ml of Buffer E. The material that passed through the column contained a l l of the supernatant (anionic) GOT while a l l of the mitochrondrial (cationic) GOT remained on the column. The separation of the two forms at this step was 21 Figure 3: Chromatography of dolphin muscle glutamate-oxalacetate trans-aminase ( 0 ) and malate dehydrogenase ( • ) on Sephadex G-200. A l l conditions are described in Results. 6 0 22 F r a c t i o n 23 complete by the criterion of starch gel electrophoresis. A linear gradient of 0 to 100 mM NaCl in Buffer E (200 ml reservoirs) was applied at 30 ml/hr and 3 ml fractions were collected (Fig. 4). The peak fractions containing the GOT were pooled, taken to 90% saturation with ammonium sulphate and centri-fuged at 30,000 x g for 30 min. The pellet was resolubilized in Buffer D. Step 6 - The material that passed through the CM-cellulose column and contained a l l of the sGOT was taken to 80% saturation with ammonium sulphate and centrifuged at 30,000 x g for 30 min. The pellet was redissolved in Buffer F (5 mM Tris-Cl, 0.75 mM a-ketoglutarate, 0.10 mM pyridoxal-5-phosphate, pH 8.0 at 25°C) and dialyzed against 1 l i t r e and 2 l i t r e s of Buffer F for 2 and 18 hr, respectively. The sample was centrifuged at 30,000 x g for 30 min, adjusted to the same pH and conductivity as Buffer F and applied to a DEAE-cellulose column (1.6 x 10 cm) equilibrated to Buffer F. The column was washed with 40 ml of Buffer F and then a gradient of 0 to 200 mM NaCl i n Buffer F (300 ml reservoirs) was applied at 30 ml/hr and 3 ml fractions were collected (Fig. 5). The peak fractions were pooled, taken to 80% saturation with ammonium sulphate and centrifuged at 30,000 x g for 30 min. The pellet was redissolved in Buffer D. Step 7 - Both sGOT and mGOT were dialyzed against 2 l i t r e s of Buffer D plus 0.02% sodium azide for 18 hr and then centrifuged at 30,000 x g for 30 min. Both forms were stable at 4°C for at least 4 months when redialyzed against Buffer D plus azide every 6 weeks to replenish the a-ketoglutarate and the pyridoxal-5-phosphate. The mGOT preparation contained no malate dehydrogenase contamination but the sGOT preparation contained malate dehydrogenase activity that had co-purified with the sGOT (Fig. 5). This preparation was used for a l l of the experiments with the exception of the determination of kinetic constants with oxalacetate and glutamate as 24 Figure 4: Chromatography of dolphin muscle mitochondrial (cationic) glutamate-oxalacetate transaminase on CM-cellulose. A l l conditions are described i n Results. 8 0 25 6 0 26 Figure 5: Chromatography of dolphin muscle supernatant (anionic) glutamate-oxalacetate transaminase ( 0 ) and malate dehydrogenase ( • ) on Whatman DEAE-cellulose. A l l conditions are described i n Results. F r a c t i o n 28 substrates. For these determinations, further purification was necessary. Step 8 - One ml of the sGOT preparation was electrofocused with the anode at the top of a 110 ml L.K.B. electrofocus column using a pH gradient of 5 to 8. The current was increased 50 volts every 15 min from 300 to 700 volts. This voltage was maintained for 43 hr and then the column was emptied at 80 ml/hr. The sGOT and malate dehydrogenase were totally separated by this procedure. The sGOT separated into two peaks with p i values of 6.55 and 6.00 while the malate dehydrogenase had a pi of 5.12 (Fig. 6). The purification procedures are summarized in Table III. Electrophoresis Dolphin muscle GPT appeared as a single band migrating 2.5 cm towards the anode in both crude and purified preparations (Fig. 7). The purification of GOT was monitored by starch gel electrophoresis and only two forms of the enzyme were observed. The cationic form, mGOT, migrated 3.0 cm towards the cathode while the anionic form, sGOT, migrated 2.5 cm towards the anode (Fig. 7). However, the sGOT was separated into 2 subforms by electrofocusing (Fig. 6). The peak fractions of each of the two forms were pooled and taken to 2 mM a-ketoglutarate, 0.25 mM pyridoxal-5-phosphate and 0.02% sodium azide. Both forms of the sGOT had identical Km values for a l l 4 substrates. Also, the Km values for a-ketoglutarate and aspartate were identical with those obtained with the sGOT preparation from Step 7 of the purification procedure. A l l of the sGOT samples were applied to polyacrylamide gels (2 mg protein/gel) and a current of 2-1/2 mamps/gel was applied for 70 min. The gels were stained for protein and GOT activity. Both forms of electrofocused sGOT and the non-electrofocused sGOT migrated through the gels at exactly the same rate. A mixture of the two electro-focused forms of sGOT showed up as one band of enzyme activity and protein 29 Figure 6: Isoelectric focusing of dolphin muscle supernatant (anionic) glutamate-oxalacetate transaminase ( 0 ) and malate dehydrogenase ( § ). A l l conditions are described i n Results. ( A ) = pH. 31 TABLE III Purification of dolphin muscle glutamate-oxalacetate transaminase. Fraction Total Total Specific Fold- Yield Activity Protein Activity Purification Crude Heat treated 50-75% ammonium sulphate G-200 CM-cellulose (mGOT) DEAE-cellulose (sGOT) Electro-focused sGOT units 18,270 18,896 16,944 12,327 5,200 1,880 mg 16,384 6,755 922 349 units/mg 1.12 2.80 17.1 35.4 22.8 228 56.9 33.0 1.0 2.5 4.34/ml 0.0432/ml 100 15 32 204 29 89 100 103 93 67 28 10 32 Figure 7: Starch gel electrophoresis of dolphin muscle glutamate-pyruvate transaminase (GPT) and glutamate-oxalacetate transaminase (GOT). A l l conditions are described i n Materials and Methods. (A) crude GPT (B) purified GPT (C) crude GOT (D) purified supernatnat GOT (E) purified mitochondrial GOT. 33 o r i g i n 3 2 1 0 I 2 3 c m 34 a t the sGOT si t e . The gels stained for the enzyme activity of the mixture were scanned at 400 nm using a Gilford spectrophotometer and again, only one form was apparent (Fig. 8). Consequently, the two electrofocused forms of sGOT were considered to be identical and were pooled for the determinations of the Km's of oxalacetate and glutamate. The acrylamide electrophoresis also revealed that the non-electrofocused sGOT preparation contained 2 contaminating proteins while the electrofocused sGOT contained only one. This probably represented the elimination of the malate dehydrogenase contamination. By this criterion the mGOT contained 2 contaminating proteins as well. Finally, crude preparations of dolphin muscle (3 ml Buffer D/g of tissue) were centrifuged at 30,000 x g for 30 min and electrophoresed according to the method of Decker and Rau (1963) . Again, both sGOT and mGOT appeared as single bands. In a l l experiments there appeared to be an approximately equal dis-tribution of mGOT and sGOT. Molecular weight determinations For comparative purposes, the apparent molecular weights of GOT and malate dehydrogenase from a variety of sources were determined (Fig. 9, Table IV). Effect of pH The optimum activity of GPT occurred between pH 7.15 and pH 7.8. At pH 6.3, 6.8 and 7.3 the relative activities were 40, 75 and 100% respectively (Fig. 10). The optimum activity of sGOT and mGOT occurred at pH 7.3-8.3 and pH 6.3-7.3, respectively. At pH 7.3 both sGOT and mGOT were capable of 95% of their maximum activity. At pH 6.8 this value was 76% and 100% for sGOT and mGOT, respectively (Fig. 11). 35 Figure 8: Spectrophotometry scan of a disc gel containing a mixture of the two forms of dolphin muscle supernatant glutamate-oxalacetate transaminase shown in Figure 6. The electrophoresis conditions and the enzyme stain are described in Materials and Methods and Results. The distance from the top on the separating gel and the absorbance at 400 nm are represented on the abscissa and the ordinate, respectively. m m 37 Figure 9: Calibration curve for Sephadex G-200 column used for molecular weight determinations. The column (1.6 x 33 cm) was equilibrated to Buffer C (see Results) plus 0.02% sodium azide and calibrated with Dextran Blue 2000 (lmg/ml), aldolase plus chymotrypsinogen A and ovalbumen plus ribonuclease A. A l l of the calibrating proteins were used at a concentration of 5 mg/ml and were from the k i t supplied by Pharmacia. Kav values were determined by the method of Granath and Kvist (1967), a line was fit t e d to the points by the method of least squares and the molecular weights of the unknowns were calculated using the least squares formula. A l l samples (0.4 ml) were subjected to a flow rate of 6 ml/hr. The reported values are the means of duplicate values and the d i f f e r -ences between duplicates did not exceed 1.5%. A l l enzyme assays were by the standard method at pH 7.3. (A) Sigma pig heart malate dehydrogenase (B) crude dolphin malate dehydrogenase (C) crude dolphin and Sigma pig heart glutamate-oxalacetate transaminases (D) purified dolphin supernatant glutamate-oxalacetate transaminase and i t s malate dehydrogenase contaminant. The molecular weights are summarized in Table IV. 39 TABLE IV Summary of molecular weights obtained on Sephadex G-200. A l l conditions are summarized in Figure 9. GOT refers to glutamate-oxalacetate transaminase. The "s" prefix refers to the supernatant form of the enzyme. Sample Apparent Molecular Weight sGOT (dolphin muscle) Malate dehydrogenase in sGOT preparation Sigma GOT (pig heart) Sigma malate dehydrogenase (pig heart) GOT (crude dolphin muscle preparation) Malate dehydrogenase (crude dolphin muscle preparation) 79,000 80,100 76,200 57,400 75,900 64,600 40 Figure 10: Effect of pH on the activity of dolphin muscle glutamate-pyruvate transaminase. A l l assays were by the standard method with 5 mM a-ketoglutarate and 10 mM alanine. V, = Relative activity. 42 Figure 1 1 : Effect of pH on the activity of dolphin muscle mitochondrial (• ) and supernatant ( 0 ) glutamate-oxalacetate transaminase. A l l assays were by the standard method with 5 mM a-ketoglutarate and 1 0 mM aspartate. V = Relative activity. 44 Enzyme k i n e t i c s For GPT, a decrease i n the assay pH from 7.3 to 6.3 caused an inc r e a s e i n the Km values of both amino a c i d s , a decrease i n the Km value of pyruvate, and no s i g n i f i c a n t change i n the Km value of a-ketoglutarate ( F i g s . 12, 13, Table V ) . T h e o r e t i c a l Km values f o r each s u b s t r a t e at i n f i n i t e concentra-t i o n s of co-substrate were obtained by p l o t t i n g 1/Km vs. 1/[co-substrate] and e x t r a p o l a t i n g to 1/[co-substrate] = 0 (Hopper and Segal, 1962). When 1/Km values f o r a - k e t o g l u t a r a t e , glutamate and pyruvate were p l o t t e d against 1 / [ a l a n i n e ] , l / [ p y r u v a t e ] and 1/[glutamate] r e s p e c t i v e l y , the expected s t r a i g h t l i n e s w i t h p o s i t i v e slopes were obtained which i l l u s t r a t e that the Km increases w i t h i n c r e a s i n g concentrations of co-substrate ( F i g s . 14-16, Table V ) . However, when the 1/Km values of ala n i n e were p l o t t e d against 1/[a-ketoglutarate] a U-shaped curve r e s u l t e d i l l u s t r a t i n g that above 2.5 mM a - k e t o g l u t a r a t e , increased a-ketoglutarate concentrations caused a sharp decrease i n the Km value of al a n i n e ( F i g . 17). The cause of t h i s e f f e c t i s not known. The GOT k i n e t i c s showed that sGOT had a much higher a f f i n i t y f o r a-ketoglutarate and glutamate, a s l i g h t l y higher a f f i n i t y f o r o x a l a c e t a t e and a much lower a f f i n i t y f o r aspartate than mGOT at both pH 6.8 and 7.3 (F i g s . 18-21, Table V I ) . When the assay pH was decreased from 7.3 to 6.8, the Km values of both amino acids increased w h i l e the Km values o f both a-keto a c i d s decreased f o r both sGOT and mGOT as observed w i t h GPT (Table V). This r e c i p r o c a t i n g r e l a t i o n s h i p was r e f e l c t e d i n the apparent e q u i l i b r i u m constants (K'eq) which remained the same f o r both sGOT and mGOT de s p i t e the change i n pH (Table V I I ) . The mGOT favoured the production of a-ketoglutarate and a s p a r t a t e more than sGOT. 45 Figure 12: Effect of amino acid substrate concentration on the activity of dolphin muscle glutamate-pyruvate transaminase. Alanine and glutamate saturation were examined in the presence of 1.0 mM a-ketoglutarate and 5.0 mM pyruvate, respectively. The Km values are summarized in Table V. The symbols are: alanine at pH 7.3 ( 0 ) and 6.3 ( • ); glutamate at pH 7.3 ( A ) and 6.3 (A ); v = AE-,n per min. 47 Figure 13: Effect of a-keto acid substrate concentration on the activity of dolphin muscle glutamate-pyruvate transaminase in the presence of 25 mM amino acid co-substrate. The Km values are summarized i n Table V. The symbols are: a-ketoglutarate at pH 7.3 ( 0 ) and 6.3 ( • ); pyruvate at pH 7.3 ( A ) and 6.3 ( A ); v = A E 3 4 0 per min. 1 I 2 3 / * - K e t o acid (mM) 49 TABLE V Summary of Km values for dolphin muscle  glutamate-pyruvate transaminase. A l l Km values at pH 7.3 were determined in duplicate. A l l Km values at pH 6.3 were determined in tri p l i c a t e at the concentration of co-substrate that was saturating or very nearly saturating at pH 7.3. A l l assays were by the standard method. Km (mM) of substrate Substrate a-ketoglutarate Alanine Pyruvate Glutamate Co-substrate Alanine a-ketoglutarate Glutamate Pyruvate Concentration of Co-sub-strate (mM) 25 1.0 25 » 5.0 °° pH 7.3 0.45 0.56 8.2 9.4* 0.87 1.1 15 17 pH 6.3 0.44 34 0.49 44 This value i s in some doubt as a plot of 1/Km (alanine) vs. 1/[a-ketoglutarate] was not linear as described in the text and in Figure 17. 50 Figure 14: Determination of the absolute Km of a-ketoglutarate for dolphin muscle glutamate-pyruvate transaminase at pH 7.3. All assays were by the standard method at 5, 10, 25 and 100 mM alanine using 0.1 to 5.0 mM a-ketoglutarate. Km on the ordinate refers to a-ketoglutar-ate (mM). S = alanine (mM). The results are summarized in Table V. 52 Figure 15: Determination of the absolute Km of glutamate for dolphin muscle glutamate-pyruvate transaminase at pH 7.3. A l l assays were by the standard method at 0.5, 1.5 and 5.0 mM pyruvate using 1.25 to 25 mM glutamate. Km on the ordinate refers to glutamate (mM). S = pyruvate (mM). The results are summarized in Table V. 53 O CM (J) \ m 6 O 54 Figure 16: Determination of the absolute Km of pyruvate for dolphin muscle glutamate-pyruvate transaminase at pH 7.3. A l l assays were by the standard method at 2.5, 7.5 and 25 mM glutamate using 0.2 to 3.0 mM pyruvate. Km on the ordinate refers to pyruvate (mM). S = gluta-mate (mM). The results are summarized in Table V. 56 Figure 17: Determination of the absolute Km of alanine for dolphin muscle glutamate-pyruvate transaminase at pH 7.3. A l l assays were by the standard method at 0.5, 1.0, 1.5, 2.5, 5.0 and 10.0 mM a-ketoglutar-ate using 3.0 to 50 mM alanine. Km on the ordinate refers to alanine (mM) . S = a-ketoglutarate (mM). The results are summarized in Table V. 0 -22 58 Figure 18: Effect of a-ketoglutarate concentration on the activity of dolphin muscle glutamate-oxalacetate transaminase. The results and the conditions of assay are summarized i n Table VI. The symbols are mitochondrial enzyme at pH 7.3 ( n ) and 6.8 ( f ) ; supernatant enzyme at pH 7.3 ( A ) and 6.8 ( A ) ; v = A E ^ Q per min. S = a-ketoglutarate (mM). o IO CO 60 Figure 19: Effect of glutamate concentration on the activity of dolphin muscle glutamate-oxalacetate transaminase. The results and conditions of assay are summarized in Table VI. The symbols are as in Figure 18 except that S = glutamate (mM). 62 Figure 20: Effect of oxalacetate concentration on the activity of dolphin muscle glutamate-oxalacetate transaminase. The results and conditions of assay are summarized i n Table VI. The symbols are as in Figure 18 except that S = oxalacetate (mM). 63 64 Figure 21: Effect of aspartate concentration on the activity of dolphin muscle glutamate-oxalacetate transaminase. The results and conditions of assay are summarized in Table VI. The symbols are as in Figure 18 except that S - aspartate (mM). 65 TABLE VI Summary of Km values for dolphin muscle glutamate-oxalacetate transaminase. A l l Km's were determined in duplicate at the concentration of co-substrate that was saturating or very nearly saturating at pH 7.3 using the standard method. Km (mM) of substrate Substrate a-ketoglutarate Aspartate Oxalacetate Glutamate Co-substrate Aspartate a-ketoglutarate Glutamate Oxalacetate Concentration mGOT sGOT mGOT sGOT mGOT sGOT mGOT sGOT of co-substrate (mM) 10 10 5 2 24 12 0.4 0.4 Mitochondrial pH 7.3 0.76 0.50 0.10 9.4 pH 6.8 0.68 0.65 0.06 11.0 Supernatant pH 7.3 0.13 2.4 0.06 3.2 pH 6.8 0.09 3.7 0.05 4.6 67 TABLE VII Apparent equilibrium constants (K'eq) for dolphin muscle  glutamate-oxalacetate transaminase Purified GOT (2 units in 0.1 ml) was added to 4.9 ml of 100 mM sodium phos-phate buffer containing 7.5 moles each of a-ketoglutarate and aspartate. Aliquots of 0.75 ml were added to 7.5 ml of 10% trichloroacetic acid (TCA) after 15 and 20 min. The precipitates were sedimented and aliquots of the supematants were transferred to cuvettes, neutralized with IN NaOH and assay-ed for a-ketoglutarate, oxalacetate and aspartate content by the standard method at pH 7.3. The a-ketoglutarate assay contained 0.2 ml of TCA super-natant, 16 mM ammonium sulphate and 6 units of glutamate dehydrogenase; the oxalacetate assay, 0.5 ml of TCA supernatant and 10 units of malate dehydro-genase; the aspartate assay, 0.2 ml of TCA supernatant, 50 mM a-ketoglutarate, 10 units of malate dehydrogenase and 5 units of Sigma GOT. The A-^bO was determined before the addition of enzyme, after the reaction had gone to com-pletion and after the addition of another aliquot of enzyme to allow correc-tion for the absorbance change caused by the addition of enzyme. Glutamate production was assumed to equal oxalacetate production. Each K'eq was deter-mined in duplicate and calculated from the equation: K'e = [ a ~ ^ e t ° g i u c a r a t e ] [aspartate]  e q [oxalacetate] [glutamate] using the 20 min values which represented equilibrium in a l l cases. Enzyme PH K'eq Mitochondrial 7.3 9.27 6.8 9.18 Supernatant 7.3 6.77 6.8 6.90 68 Inhibition of GOT The sGOT and mGOT were assayed i n both directions with the following metabolites: AMP, 0.5 mM; cyclic AMP, 0.2 mM; ADP, 1.0 mM; ATP, 1.0 mM; aspartate, 24 mM; creatine phosphate, 2.5 mM; fumarate, 1.0 mM; glutamate, 5.0 mM; glyoxylate, 1.0 mM; a-ketoglutarate, 3 mM; DL-lactate, 20 mM; L-malate, 3 mM; pyruvate, 1.0 mM; NAD, 0.5 mM; and succinate, 3 mM. Only succinate and glutamate were inhibitory in the direction of glutamate produc-tion while only malate and a-ketoglutarate were inhibitory in the direction of a-ketoglutarate production (Figs. 22-25, Table VIII). Inhibition was more effective at pH 6.8 than at pH 7.3 in a l l cases with the exception of the inhibition of mGOT by glutamate and a-ketoglutarate. The sGOT was more sensitive than mGOT to inhibition by succinate, glutamate and a-ketoglutarate while the reverse was true with malate. Substrate inhibition by oxalacetate and a-ketoglutarate was also examined (Figs. 26, 27, Table IX). Again, inhibition was greater at pH 6.8 than at pH 7.3 and sGOT was inhibited to a greater extent by both substrates. GOT specificity No measurable transamination of the a-keto acids of tyrosine, leucine or phenylalanine with glutamate was observed with the purified mGOT or sGOT preparations. 69 Figure 22: Succinate inhibition of dolphin muscle glutamate-oxalacetate transaminase. The results and conditions of assay are summarized in Table VIII. The symbols are as i n Figure 18 except that v = relative activity and I = succinate (mM). 70 71 Figure 23: Glutamate i n h i b i t i o n o f d o l p h i n muscle glutamate-oxalacetate transaminase. The r e s u l t s and c o n d i t i o n s of assay are summarized i n Table V I I I . The symbols are as i n Figure 22 except that I = glutamate (mM). 73 Figure 24: Malate inhibition of dolphin muscle glutamate-oxalacetate transamin-ase. The results and conditions of assay are summarized in Table VIII. The symbols are as i n Figure 22 except that I = malate (mM) . 74 75 Figure 25: a-ketoglutarate inhibition of dolphin muscle glutamate-oxalacetate transaminase. The results and conditions of assay are summarized in Table VIII. The symbols are as in Figure 22 except that I = a-ketoglutarate (mM). 76 TABLE VIII Summary of inhibition of dolphin muscle glutamate-oxalacetate  transaminase by dicarboxylates. The substrate concentrations used approximate the Km's listed in Table VI with the exception of oxalacetate since assays at Km concentrations of this substrate were non-linear with time. A l l assays were by the standard method except for those used to study a-ketoglutarate inhibition. These assays contained, in a f i n a l volume of 2.5 ml, the indicated concentrations of substrate but no NADH or coupling enzyme. The rate of enzyme activity was determined by following the disappearance of oxalacetate at 280 nm. The concentrations of inhibitors were varied from 0 to 25 mM and the concentration required for 50% inhibition was determined as described in Materials and Methods. Enzyme pH of assay Substrate concentration (mM) Inhibitor concentration (mM) required for 50% inhibition a-ketoglutarate Aspartate Succinate Glutamate Mitochondrial 7.3 0.73 0.50 70 24 6.8 0.73 0.50 31 24 Supernatant 7.3 0.15 3.0 40 10 6.8 0.08 3.0 14 7.2 Oxalacetate Glutamate Malate a-ketoglutarate Mitochondrial 7.3 0.10 10.0 6.0 16 6.8 0.10 10.0 2.9 19 Supernatant 7.3 0.10 4.0 9.0 3.2 6.8 0.10 4.0 5.0 2.2 78 Figure 26: Oxalacetate substrate inhibition of dolphin muscle glutamate-oxalacetate transaminase. The results and conditions of assay are summarized in Table IX. The symbols are as in Figure 22 except that S = oxalacetate (mM). 79 80 Figure 27: a-ketoglutarate substrate inhibition of dolphin muscle glutamate-oxalacetate transaminase. The results and conditions of assay are summarized in Table IX. The symbols are as i n Figure 22 except that S = a-ketoglutarate (mM). 82 TABLE IX Summary of inhibition of dolphin muscle glutamate-oxalacetate  transaminase by the a-keto acid substrates. A l l assays were conducted by the standard method at the indicated amino acid concentrations and a-keto acid concentrations of 0.1 to 15 mM. The amino acid concentrations approximate the Km values lis t e d in Table VI. Enzyme pH of Concentration of assay amino acid substrate % Inhibition at 5 mM con-centration of a-keto acid substrate Mitochondrial 7.3 6.8 7.3 6.8 Oxalacetate a-ketoglutarate Glutamate, 10 mM it II Aspartate, 0.6 mM II II 17.5 36.6 0.00 5.9 Supernatant 7.3 6.8 7.3 6.8 Glutamate, 4 mM II II Aspartate, 3 mM 21.5 41.2 18.5 38.6 83 DISCUSSION The transaminase reaction A l l transaminases are enzymes which contain a tightly bound coenzyme, pyridoxal phosphate, a derivative of Vitamin (also called pyridoxal and pyridoxamine). This coenzyme allows the transaminases to catalyze the transfer of the a-amino group of amino acids onto the a-keto carbon of an a-keto acid, such as pyruvate, via two intermediate Schiff's bases. To effect this transfer, the coenzyme must be in the pyridoxal form to combine with the amino acid to form a Schiff's base: Enzyme - C = 0 I H pyridoxal form HO <-H I H N — C — R COOH a-amino acid, H I Enzyme - C = N - C - R I I H COOH Schiff's base I The Schiff's base i s then hydrolyzed to yield the pyridoxamine form of the coenzyme and the a-keto acid of the amino acid. The pyridoxamine form of the enzyme then combines with a second a-keto acid to form the second Schiff's base: Schiff's base I H Enzyme - C - NH,, H pyridoxamine form H20 0 = C - R. I COOH a-keto acid. 84 H I Enzyme - C - NH2 H pyridoxamine form H 0 = C - R I COOH a-keto acid. -> H20 Enzyme C - N = C - R0 H COOH Schiff's base II When this second Schiff's base is hydrolyzed, the amino group is donated to the second a-keto acid and the enzyme is returned to the pyridoxal form: Schiff's base II H20 Enzyme - C = 0 H pyridoxal form H I H.N - C - R9 2. , 2 COOH a-amino acid„ The transaminase reaction i s freely reversible and most transaminases have an equilibrium constant that is close to 1. For both GPT and GOT, there is one binding site for a l l four substrates (Henson and Cleland, 1964; Hopper and Segal, 1962; Velick and Vavra, 1962) and this site functions according to a "Ping Pong Bi B i " mechanism. Simply, this means that one substrate (the amino acid) is bound, converted and released as a product before the second substrate (the a-keto acid) is bound. Comparative aspects of the transaminases The purification procedures for GPT and GOT did not provide completely homogeneous preparations since they were primarily chosen to eliminate, in a minimum number of steps, contaminating enzymes that u t i l i z e the same substrates 85 as the transaminases, namely, lactate and malate dehydrogenases. This condition was achieved and enabled the determination of kinetic constants for a l l 4 substrates for each enzyme. However, considerable purification was realized. For example, the purification of GPT (Table I) compared well with the degree of purification obtained i n other laboratories although the specific activities of our preparations were 10 to 15-fold lower (Saier and Jenkins, 1967; Gatehouse et a l . , 1967; Matsuzawa and Segal, 1968; Segal et a l . , 1962). This discrepancy could be due to dolphin muscle GPT having a lower turnover number than the rat l i v e r and pig heart GPT used i n the other laboratories. The purification of GOT (Table III) compared well with the specific activity in addition to the degree of purification achieved i n other laboratories (Martinez-Carrion et a l . , 1965; Bertland and Kaplan, 1968; Magee and Phi l l i p s , 1971; Aoki et a l . , 1972; Krista and Fonda, 1973; Shrawder and Martinez-Carrion, 1973). No subforms of GPT or the GOT isozymes from dolphin muscle were observed on starch gels (Fig. 7), polyacrylamide disc gels or ion exchange columns (Figs. 2, 4, 5). The only evidence of subforms was observed during the electrofocusing of sGOT (Fig. 6), possibly because the a-ketoglutarate and pyridoxal-5-phosphate included i n the sample for stabilization were removed by the electrofocusing. Immediately after the recovery of the sGOT from the electrofocusing column, the a-ketoglutarate and pyridoxal-5-phosphate were replaced and i t was after this addition of stabilizers that no difference could be found between the two forms of sGOT by electrophoretic or kinetic analysis. Since other workers (Magee and P h i l l i p s , 1971; Marino et a l . , 1972; Bertland and Kaplan, 1970) describe the appearance of new subforms of GOT under certain conditions i t seems possible that the two subforms observed for dolphin muscle sGOT arose as a result of the electrofocusing conditions and 86 were restored to the original form by the stabilizers (Fig. 8). If the dolphin muscle enzymes do not occur in multiple forms, this is in contrast to the results of other workers who have demonstrated up to 6 forms of sGOT (Bertland and Kaplan, 1968, 1970; Krista and Fonda, 1973; Shrawder and Martinez-Carrion, 1973; Martinez-Carrion and Teimeier, 1967; Martinez-Carrion et al., 1967; Michuda and Martinez-Carrion, 1969), four forms of mGOT (Bertland and Kaplan, 1968; Magee and Phillips, 1971; Shrawder and Martinez-Carrion, 1973; Martinez-Carrion and Teimeier, 1967; Michuda and Martinez-Carrion, 1969), and five forms of GPT (Chen et al., 1973; Gatehouse eit al., 1967), but in agreement with those who observed only one form of sGOT (Magee and Phillips, 1971), mGOT (Bertland and Kaplan, 1970), and GPT (Saier and Jenkins, 1967). The reason for the occurrence of the subforms of the GOT isozymes has not yet been elucidated but recent reports (Magee and Phillips, 1971; Bertland and Kaplan, 1970; Michuda and Martinez-Carrion, 1969) have more or less agreed that the major, i f not the only detectable difference between the subforms is the ratio of "catalytically active" to"catalytically inactive" content of pyridoxal phosphate. This fits in well x^ tith our observation that two forms of sGOT are apparently converted to one common form by the addition of pyridoxal-5-phosphate and a-ketoglutarate. However, Michuda and Martinez-Carrion (1969) felt that such coenzyme differences could not explain the occurrence of the subforms. They stated that after removal of only the "catalytically active" pyridoxal phosphate the subforms retained their electrophoretic integrity and, from this information, they concluded that different modes of binding of the coenzymes could not produce the subforms of GOT. Their reasoning is not quite clear since the "catalytically inactive" coenzyme was presumably s t i l l bound to the enzyme and possibly could have accounted for the electrophoretic differences observed. 87 The single form of dolphin muscle GPT observed is somewhat puzzling since GPT, like GOT, commonly exists in both supernatant and mitochondrial forms (Sallach and Fahien, 1969). If both forms do exist in dolphin muscle i t seems unlikely that they are electrophoretically identical but more lik e l y that the mitochondrial GPT was destroyed by freezing of the tissue since i t is known to be extremely labile (Sallach and Fahien, 1969). The other possibility is that no mitochondrial GPT exists, as in guinea pig heart (Davis, 1968). In either case, the one form observed is undoubtedly supernatant GPT. Further characterization of dolphin muscle GOT was provided by the molecular weight studies (Fig. 9, Table IV). These experiments were performed primarily to determine the reason for the co-purification of a portion of the malate dehydrogenase activity with sGOT, a situation also observed in other laboratories (Krista and Fonda, 1973; Martinez-Carrion and Teimeier, 1967). The very similar apparent molecular weights for malate dehydrogenase and GOT activities in the purified sGOT preparation at least partly establish a basis for this co-purification. The values of approximately 80,000 are unusual for both enzymes since the molecular weights of malate dehydrogenase and GOT are usually about 60,000 (Murphy et a l . , 1967) and 90,000 to 110,000 (Krista and Fonda, 1973) respectively, although Magee and Phillips (1971) obtained a value of 79,000 for rat brain sGOT. The molecular weight estimates of 60,000 obtained for malate dehydrogenase in the crude dolphin muscle preparation and the mixture of Sigma pig heart enzymes ill u s t r a t e that the high molecular weight malate dehydrogenase in the sGOT preparation either arose as an a r t i -fact during the purification of sGOT or represents a different species of this enzyme. The consistently low apparent molecular weight of sGOT from a l l sources probably results from the characteristic of molecular sieves to measure Stokes radii rather than true molecular weights. Since the dolphin muscle GOT in both the sGOT and crude preparations migrated through the gel at the same rate as the Sigma pig heart GOT i t is concluded that the true molecular weight of both sGOT and mGOT is probably identical to the value of 110,000 found for pig heart GOT (Michuda and Martinez-Carrion, 1969; Jenkins et al., 1959). It is not clear why the values found for GOT in the present study differ from those determined by other workers using G-200 (Krista and Fonda, 1973; Michuda and Martinez-Carrion, 1969). Role of the transaminases in the diving mammal A) Energy production The role of GOT in the maintenance of proper NAD+/NADH ratios (redox balance) between the cytoplasm and mitochondria and of GOT and GPT in the anaplerotic maintenance of Kreb's cycle intermediates is well established (Safer and Williamson, 1973; Krebs and Veech, 1969; Safer et al., 1971; Lowenstein, 1972; Davis et. al., 1972; Davis and Bremer, 1973; LaNoue et al., 1973). We suggest that these two functions are especially important to diving mammals for reasons which will be described below. In addition, we believe that the two transaminases catalyze the first steps in a pathway which could produce ATP from the carbon skeletons of amino acids during hypoxia (Fig. 28). The model is based on the substrate level phosphorylation of GDP at the succinyl thiokinase step of the Krebs cycle being coupled by NAD+ and NADH to the energy producing reduction of fumarate to succinate by a reversal of the succinate dehydrogenase reaction. This latter reaction has been observed in mammalian tissues (Sanadi and Fluharty, 1963; Wilson and Cascarano, 1970; Chance and Hollunger, 1960; Penney and Cascarano, 1970; Randall and Cohen, 1966) and other animals (Saz, 1971; DeZoeten and Tipker, 1969). When these two reactions are coupled as shown in Figure 28, redox balance within the mito-chondria is maintained and succinate accumulates as has been observed during 89 Figure 28: A metabolic map indicating the postulated role of glutamate-pyruvate and glutamate-oxalacetate transaminases during anaerobic excursions in the diving mammal. Aspartate mobilization is thought to lead to an energy-yielding reduction of fumarate, while a-ketoglutarate derived from the GPT reaction i s presumably con-verted to succinylCoA, a process setting the stage for a substrate-level phosphorylation. The two pathways are thought to be linked functionally by the formation of a redox couple between fumarate reduction and the a-ketoglutarate dehydrogenase reaction and by the cycling of glutamate and a-ketoglutarate between the GOT and GPT reactions. 90 ASPARTATE oxalacetate 1^-—NADH malate GLUCOSE GIP ^--^•NAD - ^J DPG » PEP NADH-1 pyruvate NADH s / \ ^ GLUTAMATE -NAD'*/ V ^ oC-ketoglutarate malate malate lactate alanine succinate succinate «-ketoglutarate P; succinate CYTOSOL ATP fumarate ADP+P. MITOCHONRION succinate oC-ketoglutarate GTP-GDP+ P.-C o A S H f FADH FAD -•succinate succinylCoA NAD N'ADH * NAD NADH hypoxia i n mammals (Sanadi and Fluharty, 1963; Wilson and Cascarano, 1970; Hochachka et a l . , 1974; Kondrashova and Chahovets, 1971; Penney and Cascarano, 1970; Hoberman and Prosky, 1967; Mensen deSilva and Cazorla, 1973; A r i l l o and deGiuli, 1971) and other animals (Saz, 1971; DeZwaan and Marrewijk, 1973; A r i l l o and deGuili, 1971; DeZoeten et a l . , 1969; Malanga and Aiello, 1972; Stokes and Apawara, 1968; DeZwaan and Zandee, 1972). The succinate can pass out of the mitochondria in an exchange for malate involving phosphate as an intermediate and in direct exchange for a-ketoglutarate (Chappell, 1968; Klingenberg, 1970a, 1970b) and may exert some control over the sGOT reaction as a product inhibitor (Fig. 22, Table VIII). Fumarate for the reversed succinate dehydrogenase reaction could be supplied by the transamination of aspartate by sGOT to form oxalacetate which is then reduced to malate by malate dehydrogenase. The malate passes into the mitochondria in exchange for succinate as previously mentioned and is converted to fumarate by fumarase, which is freely reversible. The a-ketoglutarate for the succinyl thiokinase reaction could be supplied by the transamination of glutamate by GPT while a-ketoglutarate for the sGOT reaction could come from endogenous pools u n t i l a cycling of this metabolite and glutamate between sGOT and GPT could be established. Significant amounts of aspartate and glutamate for sGOT and GPT, respectively, could be derived from the pools of free amino acids which consist largely of these two amino acids (Safer and Williamson, 1973; Brosnan et a l . , 1970). Indeed, aspartate levels have been observed to decrease during hypoxia in the tissues of reptiles and mammals (Hochachka et a l . , 1974; Brosnan et a l . , 1970; A r i l l o et a l . , 1972). On the basis of enzyme ac t i v i t i e s , pyruvate is considered to be the primary acceptor of glutamate nitrogen from the GPT reaction since, other than pyruvate and oxalacetate, only the a-keto acid of tyrosine participates in the tranaminase reaction with glutamate in crude 92 dolphin muscle preparations (see Results). This activity is very low despite the fact that the particulate fraction of the homogenate, which contains significant transaminase activities (Cammarata and Cohen, 1950; Hird and Roswell, 1950) was included in the assays. The small amount of tyrosine aminotransferase activity observed was probably not due to a non-specific reaction of GOT since, unlike GOT from other sources (Miller and Litwack, 1971; Shrawder and Martinez-Carrion, 1972), both dolphin muscle mGOT and sGOT were totally inactive with the a-keto acids of tyrosine, phenylalanine and leucine (see Results). GPT activity in dolphin muscle, however, is 5 to 15-fold higher (16 U/g of tissue) than that found in the muscle of other mammals (Zimmerman et al., 1968). This GPT activity is definitely not due to GOT as suggested by Miller and Litwack (1971) since the two activities can be totally separated by ammonium sulphate fractionation (Tables I and III) . Further support for the contention that pyruvate acts as the primary acceptor for glutamate nitrogen comes from the observation that alanine accumulates as an end product of hypoxia in reptilian and mammalian tissues (Hochachka et al . , 1974; Arillo et al., 1972; Felig and Wahren, 1971). If pyruvate is to play the key role indicated in Figure 28, a continuous source must be ensured. It is suggested that glucose is the source of pyruvate 2 since, in the dolphin muscle, there was no malic enzyme which converts malate to pyruvate. Although Reeves (1963) reported that essentially 100% of labelled glucose was converted to lactate in turtle hearts during hypoxia, Brachfeld et al, (1972) reported a lower value for the same diving species. In view of their results i t seems unlikely that such total conversion of glucose to lactate represents the only possibility. Also, i t would indicate that the only P. W. Hochachka, unpublished data 93 pathway for the regeneration of NAD+ consumed by glycolysis would be the lactate dehydrogenase reaction. In Figure 28 i t is apparent that the malate dehydrogenase reaction could provide NAD+ for glycolysis. Fatty acid synthesis is also believed to be a source of NAD+ during hypoxia (Brachfeld et al., 1972; Cherchi et al., 1970; Oedejans and Van der Horst, 1974; Van der Horst, 1974; Whereat et al., 1967). These additional oxidizing equivalents could presumably free some pyruvate for transamination with glutamate. Indeed, i t has been reported that when glycolysis is stimulated in mammalian tissues, the amino acids are in near-equilibrium with their a-keto acids, the specific radio-activity of alanine approaches that of lactate (Gailis and Benmouyal, 1973) and accumulated alanine accounts for 12-18% of the glucose consumed (Felig and Wahren, 1971). These data suggest that GPT can effectively compete with lactate dehydrogenase for pyruvate under conditions of hypoxia. As these conditions continue, the NAD+/NADH ratio and the pH both decrease (Chance et al., 1964) which drives the lactate dehydrogenase in dolphin muscle farther towards the production of lactate (Table X). This effect must be countered by mass action effects of lactate accumulation on lactate dehydrogenase. Speci-fically, since the conversion of pyruvate to lactate does not go to completion, an accumulation of lactate must be accompanied by an accumulation of pyruvate which is dictated by the equilibrium constant of lactate dehydrogenase and may approach 1/20 the concentration of lactate (White and Kaplan, 1972). Also, since the Km of pyruvate for GPT decreases with pH (Fig. 13, Table V), GPT probably effectively competes for pyruvate throughout the duration of hypoxia. This conclusion is confirmed by the previously mentioned alanine accumulation observed in vertebrate tissues (Hochachka et al., 1974; Arillo et al., 1972; Felig and Wahren, 1971). Another effect of the decrease in pH is that the Km of a-ketoglutarate for sGOT is decreased (Fig. 18, Table VI) while succinate 94 TABLE X Apparent equilibrium constants (K'eq) of dolphin  and rabbit muscle lactate dehydrogenases. Dolphin muscle was homogenized in 100 mM sodium phosphate, pH 6,8 (4 ml/g of tissue) and centrifuged at 30,000 x g for 30 min. Rabbit muscle lactate dehydrogenase from Sigma was diluted 10-fold to yield 750 units/ml. Aliquots (0.02 ml) of these preparations were added to cuvettes containing 2.25 ml of 30 mM L-lactate plus 5 mM NAD+ in 100 mM sodium phosphate buffer and the reaction was allowed to go to completion. The was recorded before the addition of enzyme, after the reaction had gone to completion and after the addition of another aliquot of enzyme to allow correction for the absorbance change caused by the addition of enzyme. K'eq was determined using the equation: , [pyruvate] [NADH] K e q [lactate] [NAlF] Source of , lactate K'eq x 10 dehydrogenase pH 6.3 pH 6.8 pH 7.0 pH 7.3 Dolphin muscle 0.21 0.62 1.08 1.96 Rabbit muscle 0.18 0.58 0.98* 1.75 This value agrees closely with that of 1.11 x 10 cited by Krebs and Veech (1969) determined at the same pH. 95 inhibition of sGOT is enhanced to a point where i t is as effective as gluta-mate as an inhibitor (Figs. 22, 23, Table VIII). Since glutamate levels would presumably f a l l as a-ketoglutarate was produced by GPT, i t seems possible that succinate and glutamate exert a reciprocal control over the sGOT reaction. The significance of the remainder of the inhibitions by metabolites shown in Figures 24-27 and Tables VIII and IX is difficult to ascertain at this time but i t seems likleythat pools of these intermediates could be instrumental in the controlling of the direction of the GOT reaction as suggested by Michuda and Martinez-Carrion (1970). To summarize the model (Fig. 28), i t is apparent that for every pair of pyruvate molecules converted to alanine, which accumulates, one glutamate molecule and one aspartate molecule are converted to succinate via a-ketoglu-tarate and oxalacetate, respectively. This process results in the production of 2 ATP molecules, the accumulation of 2 molecules of succinate and the consumption of one molecule each of aspartate and glutamate. Therefore, the conversion of glucose to pyruvate using the NAD+ provided by malate dehydro-genase and possibly, fatty acid synthesis (Brachfeld et al., 1972; Cherchi et al., 1970; Whereat et al., 1967; Oedejans and Van der Horst, 1974; Van der Horst, 1974) and the subsequent transamination of pyruvate to alanine allows 4 ATP molecules to be produced per molecule of glucose at the expense of two molecules of amino acid (glucose to pyruvate: 2 ATP/glucose, fumarate reductase: 1 ATP, and succinyl thiokinase: 1 ATP). Since the amino acids consumed are not irretrievably catabolized but rather are merely transaminated so that the concentration of total amino acids in the tissue remains the same (Gailis and Benmouyal, 1973), the organism could well afford to temporarily expend the carbon skeletons of aspartate and glutamate to double the ATP production from a percentage of the available glucose molecules. Felig and Wahren (1971) 96 ill u s t r a t e that this percentage could be as high as 18% and hence, would be of significant survival value to a diving mammal. B) Maintenance of redox balance As was previously mentioned, NAD+/NADH ratios decrease during hypoxia (Chance et a l . , 1964). After a diving mammal has returned to aerobic conditions the accumulated cytoplasmic NADH must be reoxidized to NAD+. Since glucose synthesis does not take place in muscle tissue (Krebs, 1964), the lactate and alanine accumulated during hypoxia are flushed into the blood-stream (Andersen, 1966; Scholander, 1940, 1962; Felig and Wahren, 1971) and transported to the l i v e r for gluconeogenesis. Consequently, cytoplasmic NADH cannot be oxidized via lactate dehydrogenase and presumably, i t s reducing equivalents must be transported into the mitochondria via the aspartate-malate cycle (Fig. 29) for oxidation by the electron transport chain (Safer and Williamson, 1973; Krebs and Veech, 1969). The fact that diving mammals en-counter more severe hypoxia and probably a greater accumulation of NADH than other mammals suggests that the aspartate-malate cycle should be very active in the muscle tissue of these animals. This supposition is supported by a number of facts. F i r s t , the GOT activity in dolphin muscle i s 3 to 17-fold higher (135 U/g of tissue) than that found in the muscle tissue of other mammals (Zimmerman et a l . , 1968). Next, there is an approximately equal distribution of the enzyme activity between the mitochondria and cytosol, according to the separation of the two forms on CM-cellulose (Table III) and by starch gel electrophoresis (Fig. 7). Finally, for the aspartate-malate cycle to transfer reducing equivalents (NADH) into the mitochondria, sGOT must catalyze the transamination reaction i n the direction of glutamate production while mGOT must produce aspartate. Table VII shows that although both forms favour aspartate formation, mGOT does so more strongly than sGOT and GOT from 97 Figure 29: The malate-aspartate cycle. Reducing equivalents in the form of NADH are transported into the mitochondria via aspartate derived malate. This complex cycle is necessary since the mitochondrial membrane is impermeable to NADH. 98 aspartate cc-ketoglutarate glutamate oxalacetate v malate NADH N A D + cytoplasm mitochondria malate N A D + NADH oxalacetate >aspartate -> glutamate—>cc-ketoglutarate 99 other sources (Veech et a l . , 1969). This situation would prevail during the entire period of recovery from hypoxia since, as Table VII confirms, the K'eq of GOT is independent of pH as stated by Krebs and Veech (1969). C) Augmentation of metabolites The third and fi n a l role for the transaminases that was introduced i n this discussion was one of anaplerotic maintenance of Krebs cycle intermediates. In diving mammals, carbohydrates are believed to be the preferred substrate during anaerobic metabolism (George ejt a l . , 1971; Kerem et a l . , 1973; Vallyathan et a l . , 1969). Aerobic metabolism, on the other hand, is maintain-ed essentially exclusively by lipids (Pierce, 1973). This conclusion is supported by the following observations: 1) harp seals possess an active muscle lipase and a high serum fat content (George et a l . , 1971; Vallyathan et a l . , 1969), and 2) resting and slightly active sea lions have R.Q.'s of 0.74 and 0.63, respectively (Whittow, 1974), which correspond closely to the value of 0.71 derived for fat metabolism (Prosser and Brown, 1961). It is also widely acknowledged that the marine mammals have vast blubber supplies (McGinnis et a l . , 1972) which are ut i l i z e d extensively during migrations. Fats, when ut i l i z e d for energy production, are converted to acetyl CoA which then enters the Krebs cycle. It is therefore clear that additional supplies of oxalacetate would be required for the citrate synthase reaction (which condenses oxalacetate and acetyl CoA to form citrate) during the intensive and sustained l i p i d oxidation that would be expected to occur i n the diving mammals. These supplies could be derived from the cytoplasmic trans-amination of aspartate to oxalacetate which would then pass into the mito-chondria in the form of malate as in the aspartate-malate cycle (Fig. 29). As previously described in this report and by Safer and Williamson (1973) and Safer et a l . (1971), the a-ketoglutarate required for the i n i t i a l transamina-100 tion of aspartate would be derived from endogenous pools and then regenerated by GPT. The result of this cycling of glutamate and a-ketoglutarate is the stoichiometric accumulation of alanine that equals the augmentation of Krebs cycle intermediates (Safer et al., 1971; Safer and Williamson, 1973). In conclusion, we suggest that the two transaminases, GPT and GOT, are particularly important in the intermediary metabolism of diving mammals since they perform key functions during most of the metabolic conditions that the animals are subjected to, namely, ATP production during hypoxia, restoration of redox balance after hypoxia and anaplerotic maintenance of Krebs cycle intermediates under a l l conditions but particularly during the periods of intense lipid oxidation encountered while migrating. 101 LITERATURE CITED Andersen H. T. (1966) Physiological adaptations in diving vertebrates. Physiol. Rev. 46, 212-243. Aoki K., Calva E. & Fonesca L. (1972) Crystallization and immunological identification of dog heart aspartate aminotransferase isoenzymes. J_. Biochem. 72, 511-519. Arillo A., Balletto E. & Cherchi M. A. (1972) Influenza dell'anaerobiosi sulle concentrazioni tissulari degli amino acid liberi. 1° studi su encefalo di Testudo hermanni Gmelin (Reptilia, Testudinidae). Boll. Mus. 1st. Biol. Univ. Genova 40, 5-13. Arillo A. & DeGiuli A. M. (1970) Variazioni del ciclo di Krebs in anaerobiosi - I. Studi su Testudo hermanni Gmelin (Reptilia, Testudinidae). Boll. Mus. 1st. Biol. Univ. Genova 38, 43-63. Arillo A. & DeGiuli A. M. (1971) Anaerobic synthesis of succinic acid in Testudo hermanni Gmelin and Eisenia phoetida. Boll. Zool. 38, 15. Bertland L. H. & Kaplan N. 0. (1968) Chicken heart soluble aspartate amino-transferase. Purification and properties. Biochemistry 7, 134-142. Bertland L. H. & Kaplan N. 0. (1970) Studies on the conformations of the multiple forms on chicken heart aspartate aminotransferase. Biochemistry 9, 2653-2665. Brachfeld N., Ohtaka Y., Klein I. & Kawade M. (1972) Substrate preference and metabolic activity of the aerobic and the hypoxic turtle heart. Circ. Res. 31, 453-467. Brosnan J. T., Krebs H. A. & Williamson D. H. (1970) Effects of ischemia on metabolite concentrations in rat liver. Biochem. J. 117, 91-96. 102 Camraarata P. S. & Cohen P. P. (1950) The scope of the transamination reaction in animal tissues. J_. Biol. Chem. 187, 439-452. Chance B. & Hollunger G. (1960) Energy-linked reduction of mitochondrial pyridine nucleotide. Nature 185, 666-672. Chance B., Schoener B. & Schindler F. (1964) The intracellular oxidation-reduction state. In Oxygen i n the Animal Organism (Edited by Dickens F. & Neil E.), p. 367. Permagon Press, Oxford. Chappell J. B. (1968) Systems used for the transport of substrates into mitochondria. Br. Med. Bull. 24, 150-157. Chen C. & Apawara J. (1969) Intracellular distribution of enzymes catalyzing succinate production from glucose in Rangia mantle. Comp. Biochem. Physiol. 30, 727-737. Chen S. H., Donahue R. F. & Scott C. R. (1973) The genetics of glutamic-pyruvic transaminase in mice: inheritance, electrophoretic phenotypes and postnatal changes. Biochem. Genet. 10, 23-28. Cherchi M. A., Balletto E. & DeGiuli A. M. (1970) Biosintesi di acidi grassi in Testudo hermanni Gmelin sottoposta ad anossia. Boll. Mus. 1st. Univ. Genova 38, 19-25. Christiansen J. & Penney D. (1973) Anaerobic glycolysis and l a c t i c acid accumulation in cold submerged Rana pipiens. J_. Comp. Physiol. 87, 237-245. Copenhaver J. H., McShan W. H. & Meyer R. K. (1950) The determination of glutamic acid dehydrogenase in tissue homogenates. J. Bio l . Chem. 183, 73-79. Davis B. J. (1964) Disc eletrophoresis - II . Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121, 404-427. 103 Davis E. J. (1968) On the nature of malonate-insensitive oxidation of pyruvate and glutamate by heart sarcosomes. Biochim. Biophys. Acta 162, 1-10. Davis E. J. & Bremer J. (1973) Studies with isolated surviving rat hearts. Interdependence of free amino acids and citric-acid-cycle intermediates. Eur. J. Biochem. 38, 86-97. Davis E. J., Lin R. C. & Chao D. L-S. (1972) Sources and disposition of aerobically generated intermediates i n heart muscle. In Energy Metabolism  and the Regulation of Metabolic Processes in Mitochondria (Edited by Mehlman M. A. & Hanson R. W.), pp. 211-238. Academic Press, New York, London. Decker L. E. & Rau E. M. (1963) Multiple forms of glutamic-oxalacetic trans-aminase in tissues. Proc. Soc. Exp. Biol. Med. 112, 144-149. Eisner R. W. (1968) Cardiovascular adjustments to diving. In The Biology of  Marine Mammals (Edited by Andersen H. T.), pp. 117-145. Academic Press, New York. Eisner R., Kooyman G. L. & Drabeck C. M. (1970a) Diving duration in pregnant Weddell seals. In Antarctic Ecology (Edited by Holdgate M. N.), Vol. I, pp. 477-482. Academic Press, New York. Eisner R., Shurley J. T., Hammond D. D. & Brooks R. E. (1970b) Cerebral tolerance to hypoxemia in asphyxiated Weddell seals. Resp. Physiol. 9, 287-297. Ewer R. F. (1947) Whales. New Biol. 2, 53-73. Felig P. & Wahren J. (1971) Interrelationship between amino acid and carbo-hydrate metabolism during exercise: the glucose-alanine cycle. In Muscle Metabolism During Exercise (Edited by Pernow B. & Stalin B.), pp. 205-214. Gailis L. & Benmouyal E. (1973) Endogenous alanine, glutamate, aspartate and glutamine i n the perfused guinea pig heart: effect of substrates and cardiovascular agents. Can. J. Biochem. 51, 11-20. Garber A. J. & Salganicoff L. (1973) Regulation of oxalacetate metabolism i n li v e r mitochondria. J. Biol. Chem. 248, 1520-1529. Gatehouse P. W., Hopper S., Schatz L. & Segal H. L. (1967) Further characteri-zation of alanine aminotransferase of rat l i v e r . J_. Biol. Chem. 242, 2319-2324. George J. C , Vallyathan N. V. & Ronald K. (1971) The harp seal Pagophilus groenlandicus (Erxleben, 1777) - VII. A histophysiological study of certain skeletal muscles. Can. J_. Zool. 49, 25-30. Granath K. A. & Kvist B. E. (1967) Molecular weight distribution analysis by gel chromatography on Sephadex. J_. Chroma tog. 28, 69-81. Henson C. P. & Cleland W. W. (1964) Kinetic studies of glutamic oxalacetic transaminase isozymes. Biochemistry 3, 338-345. Hird F. J. R. & Roswell E. V. (1950) Additional transaminations by insoluble particle preparations of rat l i v e r . Nature 166, 517-518. Hoberman H. D. & Prosky L. (1967) Evidence of reduction of fumarate to succinate i n perfused rat l i v e r under conditions of reduced 0^ tension. Biochim. Biophys. Acta 148, 392-399. Hochachka P. W. & Mustafa T. (1972) Invertebrate facultative anaerobiosis. Science 178, 1056-1060. Hochachka P. W., Owen T. G., Allen J. F. & Whittow G. C. (1974) Anaerobic energy sources in diving vertebrates: the role of succinate production. Comp. Biochem. Physiol, (in press). Hopper S. & Segal H. L. (1962) Kinetic studies of rat l i v e r glutamic-alanine transaminase. J. Bio l . Chem. 237, 3189-3195. 105 Hunter F. E., Jr. (1949) Anaerobic phosphorylation due to a coupled oxidation-reduction between ct-ketoglutaric acid and oxalacetic acid. J_. Biol. Chem. 177, 361-372. Jenkins W. T., Yphantis D. A. & Sizer I. W. (1959) Glutamic aspartic trans-aminase - I. Assay, purification, and general properties. J_. Bio l . Chem. 234, 51-57. Kerem D., Hammond D. D. & Eisner R. (1973) Tissue glycogen levels i n the Weddell seal, Leptonychotes weddelli: a possible adaptation to asphyxial hypoxia. Comp. Biochem. Physiol. 45A, 731-736. Klingenberg M. (1970a) Metabolite transport in mitochondria: an example for intracellular membrane function. Essays i n Biochem. 7, 125-159. Klingenberg M. (1970b) Mitochondria metabolite transport. Fed. Eur. Biochem. Soc. Lett. 6, 145-154. Kondrashova M. N. & Chahovets N. R. (1971) Succinic acid in skeletal muscle during intense activity and rest. Dokl. Biol. Sci. (Engl. Trahsl. Dokl. Akad. Nauk. S. S. S_. R.) 198, 374-376. Kooyman G. L. (1966) Maximum diving capacities of the Weddell seal, Leptonychotes weddelli. Science 151, 1553-1554. Kooyman G. L. (1972) Deep diving behaviour and effects of pressure i n reptiles, birds and mammals. Soc. Exp. Biol. Symp. 26, 295-311. Krebs H. A. (1964) Gluconeogenesis. Proc. R. Soc. Lond. Ser. B^ . Biol. Sci. 159, 545-564. Krebs H. A. & Veech R. L. (1969) Pyridine nucleotide interrelations. In The  Energy Level and Metabolic Control in Mitochondria (Edited by Papa S., Tager J. M., Quagliariello E. & Slater E. C ) , pp. 329-382. Adriatica Editrice, Bari. 106 Krista M. L. & Fonda M. L. (1973) Beef brain cytoplasmic aspartate aminotrans-ferase purification, kinetics, and physical properties. Biochim. Biophys. Acta 309, 83-96. LaNoue K. F., Walajtys E. I. & Williamson J. R. (1973) Regulation of glutamate metabolism and interactions with the citric acid cycle in rat heart mitochondria. J_. Biol. Chem. 248, 7171-7183. Lenfant C. (1969) Physiological properties of blood in marine mammals. In The Biology of Marine Mammals (Edited by Andersen H. T.), pp. 95-116. Academic Press, New York. Lowenstein J. M. (1972) Replenishment and depletion of citric acid cycle intermediates in muscle. In Energy Metabolism and the Regulation of Metabolic Processes in Mitochondria (Edited by Mehlman M. A. & Hanson R. W.), pp. 53-61. Academic Press, New York, London. McGinnis S. M., Whittow G. C, Ohata C. A. & Huber H. (1972) Body heat dissipation and conservation in two species of dolphins. Comp. Biochem. Physiol. 43A, 417-423. Magee S. C. & Phillips A. T. (1971) Molecular properties of the multiple aspartate aminotransferases purified from rat brain. Biochemistry 10, 3397-3405. Malanga C.J. & Aiello E. L. (1972) Succinate metabolism in the gills of the mussels Modiolus demissus and Mytilus edulis. Comp. Biochem. Physiol. 43B, 795-806. Marino G., Paterno M. & DeRosa M. (1972) Multiple forms of aspartate amino-transferase. The formation of I J J - A A T . Fed. Eur. Biol. Soc. Lett. 21, 53-55. Martinez-Carrion M., Riva F., Turano C. & Fasella P. (1965) Multiple forms of supernatant glutamate aspartate transaminase from pig heart. Biochem. Biophys. Res. Comm. 20, 206-211. 107 Martinez-Carrion M. & Tiemeier D. (1967) Mitochondrial glutamate-aspartate transaminase. I. Structural comparison with the supernatant isozyme. Biochemistry 6, 1715-1722. Martinez-Carrion M., Turano C, Chiancone E., Bossa F., Ciartosio A., Riva F. & Fasella P. (1967) Isolation and characterization of multiple forms of glutamate-aspartate aminotransferase from pig heart. J_. Biol. Chem. 242, 2397-2409. Matsuzawa T. & Segal H. L. (1968) Rat liver alanine aminotransferase. Crystallization, composition, and role of sulfhydryl groups. J_. Biol. Chem. 243, 5929-5934. Mensen DeSilva E. & Cazorla A. (1973) Lactate, a-glycerophosphate and Krebs cycle in sea-level and high altitude native guinea pigs. Am. J_. Physiol. 224, 669-672. Michuda C. M. & Martinez-Carrion M. (1969) Mitochondrial aspartate transamin-ase. II. Isolation and characterization of the multiple forms. Bio- chemistry 8, 1095-1105. Michuda C. M. & Martinez-Carrion M. (1970) The isozymes of glutamate-aspartate transaminase. Mechanism of inhibition by dicarboxylic acids. J_. Biol. Chem. 245, 262-269. Miller J. E. & Litwack G. (1971) Purification, properties, and identity of liver mitochondrial tyrosine aminotransferase. J. Biol. Chem. 246, 3234-3240. Murphy W. H., Kitto G. B., Everse J. & Kaplan N. 0. (1967) Malate dehydrogen-ases - I. A survey of molecular size measured by gel filtration. Biochemistry 6, 603-609. 108 Mustafa T. & Hochachka P. W. (1971) Catalytic and regulatory properties of pyruvate kinases in tissues of a marine bivalve. J. Biol. Chem. 246, 3196-3203. Mustafa T. & Hochachka P. W. (1973) Enzymes in facultative anaerobiosis of molluscs - II. Basic catalytic properties of phosphoenolpyruvate carboxykinase in oyster adductor muscle. Comp. Biochem. Physiol. 45B, 639-655. Oudejans R. C. H. M. & Van der Horst D. J. (1974) Aerobic-anaerobic biosyn-thesis of fatty acids and other lipids from glycolytic intermediates in the pulmonate land snail, Cepaea nemoralis (L.). Comp• Biochem. Physiol. 47B, 139-147. Packer B. S., Airman M., Cross C. E., Murdaugh H. V., Jr., Linta J. M. & Robin E. D. (1969) Adaptations to diving in the harbour seal: oxygen stores and supply. Am. J_. Phys i o l . 217, 903-906. Penney D. G. & Cascarano J. (1970) Anaerobic rat heart. Effects of glucose and tricarboxylic acid-cycle metabolites on metabolism and physiological performance. Biochem. J_. 118, 221-227. Pickwell G. V. (1968) Energy metabolism in ducks during submergence asphyxia: assessment by a direct method. . Comp. Biochem. Physiol. 27, 455-485. Pierce J. (1973) Personal communication. Prosser C. L. & Brown F. A., Jr. (1961) Comparative Animal Physiology. W. B. Saunders, Philadelphia, London. 688 p. Randall H. M., Jr. & Cohen J. J. (1966) Anaerobic CO^ production by dog kidney in vitro. Am. J. Physiol. 211, 493-505. Reeves R. B. (1963) Control of glycogen utilization and glucose uptake in the anaerobic turtle heart. Am. J. Physiol. 205, 23-29. Safer B., Smith C. M. & Williamson J. R. (1971) Control of the transport of reducing equivalents across the mitochondrial membrane in perfused rat heart. J. Moi. Cell. Card. 2, 111-124. Safer B. & Williamson J. R. (1973) Mitochondrial-cytosolic interactions in perfurse rat heart. Role of coupled transamination in repletion of citric acid cycle intermediates. J. Biol. Chem. 248, 2570-2579. Saier M. H., Jr. & Jenkins W. T. (1967) Alanine aminotransferase - I. Puri-fication and properties. J. Biol. Chem. 242, 91-100. Sallach H. J. & Fahien L. A. (1969) Nitrogen metabolism of amino acids. In Metabolic Pathways (Edited by Greenberg D. M.), Vol. I l l , pp. 1-94. Academic Press, New York, London. Sanadi D. R. & Fluharty A. L. (1963) On the mechanism of oxidative phosphory-lation. VII. The energy-requiring reduction of pyridine nucleotide by succinate and the energy-yielding oxidation of reduced pyridine nucleotid by fumarate. Biochemistry 2, 523-528. Saz H. J. (1971) Anaerobiosis in invertebrates. Amer. Zool. 11, 125-135. Scholander P. F. (1940) Experimental investigations on the respiratory func-tion in diving mammals and birds. Hvalradets Skr. 22, 1-131. Scholander P. F. (1962) Physiological adaptation to diving in animals and man Harvey Lectures 57, 93-110. Scholander P. F., Irving L. & Grinnell S. W. (1942) Aerobic and anaerobic changes in seal muscles during diving. J_. Biol. Chem. 142, 431-440. Segal H. L., Beattie D.S. & Hopper S. (1962) Purification and properties of liver glutamic-alanine transaminase from normal and corticoid-treated rats. J. Biol. Chem. 237, 1914-1920. Seidman I. & Entner N. (1961) Oxidative enzymes and their role in phosphory-lation in sarcosomes of adult Ascaris lumbricoides. J_. Biol. Chem. 236, 915-919. 110 Shaw C. R. & Prasad R. (1970) Stach gel electrophoresis of enzymes - a compilation of recipes. Biochem. Genet. 4, 297-320. Shrawder E. & Martinez-Carrion M. (1972) Evidence of phenylalanine transamin-ase activity in the isoenzymes of aspartate transaminase. J_. Biol. Chem. 247, 2486-2492. Shrawder E . J . & Martinez-Carrion M. (1973) Simultaneous isolation and characterization of chicken supernatant and mitochondrial isoenzymes of aspartate transaminase. J_. Biol. Chem. 248, 2140-2146. Stokes T. M. & Apawara J. (1968) Alanine and succinate as end products of glucose degradation in the clam, Rangia cuneata. Comp. Biochem. Physiol. 25, 883-892. Vallyathan N. V., George J. C. & Ronald K. (1969) The harp seal, Pagophilus groenlandicus (Erxleben, 1777). V. Levels of haemoglobin, iron, certain metabolites and enzymes in the blood. Can. J_. Zool. 47, 1193-1197. Van der Horst D. J. (1974) In vivo biosynthesis of fatty acids in the pulmonate land snail, Cepaea nemoralis (L.). Comp. Biochem. Physiol. 47B, 181-187. Veech R. L., Eggleston L. V. & Krebs H. A. (1969) The redox state of free nicotinamide-adenine dinucleotide phosphate in the cytoplasm of rat liver. Biochem. J. 115, 609-619. Velick S.F. & Vavra J. (1962) A kinetic and equilibrium analysis of the glutamic oxalacetate transaminase mechanism. J_. Biol. Chem. 237, 2109-2122. Whereat A. F., Hull F. E. & Orishimo M. W. (1967) The role of succinate in the regulation of fatty acid synthesis by heart mitochondria. J_. Biol. Chem. 242, 4013-4022. I l l White H. B., I l l & Kaplan N. 0. (1972) Separate physiological roles for two isozymes of pyridine nucleotide-linked glycerol-3-phosphate dehydrogenase in chicken. J_. Molec. Evolution 1, 158-172. Whittow G. C. (1974) Personal communication. Wilson M. A. & Cascarano J. (1970) The energy-yielding oxidation of NADH by fumarate in submitochondrial particles of rat tissue. Biochim. Biophys. Acta 216, 54-62. Zimmerman H. J., DuJovne C. A. & Levy R. (1968) The correlation of serum levels of two transaminases with tissue levels in six vertebrate species. Comp. Biochem. Physiol. 25, 1081-1089. DeZoeten L. W., Posthuma D. & Tipker J. (1969) Intermediary metabolism of the l i v e r fluke, Fasciola hepatica, I. Biosynthesis of propionic acid. Hoppe-Seyler's Z. Physiol. Chem. 350, 683-690. DeZoeten L. W. & Tipker J. (1969) Intermediary metabolism of the l i v e r fluke, Fasciola hepatica, II. Hydrogen transport and phosphorylation. Hoppe-Seyler's Z. Physiol. Chem. 350, 691-695. DeZwaan A. & VanMarrewijk W. J. A. (1973) Anaerobic glucose degradation i n the sea mussel, Mytilus edulis L. Comp. Biochem. Physiol. 44B, 429-439. DeZwaan A. & Zandee D. I. (1972) The u t i l i z a t i o n of glycogen and accumulation of some intermediates during anaerobiosis i n Mytilus edulis L. Comp. Biochem. Physiol. 43B, 47-54. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0093494/manifest

Comment

Related Items