Investigations on some enzymes involved in the anaerobic metabolism of amino acids of Arenicola marina L.

Comp. Biochem. Physiol., Vol. 66B, pp. 205 to 213

0305-0491/80/0601-0205502.00/0

© Pergamon Press Ltd 1980. Printed in Great Britain

I N V E S T I G A T I O N S O N SOME E N Z Y M E S I N V O L V E D IN THE A N A E R O B I C M E T A B O L I S M OF A M I N O ACIDS OF A R E N I C O L A M A R I N A L. HORST FELBECKand MANFREDK. GRIESHABER Zoologisches Institut der Universit~itMiinster, Lehrstuhl fiir Tierphysiologie, Hindenburgplatz 55, D-4400 MUnster, West Germany (Received 8 October 1979) Abstract--l. Glutamate oxalacetate transaminase, glutamate pyruvate transaminase, and malate de-

hydrogenase were isolated from body wall musculature of the lugworm Arenicola marina and some properties (molecular weight, pH-optima and Kin-values)were analyzed. 2. The steady state concentrations of aspartate, alanine, succinate, and pyruvate and their alterations during 6 hr of anaerobic incubation were determined. 3. A model of "reductive transamination" is discussed.

INTRODUCTION During anaerobiosis the energy metabolism of various invertebrate animals is characterized by biochemical pathways different from lactate fermentation which serves as the anaerobic energy supply in vertebrates. Meanwhile in invertebrates a considerable amount of data has accumulated which demonstrates that frequently the formation of succinate and volatile fatty acids exceeds the synthesis of lactate (for review see: Zebe, 1977; Zwaan, 1977). Besides these metabolites alanine is formed and ~4C-labelled glucose and labelled pyruvate are rapidly converted into this amino acid (Stokes et al., 1968). Alanine is synthesized by the amination of pyruvate. The enzyme probably involved in the formation of alanine is glutamate pyruvate transaminase (reaction 1 in the following scheme). In this reaction glutamate pyruvate transaminase competes with lactic dehydrogenase for pyruvate. While NADH is oxidized to NAD in the reaction mediated by lactate dehydrogenase no such involvement of NADH occurs during the formation of alanine. Therefore NADH must be oxidized in another reaction in order to maintain the flux of glycolysis during anoxia. Already in 1965 Sacktor proposed a model in which three consecutive reactions could replace the formation of lactate as the final step of glycolysis in insect flight muscle. This model comprises the following reactions: pyruvate + glutamate--~ alanine + ~-ketoglutarate (1) ~-ketoglutarate + aspartate glutamate + oxaloacetate

can proceed as long as the supply of pyruvate and aspartate is sufficient. The lugworm Arenicola marina which dwells in U-shaped burrows of the intertidal zone could be forced to anaerobic energy production during low tide. This is suggested by the formation of succinate and volatile fatty acids during prolonged exposure of submersed animals to pure nitrogen (Surholt, 1977). To evaluate whether alanine formation is possible and significant during anaerobiosis in Arenicola marina, the enzymes glutamate oxaloacetate transaminase, glutamate pyruvate transaminase and malate dehydrogenase were purified and their kinetics were investigated. In order to correlate the various Michaelis-Menten constants with actual concentrations of metabolites involved in alanine synthesis we estimated the contents of aspartate, glutamate, alanine, malate and pyruvate.

(2)

oxaloacetate + NADH ---,malate + NAD (3) pyruvate + aspartate + NADH ---7 alanine + malate + NAD (3) Reaction 1 is catalyzed by pyruvate glutamate transaminase, reaction 2 by glutamate oxaloacetate transaminase and reaction 3 by malate dehydrogenase. From this scheme it is evident that during anoxia the transamination reactions and the oxidation of NADH 205

MATERIALS AND METHODS Materials

All enzymes and coenzymes applied were purchased from Boehringer, Mannheim. Reagents for polyacrylamide gel electrophoresis and N-methylphenazinium-methylsulfate (PMS) were from Serva, Heidelberg. Dithiothreitol (DTT) and Fast Violet B Salt were obtained from Sigma, St Louis and 3,Y-(3,Y-dimethoxy-4,4'-biphenylen)-bis(2-(p-nitrophenyl)-5 phenyl-2 H-tetrazoliumchloride) (NBT) from Merck, Darmstadt. All other chemicals were purchased from local distributors. Lugworms were caught in the intertidal zone near Carolinensiel, North Sea. The estimation of enzyme activities were performed with animals that were kept in aerated seawater. For the enzyme purification muscle tissue was used which had been stored at -70°C. Protein determination

Protein concentrations of the extracts and subsequent enzyme preparations were determined according to Lowry et al. 0951) using cristalline bovine serum albumine as standard. Enzyme activities

All assays were performed according to Warburg & Christian, (1936) at 340 nm.

206

HORST FELBECKand MANFREDK. GRIESHABER

The reaction mixture for the malate dehydrogenase assay contained 50mmol/1 TRA-HCI buffer pH 7.6; 5 mmol/1 EDTA; 0.2 mmol/l NADH and 0.25 mmol/l oxaloacetate. The reaction was started by the addition of oxaloacetate (Mehler et al., 1948). Glutamate oxaloacetate transaminase assays contained 100mmol/l potassium phosphate buffer pH 7.3; 100 mmol/l aspartate, 10 mmol/l ~-ketoglutarate, 0.2mmol/l NADH, 3 U/ml malate dehydrogenase and 2.75 U/ml lactate dehydrogenase. Care was taken that malate dehydrogenase and lactate dehydrogenase were free of ammonium sulfate in order to prevent a reaction between NH 3 and ~-ketoglutarate catalyzed by glutamate dehydrogenase. The reaction mixture for glutamate pyruvate transaminase contained 100 mmol/1 potassium phosphate buffer pH 7.3; 400mmol/l alanine, 0.2mmol/1 ~-ketoglutarate and 2.75 U/ml lactate dehydrogenase when assayed in the direction of pyruvate production. The assay was started by the addition of ~-ketoglutarate. In the direction of alanine production pyruvate glutamate transaminase was assayed in a reaction mixture containing 100mmol/1 potassium phosphate buffer pH 7.3; 2 mmol/l pyruvate, 50mmol/1 glutamate, 15 mmol/l NH4C1, 0.2mmol/1 NADH and 6U/ml glutamate dehydrogenase. One unit of enzyme activity is defined as the production or removal of 1 mmol/1 NADH/min.

Electrophoresis Polyacrylamide gel electrophoresis was conducted by the method of Davis (1964) using a 7~o separating gel. Proteins were stained with Amido Schwarz or Coomassie Blue. Malate debydrogenase activity was detected by incubating the gels in a solution of 50mmol/1 Tris-HC1 pH 8.5; 3.7 mmol/l malate, 0.4 mmol/1 NADH, 3 retool/1 NBT and 0.2 retool/1 PMS. Immediately after electrophoresis the gels were incubated for 1 hr at 37°C in the above mixture without PMS. After the addition of PMS and further incubation malate dehydrogenase activity could be localized by violet bands. The staining solution for glutamate oxaloacetate transaminase consisted of 100mmol/l Tri~HCI pH 7.6; 7.5 mmol/l aspartate, 4.2 mmol/l ~t-ketoglutarate and 1.9 mmol/l pyridoxalphosphate. 25 ml of this mixture contained 50 mg Fast Violet B Salt (Decker & Rau, 1963).

Enzyme purification A summarizing outline of the procedure is given in Table 1. All steps were carried out at 4°C. Step 1: Muscle tissue was homogenized in a six-fold volume of 100 mmol/1 potassium phosphate buffer pH 7.3 containing 0.1 mmol/l EDTA, 0.1 mmol/1 dithiothreitol and 10~o glycerol (v/v) (buffer A). The first homogenization was carried out in a Sorvall Omnimix at full speed for 2 x 60sec. This treatment was followed by a second homogenization using an Ultra-Turrax at full speed for 2min. The resulting homogenate was centrifuged for 60 min at 23,000 g. Step 2: The supernatant of step 1 was brought to 45~o (w/v) saturation of ammonium sulfate, stirred for 15 min and centrifuged for 60 rain at 23,000g. Step 3: The resulting supernatant of step 2 was brought to 75~ saturation by the addition of solid ammoniumsulfate, stirred for 30 min and centrifuged. The resulting pellet was redissolved in a minimum amount of 10 mmol/1 potassium phosphate buffer pH 7.4; 0.1 mmol/1 EDTA containing 10% glycerol (buffer B) and dialyzed against the same buffer over night. Step 4: The dialyzed preparation was applied to a column (3 x 30 cm) of DEAE Sephadex equilibrated with buffer B. The column was flushed with 500 ml buffer B with a flow rate of 54 ml/hr and eluted with a linear gradient of ~500 mmol/1 KCI dissolved in buffer B.

Step 5a: Fractions of the glutamate oxaloacetate peak 1 were pooled and after concentrating by ultrafiltration (Amicon cell, membran P 10) applied to a second column of DEAE-Sephadex (3 x 5cm) which was equilibrated with 10mmol/1 potassium phosphate buffer pH 8.0; 0.1 mmol/1 EDTA containing 10~o glycerol. The column was eluted with a linear gradient of 0-500 mmol/1 KC1. Step 5h: The fractions of the glutamate oxaloacetate transaminase peak 2 were concentrated as those of peak 1, applied on a column (2.5 x 90cm) of Sephadex G-100 equilibrated with buffer A. The column was eluted with a flow rate of l0 ml/hr and 6 ml fractions were collected. Step 5c: The overlapping malate dehydrogenase, pyru° vate glutamate transaminase peaks were combined in one preparation, concentrated and applied to a column (7.5 x 150 cm) of Sephadex G- 100 equilibrated with buffer A. The column was eluted at 100 ml/hr and 20 ml fractions were collected. Step 6a: The glutamate oxaloacetate transaminase which resulted from step 5a(glutamate oxaloacetate transaminase 1) was also separated on a column (1 x 30 cm) of Sephadex G-100 equilibrated with buffer A. The elution velocity was 2 ml/hr and 1 ml fractions were collected. Step 7: Preparative gel electrophoresis with aliquots of glutamate oxaloacetate transaminase 2 (resulting from step 5c) was performed according to Nees & Richter (1972) for anionic gel systems. The separation was carried out in 7~°o slab gels using the Ultraphor equipment (Colora, Lorch). Electrophoresis on 3 x 140 x 70ram slab gels was started at 12 W and 75 pulses/sec. After the sample was concentrated the power was increased to 24 W and pulsation frequency to 250 pulses/sec. The gel was eluted with 21 ml/hr and 2.3 ml fractions were collected. The separation was performed at 4~'C. Molecular weight determination The molecular weights were determined by gel filtration on Sephadex G-100. Calibration proteins were chymotrypsine A (mol. wt 25,000), ovalbumine (mol. wt 45,000), bovine serum albumine (tool wt 67,000), and aldolase (mol. wt 158,000).

pH Optima determination The effect of pH on glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and malate de° hydrogenase was determined in citrate-phosphate borate buffer at 2Y'C.

Incubation of body wall tissue.from lugworms and extraction of metabolites The body wall musculature of 20 lugworms was dissected and each body wall was cut into five equal parts, which were distributed to give five portions. Four of these portions were incubated anaerobically in the three-fold volume of seawater at room temperature, the fifth portion was frozen immediately. Corresponding preparations were incubated under aerobic conditions. Metabolites were extracted by homogenizing the deepfrozen pulverized lugworms of body wall tissue in the fivefold volume of ice-cold 0.6 mol/l perchloric acid using an Ultra-Turrax (2 x 30 sec). After centrifugation the extract was neutralized with potassium hydroxide or potassium carbonate and centrifuged again.

Determination ~f metaholites Aspartale was quantitated using the test of Bergmeyer et al. (1974). Glutamate, alanine and malate were estimated by NAD+-linked assays using glutamate, alanine and malate dehydrogenases, respectively (Bernt & Bergmeyer, 1974; Williamson, 1974; Gutmann & Wahlefeld, 1974). Pyruvate was determined according to Czok & Lamprecht 0974) and succinate following the procedure of Williamson {1974).

Anaerobic metabolism of amino acids

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Table 2. Activities of glutamate pyruvate transaminase, glutamate oxaloacetate transaminase, and malate dehydrogenase in body wall tissue of Arenicola marina U/g fresh wt Glutamate pyruvate transaminase Glutamate oxaloacetate transaminase Malate dehydrogenase

24 22 190

RESULTS

Enzyme activities The activities of glutamate pyruvate transaminase, glutamate oxaloacetate transaminase and malate dehydrogenase are shown in Table 2. Glutamate pyruvate transaminase and glutamate oxaloacetate transaminase show similar activities while malate dehydrogenase exceeds both transaminases nine-fold.

Enzyme purification The precipitate which resulted from the addition of ammonium sulfate from 45 up to 75% saturation contained malate dehydrogenase, glutamate pyruvate transaminase and glutamate oxaloacetate transaminase.

Ion exchange chromatography on DEAE Sephadex increased the specific activity of all three enzymes nine-fold. However, a complete separation could not be achieved in this step (Fig. 1). Glutamate oxaloacetate transaminase 1 eluted with the buffer wash at 300 ml. The main peak of glutamate oxaloacetate transaminase (designated glutamate oxaloacetate transaminase 2) which comprised 95% of the total activity emerged with the gradient at 80 mmol/l potassium chloride. Malate dehydrogenase was eluted at 170mmol/l potassium chloride and glutamate pyruvate transaminase at 200 mmol/l (step 4). Rechromatography of glutamate oxaloacetate transaminase on DEAE Sephadex equilibrated at pH 8.0 (step 5a)

resulted in a single peak which eluted at 90mmol/1 potassium chloride, During the following gel chromatography this preparation emerged as a single peak (step 6a). Pooled fractions from DEAE-Sephadex treatment (step 4) which contained mainly glutamate oxaloacetate transaminase 2 were subjected to gel chromatography on a small Sephadex G-100 column (step 5b). Glutamate oxaloacetate transaminase 2 which emerged at 200 ml was still contaminated with glutamate pyruvate transaminase and malate dehydrogenase. Therefore, the further purification of glutamate oxaloacetate transaminase 2 required preparative gel electrophoresis. During this treatment glutamate oxaloacetate transaminase 2 separated into two fractions, both migrating towards the anode (Fig. 2). The fast-moving band (fraction 65-80) showed also malate dehydrogenase activity. Only the second band (fraction 80-110) which contained 90V,, of the activity applied to the gel was examined and referred to as glutamate oxaloacetate transaminase 2. The concentrated preparations of malate dehydrogenase and glutamate pyruvate transaminase from step 4 were subjected to gel chromatography on a large Sephadex G-100 column (step 5c). Glutamate pyruvate transaminase eluted at 2480 ml and malate dehydrogenase at 2380ml. The pooled and concentrated fractions of glutamate pyruvate transaminase resisted further purification. Malate dehydrogenase fractions from steps 5b and 5c were pooled. The concentrated preparation was subjected to preparative gel electrophoresis (Fig. 3). Malate dehydrogenase separated into two bands, both migrating towards the anode. The faster moving band contained 87%, the second band the remaining 13% of the applied activity. Only the fast moving band was examined and referred to as malate dehydrogenase 1.

Kinetics The K,, values of all three enzymes were determined (Table 3) in order to compare them with

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Anaerobic metabolism of amino acids

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Fig. 3. Preparative gel electrophoresis of malate dehydrogenase (__I-I__) the cellular concentrations of the metabolites involved in alanine formation. Initial velocities measured for glutamate pyruvate transaminase at four concentrations of glutamate plotted against varying concentrations of pyruvate showed parallel lines. The a p p a r e n t Km values obtained from secondary plots of intercepts against

the reciprocal of the substrate concentrations were 8 × 1 0 - S m o l / l for pyruvate and 5 x 10-3mol/1 for glutamate. In the reverse reaction the plots of initial velocities resulted in clearly intersecting lines. Km values of 3 x 1 0 - 4 m o l / l for ct-ketoglutarate and 2 x 10-2 mol/1 for alanine were obtained. The determinations of the Km values of both gluta-

Table 3. Comparison of Km values of the enzymes catalyzing reductive transamination and steady state concentrations of involved metabolites from body wall muscle of Arenicola marina kept aerobically Enzyme

Substrate

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210

HORST FELBECK and MANFRED K. GR1ESHABER

mate oxaloacetate transaminase preparations were performed as in the case of glutamate pyruvate transaminase. Two different patterns were obtained. At fixed concentrations of aspartate and varying concentrations of ~-ketoglutarate glutamate oxaloacetate transaminase 1 showed intersecting lines and glutamate oxaloacetate transaminase 2 parallel lines for the initial velocity. The K,, values were 4 x 10 -4 mol/1 for ct-ketoglutarate and 8 x 10 -4 mol/1 for aspartate in the case of glutamate oxaloacetate transaminase 1 respectively 9 x 10 -4 mol/l and 8 x 10 -4 tool/1 for glutamate oxaloacetate transaminase 2. The initial velocity of glutamate oxaloacetate transaminase 1 and 2 using glutamate and oxaloacetate as substrates showed intersecting lines for both enzyme preparations. The K,. values were 7 × 10 3 mol/l for glutamate and 3 x 10-s mol/1 for oxaloacetate in the case of glutamate oxaloacetate transaminase 1 and 1 x 10 Zmol/1 for glutamate and 1 × 10 5mol/1 for oxaloacetate in the case of glutamate oxaloacetate transaminase 2. Concerning malate dehydrogenase only the K,. value for oxaloacetate was determined. In the presence of saturating amounts of NADH it was estimated to 2 x 10-~mol/l. Estimation of metabolites connected to alanine production

The comparison of the concentrations of pyruvate, glutamate, alanine, aspartate and malate estimated from the body wall musculature of aerobically and anaerobically incubated lugworms is difficult because of large individual variations in the metabolite content. In a typical experiment whole lugworms were incubated anaerobically. At the end of the experiment the metabolite concentrations of the body wall tissue 24

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were estimated. The aspartate concentration was 10 #mol/g fresh wt and the alanine content 41 #mol/g fresh wt. Malate was estimated to 1.0/~mol/g fresh wt, glutamate to 15#mol/g fresh wt and pyruvate to 0.02/~mol/g fresh wt. After 6 hr of anaerobic incubation the concentrations of malate and glutamate did not differ significantly from those of aerobic controls. However the pyruvate content increased to 0.06/,tmol/g fresh wt and the content of alanine to 50 pmol/g fresh wt within 6 hr. During the same time the aspartate concentration decreased to 2/~mol/g fresh wt. In another experiment only body wall tissue was incubated under anaerobic and aerobic conditions for various periods (Fig. 4). In addition to the metabolites mentioned succinate was estimated since it serves as a good indicator for anaerobic metabolism (Zwaan, 1977). This compound increased from 0.8 to 2.3/~mol/g fresh wt within 6 hr. In order to reduce the scattering of the values due to individual variation pieces of muscle tissue obtained from different animals were distributed evenly to the incubation vessels prior to the experiment (for details, see Materials and Methods). The alanine content increased from 17 to 23,umol/g fresh wt and aspartate decreased from 6.8 to 2.6/~mol/g fresh wt during 6 hr of anoxia. The change of both metabolite concentrations were more pronounced during the first 2 hr (3 ~mol/g fresh wt) than during the last 2 hr of incubation. Succinate also increased during the beginning of the anaerobic treatment and rose only slowly during further incubation. Malate and glutamate concentrations remained constant, DISCUSSION

In reports which describe enhanced alanine synthesis (Stokes & Awapara, 1968) and alanine accumulation (Zwaan & Zandee, 1972) in molluscs during anoxia several models concerning the role of alanine in the anaerobic metabolism of invertebrates have been put forward. In these schemes alanine formation (1) either serves for the generation of NAD required for repeated use in glycolysis and for the reduction of fumarate to succinate (Hochachka & Mustafa, 1972; Collicut & Hochachka, 1977) or (2) alanine synthesis replenishes NAD for glycolysis only at the onset of anaerobiosis thus replacing lactate fermentation (Zwaan, 1977). All models agree that alanine is formed by a transamination reaction between pyruvate and glutamate. However, a decrease of glutamate concentration during anoxia could never be substantiated. This indicates that glutamate is only the "turntable" for the amino group supplied from other transaminating reactions. The body wall tissue of Arenicola marina shows high activities of glutamate pyruvate transaminase and glutamate oxaloacetate transaminase. From the enzymes investigated only glyceraldehydephosphate dehydrogenase and malate dehydrogenase showed higher activities than these two transaminases (Table l ; see also Zebe, (1975)). During anoxia the transaminating reactions coupled to the malate dehydrogenase catalyzed reduction of oxaloacetate might proceed in the direction of malate and alanine production. To evaluate the possible function in anaerobiosis glutamate pyruvate transaminase, glutamate oxaloacetate transaminase and malate dehydrogenase were

Anaerobic metabolism of amino acids

211

Table 4. Comparison of pH optima of the enzymes catalyzing reductive transamination Substrate Glutamate pyruvate transaminase

Glutamate oxaloacetate transaminase 1

Glutamate oxaloacetate transaminase 2

Malate dehydrogenase

pH optima

Pyruvate, glutamate Alanine, ct-ketoglutarate Aspartate, ct-ketoglutarate Oxaloacetate, glutamate Aspartate, ct-ketoglutarate Oxaloacetate, glutamate Oxaloacetate

purified. In the case of glutamate oxaloacetate transaminase and malate dehydrogenase two enzymatically active, homogeneous fractions were separated which we tentatively consider as isoenzymes. Glutamate pyruvate transaminase was only obtained in a partially purified preparation. The molecular weight of glutamate pyruvate transaminase (91,000 daltons) is lower than that of vertebrate enzymes (110,000; Barmann, 1969; Matsuzawa & Segal, 1968). The molecular weight of glutamate oxaloacetate tralasaminase (85,000 daltons) is within the range of 80,000 to 110,000 daltons estimated for several enzymes of different sources (Barmann, 1969). Malate dehydrogenase has a molecular weight (69,000) which is similar to that of this enzyme from other sources (Banaszak & Bradshaw, 1975). The pH optima vary between pH 7.3 and 7.9 for all three enzymes. If forward and reverse directions of the three reactions are compared only a difference of pH 0.3 is found in the pH optima (Table 4). So a simultaneous operation of these enzymes can be anticipated in either direction with regard to physiological pH-values. The K,, values for the three enzymes investigated were determined in order to compare the enzyme affinities for their substrates with the actual concentration of these substrates within the cell. Glutamate oxaloacetate transaminase showed K,. values for aspartate, c~-ketoglutarate, glutamate and oxaloacetate which are similar to those known for this enzyme from other sources (Table 3). In the case of glutamate pyruvate transaminase the K,, values of alanine, ~-ketoglutarate and glutamate are also similar to those found in vertebrates. But the K,, value for pyrurate is approx ten-fold lower than that from vertebrate enzymes (Hopper & Segal, 1962; Saier & Jenkins, 1967; Barmann, 1969; Owen & Hochachka, 1974). Malate dehydrogenase showed a lower affinity for oxaloacetate than the enzymes form vertebrates (Banaszak & Bradshaw, 1975). The cellular concentrations of the metabolites involved in the three steps of reductive transamination showed similar values for ~-ketoglutarate, glutamate, oxaloacetate and malate when aerobically and anaerobically kept animals were compared. Only alanine and pyruvate levels increased while the aspartate content decreased.

7.6 7.6 7.6 7.9 7.3 7.6 8.3

From the data in Table 3 it is evident that the K,, values of both transaminases approximate the steady state concentrations of the metabolites involved as substrates in the transamination reaction with the exception of ~-ketoglutarate in the case of glutamate pyruvate transaminase and aspartate in the ease of glutamate oxaloacetate transaminase. It is also known that the equilibrium constants of the two transaminating reactions is near unity (Barmann, 1969) and that they proceed near their equilibrium within the cell as indicated by the mass action ratio. Thus an increase in the pyruvate concentration as it indeed occurs during anoxia would favour alanine formation thermodynamically as well as by an increase of the catalytic activity of glutamate pyruvate transaminase. Since enhanced alanine synthesis is tantamount to an increasing speed of ~-ketoglutarate formation, the transamination of aspartate to glutamate and oxatoacetate can also proceed. This reaction is in addition geared to glutamate formation because glutamate oxaloacetate transaminase is saturated with aspartate the cellular concentration of which is about tenfold higher than the corresponding K,, value. The resulting oxaloacetate could be reduced to malate as was mentioned above; the equilibrium constant of this reaction is extremly in favour of malate synthesis. This reaction then would replenish NAD for glycolysis and in addition would favour alanine synthesis because it constantly withdraws oxaloacetate from the equilibrium. Thus according to the overall equation of reductive transamination pyruvate + aspartate + NADH alanine + malate + NAD malate should accumulate besides alanine. However, in Arenicola marina the malate pool never increased during anoxia. This is not at all surprising since malate can permeate into the mitochondria where it is transformed to succinate. The concentration of succinate is found to have increased by 2 #mol/g fresh wt already in the beginning of anoxia. NADH necessary for the reduction of fumarate to succinate (according to the scheme of Collicut & Hochachka 0977)) must not necessarily originate from reactions located in the cytoplasmic matrix but can be supplied entirely from a partially operating Krebs cycle (Schroff & SchiSttler, 1977). Therefore,

212

HORST FI~LBECKand MANFRED K. GRIESHABER

REFERENCES glucose b r e a k d o w n via the E m b d e n - M e y e r h o f pathway could proceed to pyruvate and its redox-balance BANASZAK L. J. 8z BRADSHAW R. A. (1975) The Enzymes could be maintained by the reductive t r a n s a m i n a t i o n (Edited by BOYER P. D.) Vol X1A, pp. 369-416, Acareactions. demic Press, New York. As a corollary there is no need of coupling cyto- BARMANN T. E. (1969) Enzyme Handbook, Springer, Berlin. plasmic and mitochondrial redox-balance. Only BERGMEYER H. U., BERNT E., MOLLERING H. 8,~ PFLEIDERER G. (1974) Methoden der enzymatischen Analyse (Edited malate joins both compartements. Consequently, the by BERGMEYI!RH. U.) Vol. I1, pp. 1741 1745. carbon skeleton of aspartate must be incorporated predominantely into succinate. This is indeed the case BERNT E. & BERGMEYER H. U. (1974) Methoden der enzymatisehen Analyse (Edited by BERGMEYERH. U.) Vol. If, in Arenicola marina. Zebe (1975) demonstrated that pp. 1749 1753. 60°~ of aspartate metabolized is incorporated into COLLICUT J. M. 8z HOCHACHKA P. W. (1977) The anerobic malate and succinate under anaerobic conditions. oyster heart; coupling of glucose and aspartate fermenIf the cytoplasmic N A D : N A D H ratio is to be kept tation. J. eomp. Physiol. 115, 147 157. constant by reductive transamination, 2tools of CZOK, R. & LAMPRECHTW. (1974) Methoden der enzymatisaspartate must be t r a n s a m i n a t e d per tool glucose chen Analyse (Edited by BERGMEYER H. U.) Vol. II, pp. 1491 1496. metabolized. This would mean that as much alanine is synthesized as aspartate disappears. Our results are DAVIS G. J. (1964) Disc electrophoresis. II. Method and application to human serum protein. Ann. N.E Acad. not in accordance with this assumption; the net inSci. 121,403~,27. crease of alanine always exceeds the decrease of DECKER L. E. & RAt; E. M. (1963) Multiple forms of gluaspartate. tamic oxalacetic transaminase in tissues. Proc. Soc. exp. Similar data were obtained by Knauel (1977). He Biol. Med. !12, 144 149. investigated the free a m i n o acid concentration in GUTMANN I. & WAHLEFELD A. W. (1974) Methoden der blood, coelomic fluid and body wall of Arenicola marenzymatischen Analyse (Edited by BERGMEYER H. U.) Vol. 11, pp. 1632-q636. ina and their alterations during anaerobiosis. After HOCHACRKA P. W. & MUSTAFA T. (1972) Invertebrate 24 hr of anaerobic incubation he found in body wall facultative anaerobiosis. Science 178, 1056- 1060. an average decrease of the aspartate concentration of HOCHACHKA P. W. & STOREY K. B. (1975) Metabolic con5 ,umol/g fresh wt and an increase of the alanine consequences of diving in animals and man. Science 187, centration of 7/~mol/g fresh wt. 613 621. Therefore the reduction of oxaloacetate derived HOPPI~R S. & SEGAL H. (1962) Comparative properties of from aspartate cannot entirely replenish all the N A D glutamic alanine transaminase from several sources. J. required for glycolysis. In addition, oxaloacetate synbiol. Chem. 237, 3189-3195. thesized by the carboxylation of phosphoenolpyruKNAUEL B. (1977) Untersuchungen fiber den Aminos~iuregehalt der K6rperflfissigkeiten und des Hautmuskelschvate must be reduced to malate. Malate originating lauchs yon Arenieola marina. Staatsarbeit, Mfinster. from either glucose or aspartate would then permeate LOWRY O. H., ROSEBROUGH N. J., FARR A. & RANDALL into the m i t o c h o n d r i a where it would dismutate to R. J. (1951) Protein measurements with the Folin phenol succinate and pyruvate (via the malic enzyme reacreagent J. biol. Chem. 193, 265 275. tion) (Schroff & Sch6ttler, 1977). Pyruvate in turn MATSUZAWA T. 8.~ SEGAL H. L. (1968) Rat liver alanine could be transaminated. This would also explain the amino transferase crystallization, composition, and occurrence of radioactively labelled carbon (35.°o of role of sulfhydryl groups. J. biol. Chem. 24B, 5929 5934. aspartate metabolized; (Zebe, 1975)) in alanine. The MEHLER A. H., KORNBERG A., GRISOL1A S. & OCHOA S. origin of the non-aspartate amino group necessary for (1948) The enzymatic mechanism of oxidation reductions between malate or isocitrate and pyruvate. J. biol. further pyruvate amination, however, remains unChem. 174, 961 977. known. N~Es S. & RICHT}~R K. (1972) Bedienungsanleitung fiir den The experiments on anaerobic energy metabolism UItraPhor, Colora Messtechnik, Lorch/Wtirtt., BRD. of Arenicola marina demonstrated that during the first OWEN T. G. & HOCHACHKA P. W. (1974) Purification and 6 hr of anoxia alanine is indeed accumulated. The net properties of dolphin muscle aspartate and alanine increase of this a m i n o acid exceeds the a m o u n t s of transaminase and their possible roles in the energy succinate (2.1 #mol/g fresh wt) and of propionate metabolism of diving mammals. Biochem. J. 143, (1.3/~mol/g fresh wt; (Surholt, 1977)) synthesized dur541 553. ing this time. Since the amino group of alanine preSACKTOR B. (1965) Energetics and respiratory metabolism of muscular contraction. In The Physiolo~ty q[ Inseeta dominantely originates from aspartate it can be (Edited by ROCKSTHN M.) Vol. !I, pp. 484 581. assumed that this a m i n o acid is the limiting factor for reductive t r a n s a m i n a t i o n during anoxia. Due to the SAIER M. H. ~,~ JENKINS W. T. (1967) Alanine amino transferase I. Purification and properties. J. biol. Chem. 242, rather low level of aspartate as compared to glycogen, 92 100. which is the substrate for a long lasting anaerobiosis, SCHROFF G. & SCHOTTLER U. (1977) Anaerobic reducreductive t r a n s a m i n a t i o n in Arenicola is quantitattion of fumarate in the body wall musculature of ively insignificant for the catabolism during proArenicola marina (Polychaeta). J. comp. Physiol. 116, 325-336. longed anoxia. In addition the role of amino acids as STOKES T. M. & AWAPARAJ. (1968) Alanine and succinate fuel for prolonged anaerobic energy production as it as end products of glucose degradation in the clam Ranhas been proposed by Collicut & H o c h a c h k a (1977) gia cuneata. Comp. Biochem. Physiol. 25, 883 892. appears quite doubtful. SURHOLT B. (1977) Production of volatile fatty acids in the anaerobic carbohydrate catabolism of Arenicola marina. Comp. Biochem. Physiol. 58B, 147 150. Acknowled.qenwnts We thank E. Zebe for critical advice WARaL'RG O. & CHRISTIAN W. (1936) Pyridin, der wasserand help. This work was supported by Deutsche Forsstofffibertragende Bestandteil von G~irungsfermenten. chungsgemeinschaft grant Gr. 456/6 (Pyridin-Nuclcotide). Biochem. Z. 287, 291 328.

Anaerobic metabolism of amino acids WILLIAMSON D. H. (1974) Methoden der enzymatischen Analyse (Edited by BERGMEVER"H. U,) Vol. II, pp. 1724 1727. ZEBE E. (1975) In vivo-Untersuchungen iiber den GlucoseAbbau bei Arenicola marina (Annelida, Polychaeta). J. comp. Physiol. 101, 133 145. ZEBE E. (1977) Anaerober Stoffwechsel bei wirbellosen Tieren. Rhein.-Westf. Akad. Wiss., Vortr~ige N 269.

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ZWAAN A. DE (1977) Anaerobic energy metabolism in bivalve molluscs. Oceanogr. Mar. Biol. Ann. Rev. 15, 103-187. ZWAAN A. DE & ZANDEE D. I. (1972) The utilization of glycogen and accumulation of some intermediates during anaerobiosis in Mytilus edulis L. Comp. Biochern. Physiol. 43B, 47-54.