AROMATIC AMINOTRANSFERASE ACTIVITY OF RAT BRAIN CYTOPLASMIC AND MITOCHONDRIAL ASPARTATE AMINOTRANSFERASES

Abstract— Mitochondrial and cytoplasmic forms of aspartate aminotransferase were purified from rat brain homogenates and tested for their ability to catalyze transamination of various aromatic amino acids. The mitochondrial enzyme exhibited activity toward tyrosine and phenylalanine with 2-oxoglutar-ate as acceptor, although the specific activities were less than 1% of the corresponding aspartate activity when all substrates were 10 mM. Even less activity was seen with DOPA, 5-hydroxytryptophan and tryptophan. The cytoplasmic aspartate aminotransferase was active toward tryptophan, 5-hydroxytryptophan and DOPA, but these transaminations were favored by pyruvate or oxaloacetate rather than 2-oxoglutarate as keto acid. Based on co-migration of aromatic activities with the respective aspartate aminotransferases during isoelectric focusing and based on equal sensitivities of aromatic transamination and aspartate transamination to inhibition by vinylglycine, it was concluded that all activities resided in the aspartate aminotransferase enzymes. Some doubt exists, however, as to the physiological significance of these alternate activities in view of the requirement that aromatic amino acids must compete with aspartate for transamination by these enzymes.     

Interactions of Phosphoenolpyruvate Carboxylase and Pyruvic Kinase in Developing Soybean Seeds

Developing soybean seeds contain phosphoenolpyruvate (PEP) carboxylase,pyruvic kinase, malate dehydrogenase, aspartate aminotransferase,alanine aminotransferase and malic enzyme activities. PEP carboxylasemay be important in competing with pyruvic kinase and directinga portion of glycolytic carbon towards oxaloacetate synthesis.The oxaloacetate can then be converted to aspartate and malate.Malic enzyme produces pyruvate and NADPH from malate, and thismay be an important additional source of reducing power forlipid biosynthesis. In the presence of high levels of PEP carboxylaseit is possible to demonstrate PEP formation by pyruvic kinase.PEP carboxylase and pyruvic kinase independently compete forPEP in a mixed system. Soybean seed extracts readily convertedradioactive PEP into alanine and aspartate when supplementedwith ADP, Mg²⁺, K+, HCO₃– and glutamate. Under varyingconditions of pH, metal ions, PEP, enzyme concentration andtime both alanine and aspartate were always produced. Possiblythe final products of glycolysis should be considered as pyruvateand oxaloacetate in plants. (Received April 22, 1981; Accepted June 26, 1981)     

A 1H NMR study of succinate synthesis from exogenous precursors in oxygen-deprived rat heart mitochondria

Succinate synthesis from exogenous malate, alpha-ketoglutarate, oxaloacetate and L-glutamate in isolated oxygen-deprived rat heart mitochondria was studied using 1H NMR. The highest rate of succinate synthesis was observed during incubation of mitochondria with a mixture of L-glutamate and oxaloacetate. When mitochondria were incubated with [U-13C] glutamate and oxaloacetate the [U-13C] succinate/succinate and aspartate/succinate ratios were equal to 2. This suggests that the succinate produced from [U-13C] alpha-keto-glutarate formed via transamination of [U-13C] glutamate with oxaloacetate by aspartate aminotransferase exceeds twofold that synthesized via oxaloacetate reduction. It may thus be expected that GTP yield in a reaction catalyzed by the succinic thiokinase will be 2 times higher that of ATP production coupled with NADH-dependent fumarate reduction.     

Purification,characterization and identification of tryptophan aminotransferase from rat brain

Tryptophan aminotransferase was purified from rat brain extracts. The purified enzyme had an isoelectric point at pH 6.2 and a pH optimum near 8.0. On electrophoresis the enzyme migrated to the anode. The enzyme was active with oxaloacetate or 2-oxoglutarate as amino acceptor but not with pyruvate, and utilized various L-amino acids as amino donors. With 2-oxoglutarate, the order of effectiveness of the L-amino acids was aspartate > 5-hydroxytryptophan > tryptophan > tyrosine > phenylalanine. Aminotransferase activity of the enzyme towards tryptophan was inhibited by L-glutamate. Sucrose density gradient centrifugation gave a molecular weight of approx. 55,000. The enzyme was present in both the cytosol and synaptosomal cytosol, but not in the mitochondria. The isoelectric focusing profile of tryptophan: oxaloacetate aminotransferase activity was identical with that of L-aspartate: 2-oxoglutarate aminotransferase (EC 2.6.1.1) activity, with both subcellular fractions. On the basis of these data, it is suggested that the enzyme is identical with the cytosol aspartate: 2-oxoglutarate aminotransferase.     

Coupled reaction of immobilized aspartate aminotransferase and malate dehydrogenase. A plausible model for the cellular behaviour of these enzymes

To study the effect of facilitated diffusion of the intermediate metabolite, oxaloacetate, on the coupled reaction of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) and malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37), these enzymes were co-immobilized on the surface of a collagen film. The kinetic properties of the immobilized enzymes were compared with those observed with the enzymes in solution. Since the reactions correspond to the cytosolic enzymes, they have been studied in the direction aspartate aminotransferase toward malate dehydrogenase. Coupled enzymes in solution showed classical behaviour. A lag-time was observed before they reached a steady state and this lag-time was dependent on the kinetic properties of the second enzyme, malate dehydrogenase. The same lag-time was observed when malate dehydrogenase in solution was coupled with aspartate aminotransferase bound to the film. When aspartate aminotransferase in solution was coupled with malate dehydrogenase bound to the collagen film, a very long lag-time was observed. Theoretical considerations showed that in the latter case, the lag-time was dependent on the kinetic properties of the second enzyme and the transport coefficient of the intermediate substrate through the boundary layer near the surface of the film. Then both enzymes were co-immobilized on the collagen film. The coupled activity of aspartate aminotransferase and malate dehydrogenase was compared for films with an activity ratio of 5 and 0.8. In both cases, a highly efficient coupling was observed. In the former case, where malate dehydrogenase was rate-limiting, 81% of this limiting activity was observed. In the latter case, aspartate aminotransferase was rate-limiting and 82% of its rate was obtained for the final product formation. The linear increase of product formation with time corresponded fairly well to the theoretical equations developed in the paper. To interpret these rate equations, one should assume that the intermediate substrate oxaloacetate formed by aspartate aminotransferase was used by malate dehydrogenase in the diffusion layer near the film, before diffusing in the bulk solution.     

Comparative aspects of aminotransferases in the rat, pigeon and rainbow trout.

1. The activities of aminotransferases catalysing the transfer of amino groups from aspartate, alanine and leucine to 2-oxoglutarate in different tissues of the rat, pigeon and trout have been determined. 2. Alanine-2-oxoglutarate aminotransferase was high in the liver of the rat and trout and low in that of the pigeon. 3. Aspartate-2-oxoglutarate aminotransferase was usually the dominant aminotransferase in all tissues and was highest in oxidative tissues where the TCA cycle is active. Its activity in the various livers is not correlated with the function of aspartate in nitrogen excretion. 4. The activity of aspartate-2-oxoglutarate aminotransferase in oxidative tissues argues that aspartate in conjunction with this enzyme serves as a buffer of oxaloacetate to keep the TCA cycle running and/or to mediate the transfer of reducing equivalents across mitochondrial membranes.     

Oxidation of NADH in Glyoxysomes by a Malate-Aspartate Shuttle

Glyoxysomes isolated from germinating castor bean endosperm accumulate NADH by β-oxidation of fatty acids. By utilizing the glutamate: oxaloacetate aminotransferase and malate dehydrogenase present in glyoxysomes and mitochondria, reducing equivalents could be transferred between the organelles by a malate-aspartate shuttle. The addition of aspartate plus α-ketoglutarate to purified glyoxysomes brought about a rapid oxidation of accumulated NADH, and the oxidation was prevented by aminooxyacetate, an inhibitor of aminotransferase activity. Citrate synthetase activity in purified glyoxysomes could be coupled readily to glutamate: oxaloacetate aminotransferase activity as a source of oxaloacetate, but coupling to malate dehydrogenase and malate resulted in low rates of citrate formation. Glyoxysomes purified in sucrose or Percoll gradients were permeable to low molecular weight compounds. No evidence was obtained for specific transport mechanisms for the proposed shuttle intermediates. The results support a revised model of gluconeogenic metabolism incorporating a malate-aspartate shuttle in the glyoxysomal pathway.     

Acidosis and astrocyte amino acid metabolism

The relationship between acidosis and the metabolism of glutamine and glutamate was studied in cultured astrocytes. Acidification of the incubation medium was associated with an increased formation of aspartate from glutamate and glutamine. The rise of the intracellular content of aspartate was accompanied by a significant decline in the extracellular concentration of both lactate and citrate. Studies with either [2-(15)N]glutamine or [15N]glutamate indicated that there occurred in acidosis an increased transamination of glutamate to aspartate. Studies with L-[2,3,3,4,4-(2)H5]glutamine indicated that in acidosis glutamate carbon was more rapidly converted to aspartate via the tricarboxylic acid cycle. Acidosis appears to result in increased availability of oxaloacetate to the aspartate aminotransferase reaction and, consequently, increased transamination of glutamate. The expansion of the available pool of oxaloacetate probably reflects a combination of: (a) Restricted flux through glycolysis and less production from pyruvate of acetyl-CoA, which condenses with oxaloacetate in the citrate synthetase reaction; and (b) Increased oxidation of glutamate and glutamine through a portion of the tricarboxylic acid cycle and enhanced production of oxaloacetate from glutamate and glutamine carbon. The data point to the interplay of the metabolism of glucose and that of glutamate in these cells.     

Metabolism of amino acids in germinating yellow lupin seeds. II. Pathway of conversion of aspartate to alanine during the imbibition

The incorporation of ¹⁴C-aspartate during the imbibition of yellow lupin seeds resulted in the production of ¹⁴C-alanine and ¹⁴CO₂. On the basis of tracer and enzymatic assays, conducted in vitro on the extract obtained from lupin seeds, it is postulated that aspartate can be converted to oxaloacetate, then, by phosphoenolopyruvate and pyruvate to alanine. This pathway can be catalyzed by the following enzymes: aspartate aminotransferase, phosphoenolpyruvate carboxykinase, pyruvate kinase and alanine aminotransferase.     

13C and (15)N kinetic isotope effects on the reaction of aspartate aminotransferase and the tyrosine-225 to phenylalanine mutant

Heavy atom isotope effects at C-2, C-3, and the amino nitrogen of aspartate were determined for the reaction of porcine heart cytosolic aspartate aminotransferase and the tyrosine-225 to phenylalanine mutant of Escherichia coli aspartate aminotransferase. The effects of deuteration at C-2 of aspartate and of D(2)O on the observed heavy atom isotope effects were determined. The multiple isotope effects support the contribution of C(alpha)-H cleavage, ketimine hydrolysis, and oxaloacetate dissociation to the rate limitation with the wild-type enzyme. The existence of a quinonoid intermediate could not be determined due to the kinetic complexity of the enzyme. For the tyrosine-225 to phenylalanine mutant, we are able to conclude that ketimine hydrolysis is the major rate-determining step.     

Branched-chain amino acid metabolism and alanine formation in rat muscles in vitro. Mitochondrial-cytosolic interrelationships.

Muscle branched-chain amino acid metabolism is coupled to alanine formation via branched-chain amino acid aminotransferase and alanine aminotransferase, but the subcellular distributions of these and other associated enzymes are uncertain. Recovery of branched-chain aminotransferase in the cytosol fraction after differential centrifugation was shown to be accompanied by leakage of mitochondrial-matrix marker enzymes. By using a differential fractional extraction procedure, most of the branched-chain aminotransferase activity in rat muscle was located in the mitochondrial compartment, whereas alanine aminotransferase was predominantly in the cytosolic compartment. Phosphoenolpyruvate carboxykinase, like aspartate aminotransferase, was approximately equally distributed between these subcellular compartments. This arrangement necessitates a transfer of branched-chain amino nitrogen and carbon from the mitochondria to the cytosol for alanine synthesis de novo to occur. In incubations of hemidiaphragms from 48 h-starved rats with 3mM-valine or 3mM-glutamate, the stimulation of alanine release was inhibited by 69% by 1 mM-aminomethoxybut-3-enoate, a selective inhibitor of aspartate aminotransferase. Leucine-stimulated alanine release was unaffected. These data implicate aspartate aminotransferase in the transfer of amino acid carbon and nitrogen from the mitochondria to the cytosol, and suggest that oxaloacetate, via phosphoenolpyruvate carboxykinase, can serve as an intermediate on the route of pyruvate formation for muscle alanine synthesis.     

Concentration of free oxaloacetate in the mitochondrial compartment of isolated liver cells.

The concentration of metabolically active (i.e. 'free') oxaloacetate in the mitochondrial compartment of isolated liver cells was investigated by two independent approaches. On the basis of mitochondrial aspartate aminotransferase maintaining equilibrium and the direct measurements of mitochondrial aspartate, 2-oxoglutarate and glutamate, the concentration of free oxaloacetate was calculated to be 5 microM after incubation of hepatocytes in the presence of 1.5 mM-lactate and 0.05 mM-oleate. Gradually increasing oleate up to 0.5 mM decreased the free oxaloacetate to 2 microM. Very similar results were obtained when free oxaloacetate concentration was derived from the CO2 production of hepatocytes as a measure of citrate flux through the tricarboxylic acid cycle, and the kinetic data on citrate synthase in situ. The decrease in free oxaloacetate on increasing oleate concentration was associated with lowered rates of cycle-dependent CO2 output and O2 uptake, indicating a decrease in the disposal of acetyl-CoA into the tricarboxylic acid cycle. This decrease could explain 25-30% of the increase in ketone-body production occurring at elevated fatty acid supply. This work documents on a quantitative basis the role of free oxaloacetate in the regulation of ketogenesis.     

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: pathways of C4 acid decarboxylation in bundle sheath cells of Urochloa panicoides

The mechanism of C4 acid decarboxylation was studied in bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate carboxykinase (PCK)-type C4 plant. Added malate was decarboxylated to give pyruvate and this activity was often increased by adding ADP. Added oxaloacetate or aspartate plus 2-oxoglutarate (which produce oxaloacetate via aspartate aminotransferase) gave little metabolic decarboxylation alone but with added ATP there was a rapid production of PEP. For this activity ADP could replace ATP but only when added in combination with malate. In addition, the inclusion of aspartate plus 2-oxoglutarate with malate plus ADP often increased the rate of pyruvate production from malate by more than twofold. Experiments with respiratory chain inhibitors showed that the malate-dependent stimulation of oxaloacetate decarboxylation (PEP production) was probably due to ATP generated during the oxidation of malate in mitochondria. We could provide no evidence that photophosphorylation could serve as an alternative source of ATP for the PEP carboxykinase reaction. We concluded that both PEP carboxykinase and mitochondrial NAD-malic enzyme contribute to C4 acid decarboxylation in these cells, with the required ATP being derived from oxidation-linked phosphorylation in mitochondria.     

A selective assay for prephenate aminotransferase activity in suspension-cultured cells of Nicotiana silvestris

Prephenate aminotransferase in Nicotiana silvestris Speg. et Comes is highly stable to thermal treatment. This property was exploited to obtain, by treatment at 70° C for 10 min, a residual level (1–4%) of aspartate aminotransferase activity that proved to be catalyzed exclusively by prephenate aminotransferase. The latter enzyme was the most mobile of all aspartate aminotransferase bands during polyacrylamide-gel electrophoresis conducted under non-denaturing conditions. This methodology for convenient assay of prephenate aminotransferase in crude extracts, as demonstrated for N. silvestris, may generally apply to higher plants since prephenate aminotransferase from a variety of plant sources has been found to exhibit high thermal stability.Abbreviations AGN L-arogenate - AT aminotransferase - ASP L-aspartate - GLU L-glutamate - HPP 4-hydroxyphenylpyruvate - 2-KG 2-ketoglutarate - OAA oxaloacetate - PPA prephenate - PPY phenylpyruvate Florida Agricultural Experiment Station, Journal Series No. 8286     

beta-Sulfopyruvate: chemical and enzymatic syntheses and enzymatic assay

beta-Sulfopyruvic acid (2-carboxy-2-oxoethanesulfonic acid) is prepared in greater than 90% yield by reaction of bromopyruvic acid with sodium sulfite. beta-[35S]Sulfopyruvate is prepared by transamination between [35S]cysteinesulfonate (cysteate) and alpha-ketoglutarate using mitochondrial aspartate aminotransferase isolated from rat liver. Following either chemical or enzymatic synthesis, the crude reaction product is conveniently purified by chromatography on Dowex 1; beta-sulfopyruvate is isolated as the stable, water-soluble dilithium salt. beta-Sulfopyruvate is shown to be an alternative substrate of mitochondrial malate dehydrogenase; in the presence of 0.25 mM NADH, beta-sulfopyruvate is reduced with an apparent Km of 6.3 mM and a Vmax equal to about 40% of that observed with oxaloacetate. This finding forms the basis of a convenient spectrophotometric assay of beta-sulfopyruvate.     

Characterization of a Paracoccus denitrificans mutant pleiotropically impaired in nitrogen metabolism

A Tn5 insertional prototrophic mutant of Paracoccus denitrificans (UBM219) was generated which grew on high (>1 mM) but not low (<0.5 mM) ammonium as sole nitrogen source. It did not utilize nitrate and most amino acids except glutamate and aspartate. UBM219 showed more than 10-fold lower levels of ammonium (methylammonium) transport, aspartate and alanine aminotransferase, but more than 10-fold higher activities of glutamate dehydrogenase and glutamate synthase. This pleiotropy indicates a mutation in a regulatory gene affecting nitrogen metabolism in general. — Ammonia assimilation pathways and regulation in Paracoccus resemble the patterns in enterobacteria with the exception, that alanine is generated by amino transfer from glutamate to pyruvate.Non-standard abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GluDH glutamate dehydrogenase - GPT glutamate/pyruvate aminotransferase - GOT glutamate/oxaloacetate aminotransferase     

Kinetics of the coupled aspartate aminotransferase-malate dehydrogenase reactions and instability of oxaloacetate on anion-exchange resin

The kinetic data of Bryce et al. [C. F. A. Bryce, D. C. Williams, R. A. John, and P. Fasella (1976), Biochem. J., 153, 571–577] indicated anomalous behavior of the coupled aspartate aminotransferase and malate dehydrogenase reactions. From measurements of isotope incorporation (aspartate to malate) and the fact that no enzyme associations could be detected, they concluded that the aminotransferase generates an isomer of oxaloacetate, OAA_a, which is active with the dehydrogenase. In this model, OAA_a would diffuse from the transferase to the dehydrogenase before isomerizing to the equilibrium mixture in which the inactive isomer predominates. (OAA_a was not considered to be either the keto or enol form of oxaloacetate.) We are not able to reproduce the anomalous kinetic or isotope data of these authors. The reasons for the observation of the kinetic anomaly are uncertain. Our isotope experiments, however, indicate that the anion-exchange resin used in this method induces extensive oxaloacetate decomposition making these results unreliable. We also argue that even if there were no experimental errors, the isotope measurements of Bryce et al. would not provide evidence for the oxaloacetate isomer model.     

A novel, definitive test for substrate channeling illustrated with the aspartate aminotransferase/malate dehydrogenase system.

A novel method is presented that establishes definitively the existence or nonexistence of direct metabolite transfer between consecutive enzymes in a metabolic sequence. The procedure is developed with the specific example of channeling of oxaloacetate between Escherichia coli aspartate aminotransferase (AATase) and malate dehydrogenase (MDH). The assay is carried out in the presence of a large excess of inactive variants of AATase. These mutants would outcompete the much smaller quantities of wild-type AATase for any docking sites on MDH and thus decrease the rate of the coupled L-aspartate to oxaloacetate to malate sequence only if the direct metabolite transfer mechanism is operative. The results show that oxaloacetate is not transferred directly from AATase to MDH because no decrease in rate was observed in the presence of approximately 100 microM inactive mutants. This concentration is 10 times the physiological AATase concentration, which was determined in this work. The methodology can be applied generally.     

Influence of the Malate-Aspartate Shuttle on Oxidative Metabolism in Synaptosomes

beta-Methyleneaspartate, a specific inhibitor of aspartate aminotransferase (EC 2.6.1.1.), was used to investigate the role of the malate-aspartate shuttle in rat brain synaptosomes. Incubation of rat brain cytosol, "free" mitochondria, synaptosol, and synaptic mitochondria, with 2 mM beta-methyleneaspartate resulted in inhibition of aspartate aminotransferase by 69%, 67%, 49%, and 76%, respectively. The reconstituted malate-aspartate shuttle of "free" brain mitochondria was inhibited by a similar degree (53%). As a consequence of the inhibition of the aspartate aminotransferase, and hence the malate-aspartate shuttle, the following changes were observed in synaptosomes: decreased glucose oxidation via the pyruvate dehydrogenase reaction and the tricarboxylic acid cycle; decreased acetylcholine synthesis; and an increase in the cytosolic redox state, as measured by the lactate/pyruvate ratio. The main reason for these changes can be attributed to decreased carbon flow through the tricarboxylic acid cycle (i.e., decreased formation of oxaloacetate), rather than as a direct consequence of changes in the NAD+/NADH ratio. Malate/glutamate oxidation in "free" mitochondria was also decreased in the presence of 2 mM beta-methyleneaspartate. This is probably a result of decreased glutamate transport into mitochondria as a result of low levels of aspartate, which are needed for the exchange with glutamate by the energy-dependent glutamate-aspartate translocator.     

Kinetics of the coupled reaction catalysed by a fusion protein of yeast mitochondrial malate dehydrogenase and citrate synthase.

The mechanistic implications of the kinetic behaviour of a fusion protein of mitochondrial malate dehydrogenase and citrate synthase have been reanalysed in view of predictions based on experimentally determined kinetic parameter values for the dehydrogenase and synthase activities of the protein. The results show that the time-course of citrate formation from malate in the coupled reaction catalysed by the fusion protein can be most satisfactorily accounted for in terms of a free-diffusion mechanism when consideration is taken to the inhibitory effects of NADH and oxaloacetate on the malate dehydrogenase activity. The effect of aspartate aminotransferase on the coupled reaction is likewise fully consistent with that expected for a free-diffusion mechanism. It is concluded that no tenable kinetic evidence is available to support the proposal that the fusion protein catalyses citrate formation from malate by a mechanism involving channelling of the intermediate oxaloacetate.