Aspartate: 2-oxoglutarate aminotransferase from trichomonas vaginalis. Identity of aspartate aminotransferase and aromatic amino acid aminotransferase.

Aspartate: 2-oxoglutarate aminotransferase from the anaerobic protozoon Trichomonas vaginalis was purified to homogeneity and characterized. It is a dimeric protein of overall Mr approx. 100000. Only a single isoenzyme was found in T. vaginalis. The overall molecular and catalytic properties have features in common with both the vertebrate cytoplasmic and mitochondrial isoenzymes. The purified aspartate aminotransferase from T. vaginalis showed very high rates of activity with aromatic amino acids as donors and 2-oxoglutarate as acceptor. This broad-spectrum activity was restricted to aromatic amino acids and aromatic 2-oxo acids, and no significant activity was seen with other common amino acids, other than with the substrates and products of the aspartate: 2-oxoglutarate aminotransferase reaction. Co-purification and co-inhibition, by the irreversible inhibitor gostatin, of the aromatic amino acid aminotransferase and aspartate aminotransferase activities, in conjunction with competitive substrate experiments, strongly suggest that a single enzyme is responsible for both activities. Such high rates of aromatic amino acid aminotransferase activity have not been reported before in eukaryotic aspartate aminotransferase.     

Formation of the intermediate products of the tricarboxylic acid cycle and ammonia from free amino acids in anoxic heart muscle

Glutamate and aspartate showed the highest rate of catabolism in oxygenated isolated rat heart with the formation of glutamine, asparagine and alanine. Under anoxia, the catabolism of branch chained amino acids and that of lysine, proline, arginine and methionine was inhibited. However, glutamate and aspartate catabolized at a higher rate as compared with oxygenation. Alanine was the product of their excessive degradation. During oxygenation, 70% of ammonia were produced via deamination of amino acids. Under anaerobic conditions the participation of amino acids in ammoniagenesis decreased to 4%; the principal source of ammonia was the adenine nucleotide pool. The total pool of the tricarboxylic acid cycle intermediates increased 2.5-fold due to accumulation of succinate. The data obtained suggest that the constant influx of intermediates into the cycle from amino acids is supported by coupled transamination of glutamate and aspartate. This leads to the formation of ATP and GTP in the tricarboxylic acid cycle during blocking of aerobic energy production.     

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.     

Aspartate:2-oxoglutarate aminotransferase from Trichomonas vaginalis: comparison with pig heart cytoplasmic enzyme

The aspartate:2-oxoglutarate aminotransferase from the protozoon Trichomonas vaginalis exists as a mixture of sub-forms of identical Mr and amino acid composition, and of similar catalytic properties. The amino acid composition closely resembles that of aspartate aminotransferase from prokaryotic and vertebrate sources. Some molecular and catalytic properties of the T. vaginalis aspartate aminotransferase are compared with those of the cytoplasmic pig heart enzyme. A major difference is in the ability of the trichomonal enzyme to transaminate aromatic amino acids and 2-oxo acids. A range of inhibitors have been used to compare the active-site regions of the T. vaginalis and cytoplasmic pig heart aspartate aminotransferases.     

Amino acid metabolism in Ancylostoma ceylanicum and Nippostrongylus brasiliensis

Ancylostoma ceylanicum and Nippostrongylus brasiliensis decarboxylated most of the amino acids examined, but only a few at significant rates. The former nematode in general possessed higher activities. Striking differences between the two parasites were, however, noticed regarding the metabolism of some of the amino acids. For instance, while alanine followed by aspartate produced highest amounts of 14CO2 in the presence of A. ceylanicum, proline exhibited maximum decarboxylation in case of N. brasiliensis. Tyrosine and lysine, on the other hand, did not liberate detectable CO2 with either parasite. Likewise, although large number of amino acids underwent transamination with 2-oxoglutarate, only some of them elicited appreciable activity for any of the two parasites.     

Oxygen evolution by a reconstituted spinach chloroplast system in the presence ofl-glutamine and 2-oxoglutarate

Intact chloroplasts prepared from summer-grown spinach plants supported (aspartate plus 2-oxoglutarate)-dependent O₂ evolution but not (glutamine plus 2-oxoglutarate)-dependent O₂ evolution. The former activity, which was sensitive to amino oxyacetate, was attributed to transaminase activity and reduction of the resulting oxalo-acetate to malate using H₂O as eventual electron donor. A reconstituted chloroplast system which included chloroplast stroma, thylakoid membranes, ferredoxin and NADP(H) supported O₂ evolution in the presence ofl-glutamine and 2-oxoglutarate at rates of 15–22 μmol mg^-1 chlorophyll h^-1 although lower rates were obtained with material from winter-grown plants. Activity was not observed in the absence of ferredoxin and omission of NADP(H) decreased activity by 40%. The reaction was associated with the production of 0.49 mol O₂ mol^-1 2-oxoglutarate consumed and up to 0.46 mol O₂ mol^-1 glutamine supplied. The reaction, which was inhibited by azaserine but not by methionine sulphoximine or amino oxyacetate, was attributed to light-coupled glutamate synthase (EC 1.4.1.13) with H₂O serving as eventual electron donor. Activity was not affected significantly byl-malate. The reconstituted system also supported O₂ evolution in the presence of nitrite, oxaloacetate, (aspartate plus 2-oxoglutarate) and oxidised glutathione.     

Floral Induction in a Photoperiodically Insensitive Duckweed, Lemna paucicostata LP6 : Role of Glutamate, Aspartate, and Other Amino Acids and Amides

The effects of 20 amino acids and two amides were studied on the flowering of a photoperiodically insensitive duckweed, Lemna paucicostata LP6. Alanine, asparagine, aspartate, cystine, glutamate, glutamine, glycine, lysine, methionine, proline, serine, and threonine induced flowering under a photoperiodic regime of 16 hours light and 8 hours darkness. Among these, glutamate and aspartate were found to be the most effective for flower induction. These acids could initiate flowering even at 5 × 10⁻⁷ molar level, though maximal flowering (about 80%) was obtained at 10⁻⁵ molar. Change in the photoperiodic schedule or the pH of the nutrient medium did not influence glutamate- or aspartate-induced flowering. The low concentrations at which glutamate and aspartate are effective suggests that they may have a regulatory role rather than simply acting as metabolites.     

Participation of alpha-amino group of amino acids in ammonia production in heart muscle

The ammoniogenetic capacity of some L-amino acids in rat heart muscle was studied. It was shown that [15N]leucine, [15N]glutamate and [15N]aspartate are involved in ammonia production without being the major source of this compound. The amount of [15N]ammonia produced by [15N]amino acids makes up to 0.25% of its total content. The deamination of L-[15N]leucine catalyzed by 2-oxoglutarate occurred at the highest rate. The [15N]aspartate and [15N]glutamate appeared to be less efficient precursors of ammonia. The bulk of amino nitrogen of L-[15N]amino acids was incorporated into the proteins and free amino acids in heart muscle.     

Glutamine:phenylpyruvate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8

A subfamily I aminotransferase gene homologue containing an open reading frame encoding 381 amino acid residues (Mr=42,271) has been identified in the process of the genome project of an extremely thermophilic bacterium, Thermus thermophilus HB8. Alignment of the predicted amino acid sequence using FASTA shows that this protein is a member of aminotransferase subfamily Igamma. The protein shows around 40% identity with both T. thermophilus aspartate aminotransferase [EC 2.6.1.1] and mammalian glutamine:phenylpyruvate aminotransferase [EC 2.6.1.64]. The recombinant protein expressed in Escherichia coli is a homodimer with a subunit molecular weight of 42,000, has one pyridoxal 5'-phosphate per subunit, and is highly active toward glutamine, methionine, aromatic amino acids, and corresponding keto acids, but has no preference for alanine and dicarboxylic amino acids. These substrate specificities are similar to those described for mammalian glutamine: phenylpyruvate aminotransferase. This is the first enzyme reported so far that has the glutamine aminotransferase activity in non-eukaryotic cells. As the presence of aromatic amino acid:2-oxoglutarate aminotransferase [EC 2.6.1.57] has not been reported in T. thermophilus, this enzyme is expected to catalyze the last transamination step of phenylalanine and tyrosine biosynthesis. It may also be involved in the methionine regeneration pathway associated with polyamine biosynthesis. The enzyme shows a strikingly high pKa value (9.3) of the coenzyme Schiff base in comparison with other subfamily I aminotransferases. The origin of this unique pKa value and the substrate specificity is discussed based on the previous crystallographic data of T. thermophilus and E. coli aspartate aminotransferases.     

Intestinal apical amino acid absorption during development of the pig

Amino acids originating from the diet are the principal metabolic fuels for the small intestine, and although the developing intestine is exposed to dramatic changes in the types and amounts of protein, there is little known about rates of amino acid absorption across the apical membrane during development. Therefore, rates of absorption were measured for five amino acids that are substrates for the acidic (aspartate), basic (lysine), neutral (leucine and methionine), and imino (proline) amino acid carriers using intact tissues from the proximal, mid-, and distal small intestines of pigs ranging in age from 90% of gestation to 42 days after birth (12 days after weaning). Rates of absorption (sum of carrier-mediated and apparent diffusion) were highest at birth (except for proline) and declined by an average of 30% during the first 24 h of suckling. There were continuing declines for leucine, methionine, and proline but not for aspartate and lysine. Due to rapid growth of the intestine, absorption capacities for all amino acids increased faster than predicted from gains in metabolic mass. Regional differences for rates of absorption were not detected until after birth, and only for aspartate and proline. Maximum rates of saturable absorption (nmol. min(-1). mg tissue(-1)) by the midintestine increased during the last 10% of gestation, were highest at birth, and then declined. The contribution of apparent diffusion to amino acid absorption was lowest at birth, then increased after onset of suckling.     

Glutamate Dehydrogenase Reaction as a Source of Glutamic Acid in Synaptosomes

The role of the glutamate dehydrogenase reaction as a pathway of glutamate synthesis was studied by incubating synaptosomes with 5 mM 15NH4Cl and then utilizing gas chromatography-mass spectrometry to measure isotopic enrichment in glutamate and aspartate. The rate of formation of [15N]glutamate and [15N]aspartate from 5 mM 15NH4Cl was approximately 0.2 nmol/min/mg of protein, a value much less than flux through glutaminase (4.8 nmol/min/mg of protein) but greater than flux through glutamine synthetase (0.045 nmol/min/mg of protein). Addition of 1 mM 2-oxoglutarate to the medium did not affect the rate of [15N]glutamate formation. O2 consumption and lactate formation were increased in the presence of 5 mM NH3, whereas the intrasynaptosomal concentrations of glutamate and aspartate were unaffected. Treatment of synaptosomes with veratridine stimulated reductive amination of 2-oxoglutarate during the early time points. The production of ([15N]glutamate + [15N]aspartate) was enhanced about twofold in the presence of 5 mM beta-(+/-)-2-aminobicyclo [2.2.1]heptane-2-carboxylic acid, a known effector of glutamate dehydrogenase. Supplementation of the incubation medium with a mixture of unlabelled amino acids at concentrations similar to those present in the extracellular fluid of the brain had little effect on the intrasynaptosomal [glutamate] and [aspartate]. However, the enrichment in these amino acids was consistently greater in the presence of supplementary amino acids, which appeared to stimulate modestly the reductive amination of 2-oxoglutarate. It is concluded: (a) compared with the phosphate-dependent glutaminase reaction, reductive amination is a relatively minor pathway of synaptosomal glutamate synthesis in both the basal state and during depolarization; (b) NH3 toxicity, at least in synaptosomes, is not referable to energy failure caused by a depletion of 2-oxoglutarate in the glutamate dehydrogenase reaction; and (c) transamination is not a major mechanism of glutamate nitrogen production in nerve endings.     

Glutamate-glyoxylate aminotransferase in rat liver cytosol. Purification, properties and identity with alanine-2-oxoglutarate aminotransferase

After cortisone injection, virtually identical increases in rat liver cytosol alanine-2-oxoglutarate aminotransferase and glutamate-glyoxylate aminotransferase activities were observed. The two activities were co-purified to homogeneity from rat liver cytosol. The purified enzyme was specific for L-alanine with 2-oxoglutarate as amino acceptor. With glyoxylate, however, the enzyme utilized various L-amino acids as amino donors in the following order of activity: glutamate greater than alanine greater than glutamine greater than methionine. The ratio of alanine-2-oxoglutarate aminotransferase activity to glutamate-glyoxylate aminotransferase activity remained constant during purification and was unchanged by a variety of treatments of the purified enzyme. These results suggest that glutamate-glyoxylate aminotransferase is identical with alanine-2-oxoglutarate aminotransferase. Evidence was obtained that the two enzyme activities in the cytosol of dog, cat and human liver are also properties of the same protein.     

The role of amino acid catabolism in the formation of the tricarboxylic acid cycle intermediates and ammonia in anoxic rat heart

Amino acid catabolism, the tricarboxylic acid cycle intermediates and ammonia formation were studied in isolated perfused rat heart under anoxia. The total net anaplerosis due to amino acid degradation in anoxia was equal to that in oxygenation (6.29 and 6.09 mumol/g dry weight per h, respectively) as a result of the increased transamination of glutamic and aspartic acids. During anoxic perfusion, the rate of catabolism of glutamic and aspartic acids was 1.5-times higher than in normoxia, while depletion of branched-chain amino acids, lysine, proline, arginine and methionine, was inhibited. Alanine was the product of excessive degradation of glutamic and aspartic acids. Under anaerobic conditions, in spite of inhibition of amino acid deamination, ammonia formation was increased 2.7-fold as compared to oxygenation. The principal amount of ammonia (96%) was produced at degradation of adenine nucleotides. A 2.5-fold increase in the pool of the tricarboxylic acid cycle intermediates under anoxia was associated mainly with accumulation of succinate. The data suggest that the coupling of alanine- and aspartate amino transferases is a mechanism controlling the tricarboxylic acid cycle pool size in anoxic heart.     

Biosynthesis of amino acids in Clostridium pasteurianum

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.     

Amino Acid Biosynthesis by Isolated Chloroplasts during Photosynthesis

The pool sizes of the common amino acids in purified intact chloroplasts from Vicia faba L. were measured (nanomoles per milligram chlorophyll). The three amino acids present in the highest concentrations were glutamate, aspartate, and threonine. Alanine, serine, and glycine were each present at levels between 15 and 20 nanomoles per milligram chlorophyll and 13 other amino acids were detectable at levels below 10.     

4-Aminobutyrate:2-oxoglutarate aminotransferase of Streptomyces griseus: purification and properties

4-Aminobutyrate: 2-oxoglutarate aminotransferase of Streptomyces griseus was purified to homogeneity on disc electrophoresis. The relative molecular mass of the enzyme was found to be 100 000 +/- 10 000 by a gel filtration method. The enzyme consists of two subunits identical in molecular mass (Mr 50 000 +/- 1000). The transaminase is composed of 486 amino acids/subunit containing 10 and 12 residues of half-cystine and methionine respectively. The NH2-terminal amino acid sequence of the enzyme was determined to be Thr-Ala-Phe-Pro-Gln. The enzyme exhibits absorption maxima at 278 nm, 340 nm and 415 nm with a molar absorption coefficient of 104 000, 11 400 and 7280 M-1 cm-1 respectively. The pyridoxal 5'-phosphate content was calculated to be 2 mol/mol enzyme. The enzyme has a maximum activity in the pH range of 7.5-8.5 and at 50 degrees C. The enzyme is stable at pH 6.0-10.0 and at temperatures up to 50 degrees C. Pyridoxal 5'-phosphate protects the enzyme from thermal inactivation. The enzyme catalyzes the transamination of omega-amino acids with 2-oxoglutarate; 4-aminobutyrate is the best amino donor. The Michaelis constants are 3.3 mM for 4-aminobutyrate and 8.3 mM for 2-oxoglutarate. Low activity was observed with beta-alanine. In addition to omega-amino acids the enzyme catalyzes transamination with ornithine and lysine; in both cases the D isomer is preferred. Carbonyl reagents and sulfhydryl reagents inhibit the enzyme activity. Chelating agents, non-substrate L and D-2-amino acids, and metal ions except cupric ion showed no effect on the enzyme activity.     

Aspartate aminotransferase of Pediococcus cerevisiae.

A five-step procedure is described for preparing highly purified aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC.2.6.1.1) from cell-freee enzyme extracts of Pediococcus cerevisiae. An overall purification of 130-fold was achieved. Some of P. cerevisiae aspartate aminotransferase properties were studied, i.s. pH optimum (7.8--8.0), optimum of temperature (37 degrees), Michaelis constans for 4 enzyme substrates and substrate specificity of enzyme. The enzyme is very thermolabile. During purification the enzyme was stabilizated by 2-oxoglutarate. The highly purified preparation was stored in the solution containing ammonium sulphate. The obtained aspartate aminotransferase preparation was free of alanine and aromatic amino acids aminotransferase activites and did not reveal malate dehydrogenase activity.     

Transamination pathways influencing L-glutamine and L-glutamate oxidation by rat enterocyte mitochondria and the subcellular localization of L-alanine aminotransferase and L-aspartate aminotransferase

Using analytical subcellular fractionation techniques, 12% of the total L-alanine aminotransferase activity and 26% of the total L-aspartate aminotransferase activity was localized in enterocyte mitochondria. Alanine and aspartate were products from the oxidation of glutamine and glutamate by enterocyte mitochondria. At low concentrations, malate stimulated aspartate synthesis but was inhibitory at higher concentrations. The malate inhibition of aspartate synthesis, which increased in the presence of pyruvate, was accompanied by an increase in alanine synthesis. With glutamine as substrate in the presence of pyruvate and malate, alanine synthesis was increased by 127% on addition of purified L-alanine aminotransferase, in spite of large amounts of glutamate generated. It was concluded that when pyruvate is available the important route for glutamine or glutamate oxidation by transamination was via L-alanine:2-oxoglutarate aminotransferase and not via L-aspartate:2-oxoglutarate aminotransferase. Results suggested that mitochondria may account for 50% of alanine production from glutamine in the enterocyte despite the relatively low activity of L-alanine aminotransferase therein.     

P-aminohippurate accumulation in kidney cortex slices: stimulation by dicarboxylates, amino acids and their oxoanalogues.

The effect of various amino acids and oxoacids on the accumulation of PAH in rat kidney cortex slices was determined. The following compounds were found to increase the PAH tissue to medium ratio (T/MPAH): a) dicarboxylic acids: glutarate, 2-oxoglutarate and oxaloacetate, b) amino acids: glutamate, isoleucine, leucine, valine, methionine, tryptophane, histidine, threonine and glycine, c) monocarboxylates: hydroxymethionine, oxovaline, oxoisoleucine and oxoleucine. There were no marked concentration/effect differences to glycine, glutamate, glutarate and oxovaline. Ouabain inhibited T/MPAH only slightly, but abolished its increase by pyruvate, 2-oxoglutarate and histidine. Oxygen hyposaturation abolished the T/MPAH increase caused by 2-oxoglutarate, pyruvate, glutamate and histidine. It is concluded that various substrates stimulating the organic anion transport system (OATS) do so namely by improving the energy supply, although the direct participation of dicarboxylates in OATS could be of relevance namely in short-lasting variations.     

Molecular aspects of methionine biosynthesis

Methionine, lysine and threonine are essential amino acids required in the diets of non-ruminant animals. Major crops, such as corn, soybean and rice, are low in one or more of these amino acids. Currently, these amino acids are supplemented to animal feed to allow optimal growth--a costly process for farmers and consumer, therefore there is a great deal of interest in increasing essential amino acids in crops. The metabolism of methionine in plants is linked to the regulation of the aspartate pathway and is important for plant growth. In recent years, several key steps of this pathway have been identified at the molecular level, enabling us to initiate transgenic approaches to engineer the methionine content of plants.