Purification and properties of hydroxypyruvate: l-Alanine transaminase from rabbit liver

A procedure is described for the extensive purification of hydroxypyruvate:l-alanine transaminase from rabbit liver. On the basis of gel filtration studies, the molecular weight of the enzyme is estimated to be about 41,000 daltons. A similar value was obtained when the enzyme was subjected to gel electrophoresis in the presence of sodium dodecyl sulfate indicating that the enzyme consists of a single polypeptide chain.The purified enzyme catalyzes the transamination of glyoxylate as well as hydroxypyruvate with l-alanine as the preferred amino donor for both substrates. The two enzymatic activities were not separated during purification nor by Chromatographic or electrophoretic procedures. Kinetic studies demonstrated that the two α-keto acids are competitive substrates. The above data are consistent with the fact that a single enzyme catalyzes the transamination of both glyoxylate and hydroxypyruvate. The effects of various inhibitors on enzymatic activity were investigated. The enzyme is inhibited by glyceraldehyde-3-phosphate and other aldehydes.The possible role of hydroxypyruvate:l-alanine transaminase in gluconeogenesis is discussed.     

Occurrence of hydroxypyruvate-L-glutamate transaminase in Escherichia coli and its separation from hydroxypyruvate-phosphate-L-glutamate transaminase

Blatt, L. (University of Wisconsin, Madison), F. E. Dorer, and H. J. Sallach. Occurrence of hydroxypyruvate-l-glutamate transaminase in Escherichia coli and its separation from hydroxypyruvate-phosphate-l-glutamate transaminase. J. Bacteriol. 92:668-675. 1966.-The formation of l-serine from hydroxypyruvate by a transamination reaction with l-glutamate has been demonstrated in extracts of Escherichia coli. The level of activity with hydroxypyruvate is approximately one-tenth that observed with hydroxypyruvate-phosphate in cell-free extracts. The transamination of hydroxypyruvate, but not hydroxypyruvate-phosphate, is inhibited by inorganic phosphate. No marked differences in the levels of activity with hydroxypyruvate were observed in extracts from bacteria grown under different conditions. Heat treatment of enzyme preparations at 65 C rapidly destroys the activity with hydroxypyruvate-phosphate, but not that with hydroxypyruvate. Fractionation of extracts with lithium sulfate and alumina Cgamma resulted not only in a 10-fold purification, but also in a complete separation of the two activities, thereby establishing that two different enzymes are involved in the transamination of hydroxypyruvate and hydroxypyruvate-phosphate. Hydroxypyruvate transaminase is present in two mutants that require serine for growth. The inability of hydroxypyruvate to replace the growth requirement for serine, even to a limited extent, was shown to be due to the inability of the bacteria to accumulate this compound actively.     

Kynurenine pyruvate transaminase and its inhibitor in rat intestine

Kynurenine pyruvate transaminase was found to be present in rat small intestine, partially purified and characterized. The enzyme catalysed the conversion of L-kynurenine to kynurenic acid. Transamination rates of 3-hydroxy-DL-kynurenine and 5-hydroxy-DL-kynurenine by the enzyme were 1/2.9 and 1/2.6 that of L-kynurenine. The enzyme showed higher preference for pyruvate than 2-oxoglutarate as aminoacceptor. The pH optimum of the reaction was 8.0 to 8.5. Purification of the enzyme lowered markedly apparent Km for L-kynurenine but not for pyruvate. It was shown that the inhibitor of kynurenine pyruvate transaminase was present in the intestine, on the basis of the inhibition produced by heating a portion of each purification step enzyme preparation in 50% ethanol, centrifuging, concentrating it, and adding it to an incubate of the unheated preparation. The possible interrelationship of enzyme and inhibitor was discussed and comparisons with kynurenine transaminase in liver, kidney and brains were noted.     

Affinity purification and properties of rat liver mitochondrial L-alanine:4,5-dioxovalerate transaminase and its inhibition by hemin

The present study describes a new rapid procedure for purification of L-alanine:4,5-dioxovalerate transaminase from rat liver mitochondria which was purified 243-fold with a 32% yield to apparent homogeneity. The purification procedure involved protamine sulfate treatment, followed by phenyl-Sepharose CL-4B column chromatography and alanine-Sepharose 4B affinity chromatography. The Km values for L-alanine and 4,5-dioxovalerate were 3.3 and 0.28 mM, respectively. The enzyme-bound pyridoxal phosphate content was estimated to be two molecules per enzyme molecule. The purified enzyme was inhibited by the reaction product pyruvic acid, substrate analog, methylglyoxal, and sulfhydryl inhibitors. Excess concentrations of 4,5-dioxovalerate was also found to inhibit the enzyme and our experiments failed to demonstrate reversibility of the reaction. Only hemin among the intermediate compounds of heme metabolism tested was shown to be an inhibitor of purified alanine:4,5-dioxovalerate transaminase. Hemin was further shown as an uncompetitive inhibitor of both alanine and dioxovalerate.     

Determination of the activities of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase in a microfluidic system

A microfluidic system for the analysis of the activities of glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) was fabricated. The device consists of a glass chip with a micro-electrochemical L-glutamate sensor and a polydimethylsiloxane (PDMS) sheet with a Y-shaped micro-flow channel. A sample solution and a substrate solution for the enzymes were introduced from two injection ports at the end of the flow channel. When the flows were stopped, substrates in a solution mixed immediately with either of the enzymes by diffusion in a mixing channel. L-glutamate produced by the enzymatic reaction of GOT or GPT in the flow channel was detected by using the L-glutamate sensor. A distinct current increase was observed immediately after mixing, and the initial slope of the response curve varied in proportion to the activity of GOT or GPT. The relation between the slope of the response curve and the enzyme activity was linear between 7 and 228 U l-1 for GOT and 9 and 250 U l-1 for GPT. The quality of the response curve was improved with an increase in the channel height. The measurement based on the rate analysis in the micro-flow channel facilitated the reduction of the influence of interferents. The influence of the viscosity of the sample solution was also checked for the analysis of real samples. The determination of the enzyme activities was also conducted in a system with micropumps fabricated for a sample injection. Two solutions could be mixed in the mixing channel, and the activity of the enzymes could be measured as in the experiments using microsyringe pumps.     

Cytosolic L-alanine:4,5-dioxovalerate transaminase differs from the mitochondrial form

L-Alanine:4,5-dioxovalerate transaminase was detected in the kidney cytosolic fraction with a lower specific activity than the mitochondrial enzyme. The enzyme was purified from the cytosol to homogeneity with a yield of 32%, and comparative analysis with the mitochondrial form was performed. Both forms of the enzyme have identical pH and temperature optima and also share common antigenic determinants. However, differences in their molecular properties exist. The molecular mass of the native cytoplasmic enzyme is 260 kDa, whereas that of the mitochondrial enzyme is 210 kDa. In addition, the cytoplasmic L-alanine: 4,5-dioxovalerate transaminase had a homopolymeric subunit molecular mass of 67 kDa compared to a subunit molecular mass of 50 kDa for the mitochondrial L-alanine:4,5-dioxovalerate transaminase. This is the first report of two forms of L-alanine:4,5-dioxovalerate transaminase. The different responses of cytosolic and mitochondrial L-alanine:4,5-dioxovalerate transaminases to hemin supplementation both in vitro and in vivo was demonstrated. Maximum inhibition of mitochondrial L-alanine:4,5-dioxovalerate transaminase activity was demonstrated with hemin injected at a dose of 1.2 mg/kg body mass, whereas the same dose of hemin stimulated the cytosolic enzyme to 150% of the control. A one-dimensional peptide map of partially digested cytosolic and mitochondrial L-alanine:4,5-dioxovalerate transaminase shows that the two forms of the enzymes are structurally related. Partial digestion of the cytosolic form of the enzyme with papain generated a fragment of 50 kDa which was identical to that of the undigested mitochondrial form (50 kDa). Moreover, papain digestion resulted in a threefold increase in cytosolic enzyme activity over the native enzyme, and such enhancement was comparable to the activity of the mitochondrial form of the enzyme. Therefore, we conclude that the cytosolic form of L-alanine: 4,5-dioxovalerate transaminase is different from the mitochondrial enzyme. Furthermore, immunoblot analysis indicated that the mitochondrial enzyme has antigenic similarity to the cytosolic enzyme as well as to the papain-digested cytosolic enzyme 50-kDa fragment.     

Regional and subcellular distribution of cysteine sulfinate transaminase in rat nervous system

The distribution of the cysteine sulfinate transaminase activity in adult and newborn rat central nervous system was studied and compared with the distribution of the glutamate oxaloacetate transaminase activity. The subcellular localization of both enzyme activities was also investigated. These experiments suggest that both enzymes, sometimes considered as identical, are different.     

Morphology, RNase and transaminase of root protoplasts

RNase and transaminase activities were analysed for mechanicallyand enzymatically prepared protoplasts from Allium Cepa roots.The comparative analyses at three root regions show that theenzymes were less active in the protoplasts than in the cellsfrom which they had been obtained. The enzyme gradients (fromapex to base of the root: RNase increase and transaminase decrease)noted previously in the intact roots were found to be similarin the protoplasts, however to a lesser degree. On the otherhand, the relative activity of both tested enzymes was lowerin the enzymatically prepared protoplasts than in those obtainedby the mechanical technique. In connection with their physiologicalproperties, the respective effects of the mode of preparingthe protoplasts were discussed. (Received November 22, 1971; )     

Human brain GABA transaminase tissue distribution and molecular expression.

Human brain gamma-aminobutyrate transaminase is differentially expressed in a tissue-specific manner. mRNA master dot-blot analysis for 50 different human tissues, including different brain regions and fetal tissues, provided a complete map of the tissue distribution. Genomic Southern analysis revealed that the gamma-aminobutyrate transaminase gene is a single copy, at least 15 kb in size. In addition, human brain gamma-aminobutyrate transaminase cDNA was expressed in Escherichia coli using a pGEX expression vector system. Catalytically active gamma-aminobutyrate transaminase was expressed in large quantities and the purified recombinant enzyme had kinetic parameters that were indistinguishable from those isolated from other mammalian brains. The human enzyme was inactivated by a well-known antiepileptic drug vigabatrin. Values of Ki and kinact were 1 mM and 0.35 min-1, respectively. Results from inactivation kinetics suggested that human gamma-aminobutyrate transaminase is more sensitive to the vigabatrin drug than the enzyme isolated from bovine brain.     

On the regulation of tyrosine transaminase, glutamatic dehydrogenase and aspartic transaminase in Tetrahymena

Aspartic transaminase, tyrosine transaminase, lactic dehydrogenase, and glutamic dehydrogenase were studied in Tetrahymena pyriformis in order to gain a better understanding of the control of the entrance and exit of metabolic intermediates to and from the major carbohydrate pathways. Glucose decreased the activity of aspartic transaminase, tyrosine transaminase and glutamic dehydrogenase but not lactic dehydrogenase. Actinomycin D (6 and 12 μg/ml) blocked the decrease in glutamic dehydrogenase and aspartic transaminase activity caused by glucose; 12 μg/ml partially prevented the decrease in tyrosine transaminase activity. Actinomycin D alone had little effect on enzyme activity. Uracil incorporation into RNA was doubled by 6 μg/ml actinomycin D, a concentration which did not alter the RNA content of the cells. At 12 μg/ml this drug caused a small decrease in RNA spec. act. Cycloheximide at 10 μg/ml, a concentration which inhibited protein synthesis by 70%, caused a three-fold increase in aspartic transaminase and a two-fold increase in glutamic dehydrogenase. In the presence of both cycloheximide and glucose, the drug effect predominated. Thus both actinomycin D and cycloheximide blocked the glucose-induced decrease in enzyme activity. These results suggest that the levels of aspartic transaminase, glutamic dehydrogenase, and probably tyrosine transaminase are regulated at least in part by a degradative control system.     

Identification and biochemical characterization of the Anopheles gambiae 3-hydroxykynurenine transaminase

Spontaneous oxidation of 3-hydroxykynureine (3-HK), a metabolic intermediate of the tryptophan degradation pathway, elicits a remarkable oxidative stress response in animal tissues. In the yellow fever mosquito Aedes aegypti the excess of this toxic metabolic intermediate is efficiently removed by a specific 3-HK transaminase, which converts 3-HK into the more stable compound xanthurenic acid. In anopheline mosquitoes transmitting malaria, xanthurenic acid plays an important role in Plasmodium gametocyte maturation and fertility. Using the sequence information provided by the Anopheles gambiae genome and available ESTs, we adopted a PCR-based approach to isolate a 3-HK transaminase coding sequence from the main human malaria vector A. gambiae. Tissue and developmental expression analysis revealed an almost ubiquitary profile, which is in agreement with the physiological role of the enzyme in mosquito development and 3-HK detoxification. A high yield procedure for the expression and purification of a fully active recombinant version of the protein has been developed. Recombinant A. gambiae 3-HK transaminase is a dimeric pyridoxal 5'-phosphate dependent enzyme, showing an optimum pH of 7.8 and a comparable catalytic efficiency for both 3-HK and its immediate catabolic precursor kynurenine. This study may be useful for the identification of 3-HK transaminase inhibitors of potential interest as malaria transmission-blocking drugs or effective insecticides.     

Induction and derepression of arginase and ornithine transaminase in different strains ofSaccharomyces cerevisiae

The syntheses of arginase and ornithine transaminase were studied in two strains ofSaccharomyces cerevisiae, viz. strain B and strain α-Σ1278b. Derepression of both enzymes during nitrogen starvation was shown only by strain B, non-specific induction of arginase only by strain α-Σ1278b. This different response of both strains studied reveals substantial differences in the regulation of enzyme synthesis among yeast strains of one and the same species. The specific enzyme activities observed in chemostat cultures with arginine as the nitrogen source and different sugars, at variable carbon to nitrogen ratios, did not indicate the involvement of carbon catabolite repression in the regulation of arginase and ornithine transaminase syntheses. Specific arginase activities observed in the continuous cultures varied widely and did not show a correlation with the intracellular arginine concentration. Extracellular steady-state arginine concentrations higher than about 0.1mm, in addition to abundant energy supply, were found to be required for high production of arginase. It is suggested that, besides intracellular arginine, extracellular arginine may provide an induction signal necessary for full-scale induction of arginase synthesis. A possible intermediary role of arginine permeases or of other membrane proteins is discussed.     

Glutamic-pyruvic transaminase activity related to red blood cell age.

Red blood cell glutamic-pyruvic transaminase (GPT) phenotypes and catalytic activities were studied in normal subjects and in patients with various hemolytic syndromes associated with reticulocytosis. To assess the effect of cell age of GPT activity, young cells were separated from older cells by centrifugation, and the catalytic activities were compared. In normal blood, there was a progressive fall in GPT activity from the top layer (younger cells) to the bottom layer (older cells), with a mean ratio of 1.90 plus or minus 0.42. Similarly, in the blood of patients with reticulocytosis, the enzyme activity of the reticulocyte-rich layer was higher than that of the layer containing older cells (mean ratio 1.94 plus or minus 0.95).     

The ilvEDA operon of Escherichia coli K12 encodes only one valine-alpha-ketoglutarate transaminase activity.

Transaminase B of $\text{E. coli}$ K12 was purified to apparent homogeneity as measured by SDS acrylamide gel electrophoresis, immunoelectrophoresis, and amino terminal sequence analysis. The valine- and isoleucine-α-ketoglutarate dependent transaminase activities of pure enzyme as well as crude extracts were characterized by immunologic and kinetic methods. The data disprove the existence of a separate valine-α-ketoglutarate transaminase within the $\text{ilvEDA}$ operon.     

Histidine:pyruvate transamination and phyenylalanine:pyruvate transamination may be catalyzed by the same enzyme.

Some properties of histidine:pyruvate transaminase (HPT) and phenylalanine:pyruvate transaminase (PPT) in the cytosol of rat liver were studied. HPT and PPT activity could not be separated by DEAE-Sephadex A-50 or hydroxylapatite column chromatography, and the ratio of $\text{HPT}\text{PPT}$ activity remained constant during these purification procedures. The two enzyme activities also showed similar heat stability and responses to glucagon injection. Based on these findings, we suggest that a single enzyme may specifically catalyze histidine:pyruvate and phenylalanine:pyruvate transamination.     

Glutamine transaminase K and cysteine S-conjugate beta-lyase activity stains.

An activity stain to detect glutamine transaminase K subjected to nondenaturing polyacrylamide gel electrophoresis (ND-PAGE) was developed. The gel is incubated with a reaction mixture containing L-phenyl-alanine, alpha-keto-gamma-methiolbutyrate (alpha KMB), glutamate dehydrogenase, phenazine methosulfate (PMS) and nitroblue tetrazolium (NBT). Glutamine transaminase K catalyzes a transamination reaction between phenylalanine and alpha KMB. The resultant methionine is a substrate of glutamate dehydrogenase. The NADH formed in the oxidative deamination of methionine reacts with PMS and NBT to form a blue band on the surface of the gel coincident with glutamine transaminase K activity. Cysteine S-conjugate beta-lyase activity is detected in the gel by incubating the gel with a reaction mixture containing alpha KMB (to ensure maintenance of the enzyme in the pyridoxal 5'-phosphate form), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), PMS, and NBT. The products of the lyase reaction interact with PMS and NBT to form a blue dye coincident with the lyase activity. In addition, a new assay procedure for measuring cysteine S-conjugate beta-lyase activity was devised. This procedure couples pyruvate formation from DCVC to the alanine dehydrogenase reaction. Preparations of purified rat kidney glutamine transaminase K yield a single protein band on ND-PAGE (apparent Mr approximately 95,000). This band coincides with both the cysteine S-conjugate beta-lyase and glutamine transaminase K activities. Activity staining showed that homogenates of rat kidney, liver, skeletal muscle, and heart possess a glutamine transaminase K/cysteine S-conjugate beta-lyase activity with an Rf value on ND-PAGE identical to that of purified rat kidney glutamine transaminase K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of two forms of hepatic phenylalanine:pyruvate transaminase from glucagon treated rats.

Two forms of phenylalanine:pyruvate transaminase (EC 2.6.1. aminotransferases, the exact EC number has not been assigned) termed A and B were obtained from the liver supernatant fraction of glucagon-treated rats by DEAE-Sephadex A-50 column chromatography. Each of the two forms was further purified by hydroxylapatite, Sephadex G-100 chromatography, and preparative gel electrophoresis. Both the A and B forms have been purified to homogeneity as judged by analytical and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Moreover, histidine was found to be a competitive inhibitor of phenylalanine with both purified proteins. These findings conclusively support the view that phenylalanine:pyruvate transaminase and histidine:pyruvate transaminase reactions are catalyzed by the same protein. The overall purification was 710-fold for the A form and 1200-fold for the B form. The apparent molecular weight for both A and B are 74,000 ±6000 as determined by gel filtration. Sodium dodecyl sulfate gel electrophoresis revealed that the A form has two identical subunits of molecular weight 42,000, whereas the B form has two nonidentical subunits of molecular weight 42,000 and 44,000. The amino acid composition for the A and B forms of the enzyme are different. The major differences are in glycine, alanine and leucine. The isoelectric point for A was 7.8 and for B was 7.3. However, the A and B forms of the enzyme are of immunological identity. The substrate specificity determined for both the A and B form was phenylalanine >asparagine >alanine >leucine >histidine. The K_m for phenylalanine was 7.70 mm for the A form, 6.00 mm for the B form. For histidine, the K_m was 13.70 mm for the A form, 12.50 mm for the B form.     

Excess generation of endogenous heme inhibits L-alanine:4,5-dioxovalerate transaminase in rat liver mitochondria

In the present study, we examined the possibility that the excess heme generation within mitochondria may provide a local concentration, sufficient to inhibit the activity of L-alanine:4,5-dioxovalerate transaminase, the enzyme proposed for an alternate route of delta-aminolevulinic acid biosynthesis in mammalian system. This was accomplished by assaying together L-alanine:4,5-dioxovalerate transaminase and heme synthetase activities in intact mitochondria isolated from rat liver. Endogenous heme in intact mitochondria has been generated in excess, by increasing the concentration of the substrate of heme synthetase. Our studies showed that the activity of L-alanine:4,5-dioxovalerate transaminase decreased as the rate of heme formation increased. In intact mitochondria, almost 50% inhibition of alanine:4,5-dioxovalerate transaminase was obtained with 4.0 mumole of heme generation. We conclude that end product inhibition of L-alanine:4,5-dioxovalerate transaminase by hemin, which was proposed in earlier report by us (FEBS Letter (1985), 189, 129), is an important physiological mechanism for the regulation of hepatic heme biosynthesis.     

Microphotometric determination of enzymes in brain sections. II. GABA transaminase

The tetrazolium salt procedure of van Gelder (1965) for the demonstration of GABA transaminase (GABAT; the most important GABA degrading enzyme) was adapted for microphotometric measurements of GABAT activities in brain sections using the hippocampus of rats as selected brain region. The final incubation medium consisted of 50 mM GABA, 5 mM alpha-ketoglutarate, 7 mM NAD, 10 mM sodium azide, 6 mM nitroblue tetrazolium chloride, 20 mM malonate and 15% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 8.0. There was a linear relationship between GABAT activity and section thickness up to 14 microns and between GABAT activity and reaction time at least up to 20 min (kinetic and end-point measurements). Phenazine methosulfate as an exogenous electron carrier and pyridoxal-5-phosphate as coenzyme of GABAT did not enhance the demonstrable GABAT activities, whereas sodium azide as a blocker of the respiratory chain resulted in an increase of demonstrable enzyme activities. A coreaction of succinate dehydrogenase was excluded by the use of malonate (competitive inhibitor). Using the incubation medium described GABAT activities were demonstrated via the endogenous enzymes succinic semialdehyde dehydrogenase and NADH tetrazolium reductase which were shown to be not rate limiting and seems to be similarly localized as GABAT.