L-alanine:2-oxoglutarate aminotransferase isoenzymes from Arabidopsis thaliana leaves

The occurrence of four l-alanine:2-oxoglutarate aminotransferase (AOAT) isoenzymes (AOAT-like proteins): alanine aminotransferase 1 and 2 (AlaAT1 and AlaAT2, EC 2.6.1.2) and l-glutamate:glyoxylate aminotransferase 1 and 2 (GGAT1 and GGAT2, EC 2.6.1.4) was demonstrated in Arabidopsis thaliana leaves. These enzymes differed in their substrate specificity, susceptibility to pyridoxal phosphate inhibitors and behaviour during molecular sieving on Zorbax SE-250 column. A difference was observed in the electrostatic charge values at pH 9.1 between GGAT1 and GGAT2 as well as between AlaAT1 and AlaAT2, despite high levels of amino acid sequence identity (93 % and 85 %, respectively). The unprecedented evidence for the monomeric structure of both AlaAT1 and AlaAT2 is presented. The molecular mass of each enzyme estimated by molecular sieving on Sephadex G-150 and Zorbax SE-250 columns and SDS/PAGE was approximately 60 kDa. The kinetic parameters: K_m (Ala)=1.53 mM, K_m (2-oxoglutarate)=0.18 mM, k_cat=124.6 s⁻¹, k_cat/K_m=8.1 × 10⁴ M⁻¹·s⁻¹ of AlaAT1 were comparable to those determined for other AlaATs isolated from different sources. The two studied GGATs also consisted of a single subunit with molecular mass of 47.3–70 kDa. The estimated K_m values for l-glutamate (1.2 mM) and glyoxylate (0.42 mM) in the transamination catalyzed by putative GGAT1 contributed to indentification of the enzyme. Based on these results we concluded that each of four AOAT genes in Arabidopsis thaliana leaves expresses different AOAT isoenzyme, functioning in a native state as a monomer.     

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.     

The purification and properties of L-histidine--2-oxoglutarate aminotransferase from Pseudomonas testosteroni.

1. Inducible L-histidine--2-oxoglutarate aminotransferase was purified some 170-fold from extracts of Pseudomonas testosteroni. 2. The preparation showed only one major component after electrophoresis on polyacrylamide gels, though additional minor bands were observed when samples concentrated on a DEAE-cellulose column were used. 3. The molecular weight of the enzyme was found to be approx. 70000 by chromatography on Sephadex G-200. 4. The purification scheme produced enzyme that was inactive in the absence of pyridoxal 5'-phosphate. 5. The equilibrium constant for the reaction L-histidine+2-oxoglutarate equilibrium imidazolylpyruvate+L-glutamate was 0.49. 6. The reaction mechanism was Ping Pong. 7. The enzyme was shown to have only low activity towards aromatic amino acids and was highly specific for 2-oxoglutarate.     

L-alanine: 4,5-dioxovalerate aminotransferase from Pseudomonas riboflavina: purification and inactivation by methylglyoxal

L-Alanine:4,5-dioxovalerate aminotransferase, which catalyzes transamination between L-alanine and 4,5-dioxovalerate to yield delta-aminolevulinate and pyruvate, has been purified from Pseudomonas riboflavina IFO 3140. The enzyme had a molecular weight of 190,000 and consisted of four identical subunits. It was crystallized as pale yellow needles. The enzyme used L-alanine (relative activity 100), beta-alanine (39), and L-ornithine (14) as amino donors. gamma-aminobutyrate (55) and epsilon-aminocaproate (34) were also effective as amino donors. The reaction proceeded according to a ping-pong mechanism and the Km values for L-alanine and 4,5-dioxovalerate were 1.7 and 0.75 mM, respectively. The activity of the enzyme is strongly inhibited by pyruvate, hemin, and methylglyoxal. Methylglyoxal interacted with the enzyme and brought about a complete inactivation.     

Analysis ofl-alanine:2-oxoglutarate aminotransferase isozymes in maize

Isozyme analysis ofl-alanine:2-oxoglutarate aminotransferase (ALT) in maize indicates that there are three genes encoding this enzyme activity. Two of the gene products interact with each other to form heterodimers, while the third gene product does not interact with the other two. Another isozyme that appears after gel electrophoresis and ALT staining is shown to be glutamate dehydrogenase-1. Anaerobic treatment does not result in increased ALT levels, indicating that the previously reported increase in alanine levels caused by this treatment may be due to increases in the level of pyruvate, a substrate of ALT.D. A. Russell was partially supported by a graduate student fellowship from the Division of Biology and Biomedical Sciences, Washington University. V. M. Peschke was partially supported by a postdoctoral fellowship from Monsanto. This research was supported by NIH Grant R01 GM34740.     

Partial purification and properties of Halobacterium cutirubrum L-alanine dehydrogenase.

1. Halobacterium cutirubrum L-alanine dehydrogenase was purified approx. 100-fold. 2. It has a mol. wt. of 72 500, about one-third that of two well-studied alanine dehydrogenases from non-halophiles. 3. The activity of the enzyme increases with temperature up to 70 degrees C, but the protein itself is not thermostable. 4. In the reductive amination reaction, the enzyme is fully active in the presence of high concentrations of K+, Na+ or NH4+ and partially active with Cs+ or Li+, but for oxidative deamination it has an absolute requirement for K+.     

Effect of glucagon on alanine 2-oxoglutarate aminotransferase

Alanine-2-oxoglutarate aminotransferase activity in mouse liver is stimulated by the intravenous injection of glucagon. The stimulation is abolished by pretreatment with actinomycin D indicating that the increased activity is probably due to new enzyme formation. Administration of dibutyryl cyclic AMP, isoproterenol, an activator of adenyl cyclase and theophylline, an inhibitor of phosphodiesterase also increases the enzyme activity suggesting the involvement of cyclic AMP in glucagon-mediated increase of enzyme activity.     

A new assay for L-aspartate:2-oxoglutarate aminotransferase.

A new enzymatic assay for aspartate aminotransferase is presented. The 2-oxoglutarate formed in transamination between l-glutamate and oxalacetate was determined in a system coupled with hydroxyglutarate dehydrogenase and NADH by following a decrease in absorbance at 340 nm. The method allowed accurate determination of the initial velocity of the reaction, which was proportional to the enzyme concentration. The Michaelis constants of pig heart cytosolic aspartate aminotransferase for l-glutamate and oxalacetate and the amino acceptor specificity using l-glutamate as an amino donor were determined. The method was applicable to the determination of the enzyme activity in various materials including rat serum and bacterial crude extract.     

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.     

Identity of alanine:glyoxylate aminotransferase with alanine:2-oxoglutarate aminotransferase in rat liver cytosol

Rat liver soluble fraction contained 3 forms of alanine: glyoxylate aminotransferase. One with a pI of 5.2 and an Mr of approx. 110,000 was found to be identical with cytosolic alanine:2-oxoglutarate aminotransferase. The pI 6.0 enzyme with an Mr of approx. 220,000 was suggested to be from broken mitochondrial alanine:glyoxylate aminotransferase 2 and the pI 8.0 enzyme with an Mr of approx. 80,000 enzyme from broken peroxisomal and mitochondrial alanine:glyoxylate aminotransferase 1. These results suggest that the cytosolic alanine: glyoxylate aminotransferase activity is due to cytosolic alanine: 2-oxoglutarate aminotransferase.     

Production of delta-aminolevulinate: subcellular localization and purification of murine hepatic L-alanine: 4,5-dioxovaleric acid aminotransferase

1. L-Alanine: 4,5-dioxovaleric acid aminotransferase (DOVA transaminase) activity was measured in murine liver, kidney and spleen homogenates. 2. Among the organs examined, the specific activity of the enzyme was highest in kidney, followed by liver then spleen. 3. No differences in DOVA transaminase activity in kidney, liver and spleen homogenates were detected between mouse strains C57BL/6J and DBA/2J. 4. Based on enzyme activity, the capacity of DOVA transaminase to catalyze the formation of delta-aminolevulinic acid (ALA) in liver appeared much greater than the capacity of ALA synthase. 5. In DBA/2J animals, DOVA transaminase activity in liver mitochondrial fractions prepared by differential centrifugation was 24 nmol ALA formed/hr/mg protein compared with 0.63 nmol ALA formed/hr/mg protein for ALA synthase. 6. Cell fractionation analyses indicated that liver DOVA transaminase is located in the mitochondrial matrix. 7. The liver enzyme was purified from mitoplasts by chromatography on DEAE-Sephacel followed by affinity chromatography on L-alanine-AH-Sepharose. 8. The specific activity of the purified DOVA transaminase was 1600 nmol ALA formed/hr/mg protein. 9. The yield of the purification was ca 90 micrograms of protein per gram liver wet weight. 10. The purified enzyme had a subunit mol. wt of 146,000 +/- 5000 as determined by electrophoresis under denaturing conditions.     

Distribution and tissue specificity of 4-aminobutyrate-2-oxoglutarate aminotransferase

A rapid and specific method for assaying 4-aminobutyrate-2-oxoglutarate aminotransferase was developed. The method was based on the selectivity of ion exchange resin and the speed of vacuum filtration. With this new method, the aminotransferase activity in various tissues has been determined as follows: brain, 10.2; spinal cord, 11.8; liver, 5.7; kidney, 4.6; heart, 0.5; lung, 0.4 nmol glutamate formed/min/mg. No activity could be detected in muscle preparations. When the aminotransferases were tested with the antibody, against the purified 4-aminobutyrate aminotransferase from brain, no difference could be detected among brain, spinal cord, and kidney preparations as judged from the results of immunodiffusion, inhibition of enzyme activity by antibody, and microcomplement fixation. It is concluded that 4-aminobutyrate aminotransferases from various tissues of the mouse are probably identical or closely related.This work was supported in part by Grant No. NS 13224 and P01 NS 12116 from the National Institutes of Health, U.S.A. and Grant from Huntington's Chorea Foundation.All correspondence should be addressed to Dr. Wu.     

Purification and properties of 2-aminoadipate: 2-oxoglutarate aminotransferase from bovine kidney.

Escherichia coli endonuclease IV hydrolyses the C(3')-O-P bond 5' to a 3'-terminal base-free deoxyribose. It also hydrolyses the C(3')-O-P bond 5' to a 3'-terminal base-free 2',3'-unsaturated sugar produced by nicking 3' to an AP (apurinic or apyrimidinic) site by beta-elimination; this explains why the unproductive end produced by beta-elimination is converted by the enzyme into a 3'-OH end able to prime DNA synthesis. The action of E. coli endonuclease IV on an internal AP site is more complex: in a first step the C(3')-O-P bond 5' to the AP site is hydrolysed, but in a second step the 5'-terminal base-free deoxyribose 5'-phosphate is lost. This loss is due to a spontaneous beta-elimination reaction in which the enzyme plays no role. The extreme lability of the C(3')-O-P bond 3' to a 5'-terminal AP site contrasts with the relative stability of the same bond 3' to an internal AP site; in the absence of beta-elimination catalysts, at 37 degrees C the half-life of the former is about 2 h and that of the latter 200 h. The extreme lability of a 5'-terminal AP site means that, after nicking 5' to an AP site with an AP endonuclease, in principle no 5'----3' exonuclease is needed to excise the AP site: it falls off spontaneously. We have repaired DNA containing AP sites with an AP endonuclease (E. coli endonuclease IV or the chromatin AP endonuclease from rat liver), a DNA polymerase devoid of 5'----3' exonuclease activity (Klenow polymerase or rat liver DNA polymerase beta) and a DNA ligase. Catalysts of beta-elimination, such as spermine, can drastically shorten the already brief half-life of a 5'-terminal AP site; it is what very probably happens in the chromatin of eukaryotic cells. E. coli endonuclease IV also probably participates in the repair of strand breaks produced by ionizing radiations: as E. coli endonuclease VI/exonuclease III, it is a 3'-phosphoglycollatase and also a 3'-phosphatase. The 3'-phosphatase activity of E. coli endonuclease VI/exonuclease III and E. coli endonuclease IV can also be useful when the AP site has been excised by a beta delta-elimination reaction.