The subcellular distribution of rat liver l-alanine–glyoxylate aminotransferase in relation to a pathway for glucose formation involving glyoxylate

1. The distribution of l-alanine-glyoxylate aminotransferase activity between subcellular fractions prepared from rat liver homogenates was investigated. The greater part of the homogenate activity (about 80%) was recovered in the ;total-particles' fraction sedimented by high-speed centrifugation and the remainder in the cytosol fraction. 2. Subfractionation of the particles by differential sedimentation and on sucrose density gradients revealed a specific association between the aminotransferase and the mitochondrial enzymes glutamate dehydrogenase and rhodanese. 3. The aminotransferase activities in the cytosol and the mitochondria are due to isoenzymes. The solubilized mitochondrial enzyme has a pH optimum of 8.6, an apparent K(m) of 0.24mm with respect to glyoxylate and is inhibited by glyoxylate at concentrations above 5mm. The cytosol aminotransferase shows no distinct pH optimum (over the range 7.0-9.0) and has an apparent K(m) of 1.11mm with respect to glyoxylate; there is no evidence of inhibition by glyoxylate. 4. The mitochondrial location of the bulk of the rat liver l-alanine-glyoxylate aminotransferase activity is discussed in relation to a pathway for gluconeogenesis involving glyoxylate.     

Some structural properties of plant serine:glyoxylate aminotransferase?

The structural properties of photorespiratory serine:glyoxylate aminotransferases (SGAT, EC 2.6.1.45) from maize (Zea mays L.) and wheat (Triticum aestivum L.) leaves were examined. By means of molecular sieving on Zorbax SE-250 column and filtration through centrifugal filters it was shown that dimers of wheat enzyme (molecular mass of about 90 kDa) dissociate into component monomers (molecular mass of about 45 kDa) upon decrease in pH value (from 9.1 or 7.0 to 6.5). At pH 9.1 a 50-fold decrease of ionic strength elicited a similar effect. Under the same conditions homodimers of the maize enzyme (molecular mass similar to that of the wheat enzyme) remained stable. Immunoblot analysis with polyclonal antiserum against wheat seedling SGAT on leaf homogenates or highly purified preparations of both enzymes showed that the immunogenic portions of the wheat enzyme are divergent from those of the maize enzyme. The sequence of 136 amino acids of the maize enzyme and 78 amino acids of the wheat enzyme was established by tandem mass spectrometry with time of flight analyzer. The two enzymes likely share similarity in tertiary and quaternary structures as well as high level of hydrophobicity on their molecular surfaces. They likely differ in the mechanism of transport from the site of biosynthesis to peroxisomes as well as in some aspects of secondary structure.     

Properties of serine：glyoxylate aminotransferase purified from Arabidopsis thaliana leaves

The photorespiratory enzyme L-serine：glyoxylate amino- transferase （SGAT; EC 2.6.1.45） was purified from Arabidopsis thaliana leaves. The f＇mal enzyme was approximately 80 % pure as revealed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis with silver staining. The identity of the enzyme was confirmed by LC/MS/MS analysis. The molecular mass estimated by gel filtration chromato- graphy on Sephadex G-150 under non-denaturing conditions, mass spectrometry （matrix-assisted laser desorption/ ionization/time of flight technique） and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 82.4 kDa, 42.0 kDa, and 39.8 kDa, respectively, indicating dimer as the active form. The optimum pH value was 9.2. The enzyme activity was inhibited by aminooxyacetate and β-chloro-L-alanine both compounds reacting with the carbonyl group of pyridoxal phosphate. The enzyme＇s transaminating activity with L-alanine and glyoxylate as substrates was approximately 55 % of that observed with L-serine and glyoxylate. The lower Kmvalue （1.25 mM） for L-alanine, compared with that of other plant SGATs, and the kcat/Km（Ala） ratio being approxi- mately 2-fold higher than kcat/Km（Ser） suggested that, during photorespiration, Ala and Ser are used by Arabidopsis SGAT with equal efficiency as amino group donors for glyoxylate. The equilibrium constant （Keq）, derived from the Haldane relation, for the transamination reaction between L-serine and glyoxylate with the formation of hydroxypyruvate and glycine was 79.1, strongly favoring glycine synthesis. However, it was accompanied by a low Km value of 2.83 mM for glycine. A comparison of some kinetic properties of the studied enzymes with the recombinant Arabidopsis SGATs previously obtained revealed substantial differences. The ratio of the velocity of the transamination reaction with L-alanine and glyoxylate as substrates versus that with L-serine and glyoxylate was 1：1.8 for the native enzyme, whereas it was 1：7 for the recombinant SGAT. Native SGAT showed a much lower Km value for L-alanine compared to the recombinant enzyme.     

Detection of a gem-diamine and a stable quinonoid intermediate in the reaction catalyzed by serine–glyoxylate aminotransferase from Hyphomicrobium methylovorum

Background

The enzyme l-serine–glyoxylate aminotransferase (SGAT) from Hyphomicrobium methylovorum is a PLP-containing enzyme that catalyzes the conversion of l-serine and glyoxylate to hydroxypyruvate and glycine. The cloned enzyme expressed in Escherichia coli is isolated as a mixture of the E:PLP and E:PMP forms. The PLP form of the enzyme has a maximum absorbance at 413 nm.

Methods

Uv–visible spectra of SGAT were obtained using an HP-8453 diode array spectrophotometer in the absence and presence of substrates and substrate analogs. Pre-steady state kinetic studies were carried out using an OLIS rapid scanning spectrophotometer in the rapid scanning mode.

Results

Incubation of the enzyme with a saturating concentration D-serine leads to a shift in the 413 nm peak to 421 nm that is ascribed to the external aldimine. The reverse stereochemistry of D-serine does not allow for abstraction of the Cα proton by the ε-amine of the active site lysine residue leading to an abortive external aldimine intermediate. Pre-steady state studies pushing SGAT against d-serine leads to a rapid decrease in the 413 nm peak and an increase at ∼ 330 nm with an associated rate constant of 47 s^− 1 at pH 7.6. This is followed by a slower decrease (0.26 s^− 1) at 330 nm and an increase and shift of the 413 nm peak to 421 nm. The intermediate species that absorbs at ∼ 330 nm is attributed to the gem-diamine intermediate. The rate of the fast phase increases with pH and increase in rate is likely due to the deprotonation of an enzymatic group that accepts a proton from the α-amine of d-serine. In the presence of hydroxypyruvate and ammonia the enzyme spectra display an increase in absorbance at 521 nm that occurs on the order of minutes. The shape and position of the 521 nm species is consistent with a quinonoid intermediate.

General significance

The data suggest a non-enzymatic reaction between hydroxypyruvate and ammonia to form an imine which will be in equilibrium with the enamine. A mechanism is proposed by which the enamine reacts with the PLP form of SGAT to generate the stable highly conjugated quinonoid intermediate.     

Identity of β-alanine-oxo-glutarate aminotransferase and L-β-aminoisobutyrate aminotransferase in rat liver

L-β-Aminoisobutyrate served as an amino donor for purified β-alanine-oxo-glutarate aminotransferase from rat liver when 2-oxoglutarate was employed as an amino acceptor, but he D-isomer did not. L-β-Aminoisobutyrate acted as a competitive inhibitor with respect to β-alanine and had a K_i of approximately 2.6 mM, which is the same value as the K_m of 2.7 mM. When the crude extract was applied to a DEAE-Sepharose CL-6B column, L-β-aminoisobutyrate aminotransferase and β-alanine-oxo-glutarate aminotransferase activities were found in the same fractions with a single peak. Antiserum to rat liver β-alanine-oxo-glutarate aminotransferase inhibited L-β-aminoisobutyrate aminotransferase activity in rat liver in the same way as β-alanine-oxo-glutarate aminotransferase activity.     

The interaction between α2-macroglobulin and cationic aspartate aminotransferase

On starch-gel or polyacrylamide-gel electrophoresis of human serum, a supernumerary zone of aspartate aminotransferase activity may be demonstrated, migrating with the slow alpha(2) protein zone. This appearance is due only to cationic aspartate aminotransferase, bound by alpha(2)-macroglobulin. The binding is strongly potentiated by dilute borate buffers.     

The effects of salicylate on the activity of rat liver tyrosine–2-oxoglutarate aminotransferase in vitro and in vivo

1. Salicylate, in concentrations of 0.25mm and above, enhances the basal activity of tyrosine–2-oxoglutarate aminotransferase in homogenates of rat liver incubated in the absence of added pyridoxal 5′-phosphate (endogenous activity). The effect is decreased by increasing the concentration of the cofactor. 2. The intraperitoneal administration of sodium salicylate enhances the activity of rat liver tyrosine aminotransferase; the major effect during the first hour being on the enzyme in the absence of added pyridoxal phosphate. Actinomycin D prevents the induction of the enzyme by cortisol and tryptophan. Induction by pyridoxine or salicylate is 50% inhibited by actinomycin D. The effects of the injections of various combinations of cortisol, pyridoxine and salicylate were also studied in the absence or presence of actinomycin D. 3. It is suggested that salicylate induces rat liver tyrosine aminotransferase by displacing its protein-bound cofactor and that a cofactor-type induction of the hepatic enzyme occurs in pyridoxine-treated rats.     

Fungal thermostable α-dialkylamino acid aminotransferase: occurrence,purification and characterization

-Dialkylamino acid aminotransferase was found in various fungi; this is the first evidence for the occurrence of the enzyme in eukaryotes. The enzyme was purified from Fusarium solani and shown to be composed of four subunits with an identical molecular weight of 42,000. -Aminoisobutyrate and cycloleucine served as amino donors, and pyruvate, -ketobutyrate, -ketovalerate, -ketoisovalerate, and glyoxylate as amino acceptors. The K _m values for -aminoisobutyrate and -ketobutyrate were 28 and 0.3 mM, respectively. -Ketobutyrate inhibited the enzyme noncompetitively with -aminoisobutyrate, and showed K _i value of 8 mM. The significant inhibitory effect of l-cycloserine was observed, but d-cycloserine did not inhibit the enzyme. The pH and temperature optima for transamination of -aminoisobutyrate with pyruvate were about 8.0 and 60°C, respectively. Despite the production of this enzyme by the mesophile, the enzyme was thermostable; it retained its full activity upon heating at 60°C for 30 min.Abbreviations ACPC 1-aminocyclopropane-1-carboxylic acid - AIB -aminoisobutyrate - PLP pyridoxal 5-phosphate     

Does leucine- and norleucine-induced insulin release depend on amino acid aminotransferase activity?

In the absence of another exogenous nutrient, L-leucine but not L-norleucine stimulates insulin release from rat pancreatic islets, although the corresponding keto acids, 2-ketoisocaproate and 2-ketocaproate, are equally potent secretagogues. Such a situation cannot be ascribed to the preferential transamination of L-leucine as compared to L-norleucine in islet homogenates. Indeed, in the presence of a suitable activator of glutamate dehydrogenase, L-leucine and L-norleucine stimulate secretion to the same extent. It is concluded that the rate of transamination of these amino acids in intact islet cells depends on the availability of a 2-keto acid partner rather than on the assayed amino acid aminotransferase activity.     

Increased liver l-serine–pyruvate aminotransferase activity under gluconeogenic conditions (Short Communication)

Rat liver l-serine-pyruvate aminotransferase activity exceeds markedly the normal adult value (a) in the neonatal period, (b) after glucagon injection and (c) after alloxan injection, observations that reinforce the suggestion from comparative findings that the aminotransferase has a role in gluconeogenesis. Some findings, however, argue in favour of l-serine dehydratase as the enzyme of gluconeogenesis from l-serine.     

Taurine-like GABA aminotransferase inhibitors prevent rabbit brain slices against oxygen–glucose deprivation-induced damage

The activation of the GABAergic system has been shown to protect brain tissues against the damage that occurs after cerebral ischaemia. On the other hand, the taurine analogues (±)Piperidine-3-sulphonic- (PSA), 2-aminoethane phosphonic- (AEP), 2-(N-acetylamino) cyclohexane sulfonic-acids (ATAHS) and 2-aminobenzene sulfonate-acids (ANSA) have been reported to block GABA metabolism by inhibiting rabbit brain GABA aminotransferase and to increase GABA content in rabbit brain slices. The present investigation explored the neuroprotection provided by GABA, Vigabatrin (VIGA) and taurine analogues in the course of oxygen–glucose deprivation and reperfusion induced damage of rabbit brain slices. Tissue damage was assessed by measuring the release of glutamate and lactate dehydrogenase (LDH) during reperfusion and by determining final tissue water gain, measured as the index of cell swelling. GABA (30–300?μM) and VIGA (30–300?μM) significantly antagonised LDH and glutamate release, as well as tissue water gain caused by oxygen–glucose deprivation and reperfusion. Lower (1–10?μM) or higher concentrations (up to 3,000?μM) were ineffective. ANSA, PSA and ATAHS significantly reduced glutamate and LDH release and tissue water gain in a range of concentrations between 30 and 300?μM. Lower (0–10?μM) or higher (up to 3,000?μM) concentrations were ineffective. Both mechanisms suggest hormetic (“U-shaped”) effects. These results indicate that the GABAergic system activation performed directly by GABA or indirectly through GABA aminotransferase inhibition is a promising approach for protecting the brain against ischemia and reperfusion-induced damage.     

The level of β-alanine aminotransferase activity in regenerating and differentiating rat liver

β-Alanine aminotransferase from rat liver was purified to electrophoretic homogeneity. The immunological and kinetic properties of this enzyme were similar to those of the enzyme from rat brain. However, the liver enzyme transaminates from β-alanine to 2-oxoglutaric acid, while the brain enzyme transaminates from γ-aminobutyric acid. β-Alanine aminotransferase activity in regenerating rat liver was lower than that in control rat liver. Activity of this enzyme, as well as of other uracil-catabolizing enzymes (Weber, G., Queener, S.F. and Ferdinandus, A. (1970) in Advances in Enzyme Regulation (Weber, G., ed.), Vol. 9, pp. 63–95, Pergamon Press, Oxford), was low in newborn rat liver and increased about 5-fold, reaching the level observed in adult rat liver. β-Alanine and prednisolone induced β-alanine aminotransferase in rat liver.     

Depletion of cultured human fibroblasts of pyridoxal 5′-phospate: Effect on activities of aspartate aminotransferase,alanine aminotransferase,and cystathionine β-synthase

Human skin fibroblasts were grown in culture medium containing virtually no pyridoxal. Cells cultured under these conditions grew to confluence for several passages without morphologic signs of degeneration and without changes in activity of two control enzymes, hexokinase and lactate dehydrogenase. The pyridoxal 5′-phosphate content of these fibroblasts fell to about 3% of values obtained during growth in pyridoxal-supplemented medium. The effect of such depletion on the activities of three pyridoxal 5′-phosphate-dependent enzymes was assessed during four consecutive passages. Total activity of cystathionine β-synthase and of aspartate aminotransferase in cell extracts fell to a mean of 50% of control values whereas total activity of alanine aminotransferase remained unchanged. Saturation of these enzymes with cofactor differed as well. The ratio of holoenzyme activity to total enzyme activity fell to less than 15% or predepletion values for cystathionine β-synthase and to 60% for aspartate aminotransferase. In contrast, alanine aminotransferase remained completely saturated with cofactor. Maximal saturation of aspartate amino-transferase with pyridoxal 5′-phosphate was achieved when pyridoxal 5′-phosphate-depleted fibroblasts were grown in medium containing as little as 1 ng/ml of pyridoxal, but addition of 10 ng/ml of pyridoxal was required for maximal saturation of cystathionine β-synthase. Maximal intracellular content of pyridoxal 5′-phosphate was achieved only when 100 ng/ml of pyridoxal was added to the growth medium. Interestingly, the activity of pyridoxine kinase remained constant during all depletion and repletion experiments. We conclude that the ability to grow human fibroblasts under these conditions provides a system for the study of apoenzyme-coenzyme interactions both in intact cultured cells and in cell extracts.     

Fatty acid β-oxidation and glyoxylate cycle enzyme activities of induced glyoxysomes from anise suspension cultures

Homogenates of dedifferentiated anise (Pimpinella anisum L.) suspension cultures grown in B-5 medium with sucrose as source of carbon show all but 3 glyoxysomal enzyme activities: NAD-dependent oxidation of palmitoyl-CoA, isocitrate lyase, and malate synthase are lacking. Substitution of 20 mmol/l acetate for sucrose leads to the appearance of these enzyme activities. Only then glyoxysomes with a buoyant density of 1.23 kg/l in sucrose gradients are formed showing the enzyme activities for both ß-oxidation of fatty acids and glyoxylate cycle. Quantitatively and qualitatively they resemble glyoxysomes isolated from endosperm of 4 d old anise seedlings. Therefore, the suspension cultures constitute a valuable system for the study of both mechanisms and regulation of glyoxysome formation in anise.     

Non-coordinate expression of peroxisome biogenesis, β-oxidation and glyoxylate cycle genes in mature Arabidopsis plants

The expression of three genes that encode proteins involved in peroxisome biogenesis, -oxidation and the glyoxylate cycle was studied in Arabidopsis plants by fusing their promoter regions to the reporter gene luciferase. Malate synthase showed an extremely restricted pattern of expression, being detected only in young seedlings and the root tips of older plants. PEX1 and 3-ketoacyl thiolase (PED1) were expressed in roots, mature leaves, stems and flowers. However, only thiolase was up-regulated by starvation. Immunoblotting confirmed that neither malate synthase nor the other unique glyoxylate cycle enzyme isocitrate lyase are expressed in senescent leaves. These results indicate that, in contrast to cucumber, pumpkin and barley, the glyoxylate cycle does not play a role in the recycling of carbon from the turnover of membrane lipids during senescence and starvation in Arabidopsis.