Ryo-Hei Yamada

Presence of d-aspartate oxidase in rat liver and mouse tissues

Rat liver D-aspartate oxidase activity, which had been reported to be undetectable, was found to be well detectable in dialyzed liver homogenate. The requirements of the enzyme for activity and its sensitivity to inhibitors were identical with the known properties of the enzyme from other sources. We also demonstrated for the first time the presence of the enzyme activity in mouse tissues and some other rat tissues using dialyzed tissue homogenates.

Administration of D-aspartate increases D-aspartate oxidase activity in mouse liver

Oral administration of D-aspartate to mice for 2 weeks by addition of the amino acid to drinking water produced a nearly 4-fold increase in liver D-aspartate oxidase (EC 1.4.3.1) activity, whereas no increase was induced by L-aspartate administered in the same way. Administration of D-aspartate also produced a small significant increase in the kidney enzyme activity, but L-aspartate administration increased the activity as well. The enzyme activity in the brain and muscle was not affected by administration of either D- or L-aspartate. Intraperitoneal administration of D-aspartate increased the enzyme activity only in the liver, and other compounds tested, including D-glutamate and D-alanine, could not replace D-aspartate. The results indicate a specific relationship between D-aspartate and D-aspartate oxidase and suggest that the amino acid is, in fact, a physiological substrate of the enzyme.

D-Aspartate oxidase activity and D-aspartate content in a mutant mouse strain lacking D-amino acid oxidase

A mutant mouse strain ddY/DAO− lacks D-amino acid oxidase activity and accumulates free neutral D-amino acids in its tissues. In this study, D-aspartate oxidase activity and D-aspartate content in the tissues of these mutant mice were compared with those of normal mice. No significant difference was observed, indicating that the metabolism of acidic D-amino acids was unaffected in the mutant.

Presence of two DNA polymerases in Tetrahymena pyriformis

Two DNA polymerases were detected in Tetrahymena pyriformis, strain GL. One (enzyme I) was sensitive to N-ethylmaleimide, while the other (enzyme II) was insensitive. The molecular weight of the enzymes, as determined by glycerol gradient centrifugation analysis, were approximately 130,000 and 70,000, respectively. Optimal concentration of MgCl2 was 10mM for enzyme I and 18mM for enzyme II. KCl inhibited enzyme I but stimulated enzyme II. Poly (dA-dT) served effectively as a template for enzyme I, while poly(dA).(dT)12-18 was an effective template for enzyme II. Enzyme I activity increased with cell growth and sharply declined after the cells reached the stationary phase. On the other hand, enzyme II activity appeared only at the end of log phase. In cells synchronized by starvation-refeeding technique enzyme I was markedly stimulated in correspondence to the rate of DNA synthesis, whereas the level of enzyme II activity changed to lesser extent. By ethidium bromide treatment, only enzyme I activity was induced.

Slow- and tight-binding inhibition of aspartate aminotransferase by l-hydrazinosuccinate

The inhibition of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) by L-hydrazinosuccinate has been studied. The velocity of the enzyme reaction decreased with time when the reaction was initiated by the addition of enzyme to a mixture of the assay components and L-hydrazinosuccinate, while it increased slowly from a low level when a preincubated mixture of the enzyme and the inhibitor was added to the reaction mixture to initiate the reaction. Nearly 50% decrease in the initial reaction velocity was produced by a prolonged preincubation of the enzyme with the inhibitor, both at low concentrations of about 2 nM. These findings indicate that the inhibition is of the slow- and tight-binding type. The time-course of the reaction of the enzyme and the inhibitor, examined by the change in activity, was not in accord with single-step mechanisms, but rather appeared to follow biphasic kinetics. The inhibition could be fully reversed only in the presence of L-cysteine sulfinate or large excess of L-aspartate to convert the regenerated enzyme to its pyridoxamine form. The time-course of the reversal followed pseudo-first-order kinetics. Quantitative analysis of the experimental data has shown that the results are consistent with a mechanism of enzyme-inhibitor interaction which involves a reaction of two consecutive, reversible steps. The overall inhibition constant for L-hydrazinosuccinate was calculated to be approx. 0.2 nM.

Inhibition of aspartate aminotransferase by hydrazinosuccinate

DL-Hydrazinosuccinic acid was synthesized by the reaction of DL-bromosuccinic acid with hydrazine. The compound strongly inhibited aspartate aminotransferase and gave 50% inhibition at 1.3 microM when added simultaneously with L-aspartate to an assay mixture containing enzyme. Incubation of the enzyme with the compound prior to assay resulted in a much stronger inhibition, which proceeded time-dependently. The inhibition was protectable with L-aspartate and was substantially reversed by dialysis.

Temperature- and time-dependent inactivation of pyrroline-5-carboxylate synthase: suggestive evidence for an allosteric regulation of the enzyme

When pyrroline-5-carboxylate (PC) synthase activity in the membrane of mitochondria of rat small intestine mucosa was assayed in the presence of 0.5 mM ornithine, the time course of inactivation showed that the activity disappeared entirely by about 8 min at 30 degrees C, whereas there was no decrease in the activity at 15 degrees C. A prior incubation of the enzyme with ornithine at 30 or 37 degrees C in the presence of 50% sorbitol as a thermal stabilizer resulted in a marked loss of the activity, while that at 0 or 15 degrees C did not lose any. This suggests that PC synthase is inactivated by ornithine regardless of the presence of substrates. The inactivation at 30 degrees C proceeded gradually for about 7 h, until an equilibrium was attained. Extensive dialysis allowed the inactivated enzyme to regain about 60% of the original activity. These results suggest that the inactivation is reversible. The concentration of ornithine and the percentage of inactivation at equilibrium was correlated by the Hill equation and displayed a sigmoidicity with n = 1.47 and [S]50 = 0.036 mM. In the presence of sorbitol, the inactivation was prevented by 0.2 mM ATP or ADP. The role of the nucleotides in PC synthase regulation is discussed.

Apparent pyridoxine transport mutants of Escherichia coli with pyridoxal kinase deficiency

By nitrosoguanidine treatment of a vitamin B-6 auxotroph (KG980) of Escherichia coli, mutants were isolated that require for growth markedly higher concentrations of pyridoxine than the parent strain. One of the mutants, strain HN1, exhibited a severely reduced ability to take up extracellular pyridoxine. Besides, cell-free extracts of HN1 showed an extremely low activity to phosphorylate pyridoxine compared to that of KG980. These findings together with other results suggest that phosphorylation of pyridoxine is essential for the concentration uptake of the vitamin.

A one-step assay for pyroline-5-carboxylate synthase using a short AG1-X8 column

A rapid and simple method for assay of pyrroline-5-carboxylate synthase is presented. In this method, the incubation is terminated by raising the pH of incubation mixture to 10, and [14C]pyrroline 5-carboxylate produced from the substrate, [14C]glutamate, is first converted quantitatively to [14C]proline by reduction with NaBH4 at pH 10 and then the proline is allowed to pass through column of AG1-X8 anion exchanger under the conditions where the glutamate is completely retained by the column. Radioactive counting of the eluate gives the synthase activity. The entire procedure takes only one hour.

Citrulline accumulation in mice induced by administration of l-hydrazinosuccinate

L-Hydrazinosuccinate has been shown to induce a marked inhibition of liver aspartate aminotransferase isoenzymes in mice. The effects of the drug on the amino acid content of liver were studied. Intraperitoneal administration of L-hydrazinosuccinate enormously increased the citrulline content of liver and plasma in 6 hr and, less markedly, increased the glutamate and ammonia content of liver with a simultaneous decrease in the aspartate content. Drug administration also induced a marked increase in the liver mitochondrial activity of citrulline formation from ornithine, ammonia and carbon dioxide, with a similar increase in N-acetylglutamate content; a prominent increase in liver tryptophan dioxygenase activity; and an elevated level of plasma corticosterone. The increase of citrulline was interpreted to be produced by decreased conversion of citrulline to argininosuccinate due to a lack of aspartate because of inhibition of aspartate aminotransferase by the drug and increased formation of citrulline due to increases of glutamate and ammonia, which further induced the increase of N-acetylglutamate, because of inhibition of aminotransferase as well as stimulation of amino acid degradation by glucocorticoids.