Rat and human membrane dipeptidase: tissue distribution and developmental changes.

Distribution and developmental changes in membrane dipeptidase activity were examined in rat and human tissues. The activity to hydrolyze glycyl-D-alanine in rat and human tissues was completely or almost completely inhibited by 5 mM cilastatin, suggesting that the activity was due to membrane dipeptidase and that the contribution of leucine aminopeptidase to the activity was minor. In 8-week-old rats, the activity was high in lung, kidney, pancreas and testis, and in each pooled sample of ileal mucosa, duodenal mucosa, jejunal mucosa and adrenal mucosa. A low activity was found in spleen, liver, serum and heart. The activity in lung, kidney, adrenal and intestinal mucosa increased up to the age of 5 or 8 weeks, while that in pancreas, testis and spleen reached a maximal level at around 3 weeks and declined thereafter. The distribution profile of the enzyme in postmortem tissues of adult humans was similar to that in rat, except for an extremely low activity in lung. The enzyme was also found in serum and urine from healthy volunteers. In urine, the activity was significantly correlated to the creatinine content. No clear dependence of the activity on gender or age was observed in urine and serum.

Purification and kinetic properties of a D-amino-acid peptide hydrolyzing enzyme from pig kidney cortex and its tentative identification with renal membrane dipeptidase.

We previously reported the presence of an enzyme activity which hydrolyzes Gly-D-Asp in pig kidney cortex. In the present study, an enzyme which hydrolyzes this peptide and other peptides having a low number of D- and L-amino acids has been purified from the brush border membranes of the same tissue. The native enzyme, having a molecular weight of 99,000, was apparently a homodimer of a subunit with a molecular weight of 48,000 and its optimum pH was 7.8 with Gly-D-Ala as substrate. The enzyme hydrolyzed many dipeptides, but not most of the tripeptides tested with a few exceptions, from which the carboxyl-terminal amino acid was liberated by the enzyme. Of the dipeptides examined, those having a D-amino acid at the amino-terminal were poor substrates, whereas those bearing a D-amino acid at the carboxyl-terminal were good substrates, comparable with their diastereomers with a L-amino acid at the same position. The enzyme was potently inhibited by cilastatin but not by amastatin and bestatin. Metal ion chelators and dithiothreitol were also inhibitory. Comparison of the properties of present enzyme with those of other known enzymes suggests that this should be tentatively identified with renal membrane dipeptidase. The demonstrated high activity toward dipeptides containing various D-amino acids at the carboxyl-terminal suggests a possibility that the enzyme in fact plays a role in degradation in vivo of D-amino-acid-containing peptides.

Distribution of D-aspartate oxidase and free D-glutamate and D-aspartate in chicken and pigeon tissues.

The presence of D-aspartate oxidase activity and D-glutamate was demonstrated for the first time in several tissues of chicken and pigeon. 2. The identification of the enzyme was based on substrate and inhibitors specificity of the activity as well as other requirements for the activity, and the amino acid was identified with HPLC analysis. 3. In each animal, the highest activity was found in the kidney. In chicken, the hepatic, renal and pancreatic activities were significantly higher in male than female. In pigeon, however, any significant gender difference was not observed. 4. A substantial amount of D-glutamate, as well as D-aspartate, was detected in each tissue, irrespective of species. 5. The contents of the free D-enantiomers were significantly different between two genders in the chicken kidney, heart and pancreas and pigeon liver and kidney. However, any common relationship was not observed between the contents and the D-aspartate oxidase activity.

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.

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.

Inhibition of serum aspartate aminotransferase induced by isoniazid administration in mice.

Intraperitoneal administration of isoniazid (IN), an antituberculous drug, to mice at a dose of 100 mg/kg body weight induced significant inhibition of serum aspartate aminotransferase (AAT) in several hours. The original activity was not restored readily by in vitro treatment of the sera with pyridoxal phosphate (PLP), in contrast to the rapid activation, by the same treatment, of apoAAT in the sera from control mice. Prolonged incubation with PLP prior to the assay was required to restore most of the lost activity. Serum AAT activity enhanced by experimental and the reversal of the inhibition similarly required prolonged incubation with PLP. The results suggest the possibility that human serum AAT activity measured for clinical diagnosis may be underestimated in cases of IN overdose even if the conditions for in vitro PLP treatment of samples are sufficient for conversion of the apoenzyme to the holoenzyme.

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.

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.

Uptake and utilization of vitamin B6 and its phosphate esters by Escherichia coli.

Escherichia coli KG980, a vitamin B6 auxotroph derived from E. coli K12, utilized the three unphosphorylated forms of vitamin B6, more or less effectively, for growth, but did not utilize the three phosphate forms at concentrations ranging from 10(-7) to 10(-5) M in the minimum medium of Davis and Mingioli. The bacterium, however, utilized the phosphate forms, although less effectively than the unphosphorylated forms, in the phosphate starving medium of Garen and Levinthal which is known to derepress alkaline phosphatase. Correspondingly, the phosphate forms of 3H-labeled vitamin B6 in the minimum medium were not taken up by the bacterial cells grown in the same medium, but were taken up when the phosphate starving medium was used for growth and uptake experiments; the unphosphorylated forms were taken up with either of the media used. After 30-min incubation of the cells grown in the phosphate starving medium with 3H-pyridoxine phosphate added to the same medium, the main extracellular 3H-vitamin B6 was found to be pyridoxine, evidently indicating an involvement of alkaline phosphatase action. It is concluded from these results that the phosphate forms of vitamin B6 can be taken up and utilized only after dephosphorylation but not taken up in their intact form. The initial rate of 3H-pyridoxal and 3H-pyridoxamine uptake in the minimum medium showed saturation kinetics. The Km and Vmax values were 1.2 X 10(-6) M and 62 pmoles/min/mg (dry cell weight) for pyridoxal; 11 X 10(-6) M and 65 pmoles/min/mg for pyridoxamine. 3H-Pyridoxine uptake apparently consisted of a high-affinity saturable component, whose Km value was tentatively estimated to be 2.2 X 10(-6) M, and an unsaturable component. The uptake rates of these three unphosphorylated vitamin B6 compounds compared at limiting concentrations for the growth of E. coli KG980 appear to be essentially in good agreement with the response pattern of this bacterium to the three compounds.