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ABSTRACTA liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the separation and quantification of the enantiomers of N-methylaspartate and N-methylglutamate, after derivatization with Nα-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide was established. The time required for the LC-ESI-MS/MS analysis was within 20 min and the detection limit was approximately 10 fmol per injection, demonstrating that this method can be used for the rapid determination of D-aspartate N-methyltransferase activity in the ark shell clam Scapharca broughtonii.Abbreviations:NMDA: N-methyl-D-aspartate; NMLA: N-methyl-L-aspartate; NMDG: N-methyl-D-glutamate; NMLG: N-methyl-L-glutamate; NMA: N-methylaspartate; NMG: N-methylglutamate; HPLC: high-performance liquid chromatography; SAM: S-adenosyl-L-methionine; OPA: o-phthalaldehyde; LC-ESI-MS/MS: liquid chromatography-electrospray ionization-tandem mass spectrometry; FDLA: Nα-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide; FDAA: Nα-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide; ESI: electrospray ionization; LC-ESI-MS: liquid chromatography-electrospray ionization-mass spectrometry; MS/MS: tandem mass spectrometry

ABSTRACTD-Aspartate, aspartate racemase activity, and D-aspartate oxidase activity were detected in tissues from several types of starfish. Aspartate racemase activity in male testes of Patiria pectiniferawas significantly elevated in the summer months of the breeding season compared with spring months. We also compared aspartate racemase activity with the gonad index and found that activity in individuals with a gonad index ≥6% was four-fold higher than that of individuals with a gonad index <6%. The ratio of the D-form of aspartate to total aspartate was approximately 25% in testes with a gonad index <6% and this increased to approximately 40% in testes with a gonad index ≥6%. However, such changes were not observed in female ovaries. Administration of D-aspartate into male starfish caused testicular growth. These results indicate the possible involvement of aspartate racemase and D-aspartate in testicular maturation in echinoderm starfish.

d-Aspartate, aspartate racemase activity, and d-aspartate oxidase activity were detected in tissues from several types of starfish. Aspartate racemase activity in male testes of Patiria pectiniferawas significantly elevated in the summer months of the breeding season compared with spring months. We also compared aspartate racemase activity with the gonad index and found that activity in individuals with a gonad index =6% was four-fold higher than that of individuals with a gonad index <6%. The ratio of the D-form of aspartate to total aspartate was approximately 25% in testes with a gonad index <6% and this increased to approximately 40% in testes with a gonad index =6%. However, such changes were not observed in female ovaries. Administration of d-aspartate into male starfish caused testicular growth. These results indicate the possible involvement of aspartate racemase and d-aspartate in testicular maturation in echinoderm starfish.

D-Amino acid oxidase (DAO) is a flavin enzyme that catalyzes the oxidative deamination of d-amino acids. This enzyme has been studied extensively both biochemically and structurally as a model for the oxidase-dehydrogenase class of flavoproteins. This enzyme also has various applications, such as the determination of d-amino acids and production of building blocks for a number of pharmaceuticals. DAO has been found mainly in eukaryotic organisms and has been suggested to play a significant role in various cellular processes, one of which includes neurotransmission in the human brain. In contrast, this enzyme has not been identified in prokaryotic organisms. Some studies have recently identified and characterized DAO enzyme in some actinobacteria. In addition, a genome database search reveals a wide distribution of DAO homologous genes in this bacterial group. The bacterial DAOs characterized so far have certain distinct properties in comparison to eukaryotic DAOs. These enzymes also exhibit some important applicable properties, suggesting that bacteria could be used as a source for obtaining novel and useful DAOs. The physiological function of bacterial DAO have been proposed to include the degradation of non-canonical d-amino acids released from cell wall, but is still largely unknown and need to be studied in depth.

Renal dipeptidase (EC 3.4.13.19) activity in serum and urine from healthy volunteers (n= 20), patients with diabetes (n= 18) and patients with chronic renal failure (n= 5) was measured using glycyl-d-alanine as substrate. The assay was highly specific for the enzyme and was not affected by the various aminopeptidases present in serum and urine. No difference in serum renal dipeptidase activity was observed between the groups. The enzyme activity (U/L) in urine was higher than that in serum, irrespective of the group, suggesting the urine concentration was not affected by the serum concentration. The mean renal dipeptidase activities in urine were 2·56, 2·46 and 0·78 U/mol creatinine for healthy subjects, patients with diabetes and patients with chronic renal failure, respectively. The renal dipeptidase activity was significantly lower in the chronic renal failure group. The urinary excretion of dipeptidase (U/mmol creatinine) showed significant inverse correlations with that of β2-microglobulin, albumin and α1-microglobulin, and with serum concentrations of creatinine, β2-microglobulin and α1-microglobulin. We suggest that urine dipeptidase may be a useful marker of renal diseases.

We describe the occurrence of d-aspartate oxidase activity and free d-aspartate and d-glutamate in several tissues of five fish species, carp (Cyprinus carpio), crucian carp (Carassius auratus langsdorfii), rainbow trout (Oncorhynchus mykiss), yellowtail (Seriola quinqueradiata) and sea bream (Pagrus major). N-methyl-d-aspartate was the best enzyme substrate, followed by d-aspartate and d-glutamate. The highest specific activity was observed in the liver, followed by the kidney and brain in the crucian carp and rainbow trout. In the other fishes, enzyme activities were similar in the liver and kidney and lower in the brain except the yellowtail. d-Aspartate (% of total) and d-glutamate (%) were detected with HPLC analysis in the tissues. With the exception of spleen of some species, d-glutamate concentration tended to be higher than those for d-aspartate, but d/(d+ l) ratios were higher for aspartate.

Glutathione and its metabolites were examined for reactivity to acetaldehyde. When acetaldehyde was incubated with glutathione alone, there was only a slight decrease of acetaldehyde, while an apparently equimolar reaction between acetaldehyde and free sulfhydryl was observed with the addition of γ-glutamyltranspeptidase. Cysteinylglycine, the first metabolite in the glutathione breakdown by γ-glutamyltranspeptidase, showed a rapid and equimolar reactivity to acetaldehyde and such was comparable to the reaction seen withl-cysteine ord-penicillamine. In light of the chemical structure, cysteinylglycine probably conjugates with acetaldehyde to form thiazolidinecarboxylic acid derivatives, 2-methyl-thiazolidine-4-carbonyl-glycine, and if so, the alteration of glutathione metabolism by acetaldehyde during ethanol intoxication warrants further attention.

SummaryGenetic polymorphism of the glucose dehydrogenase in white cells extracts from random adult Japanese was investigated using polyacrylamide gel isoelectric focusing or agarose gel isoelectric focusing, followed by a specific zymogram technique. Three common phenotypes, which might correspond to GDH 1, GDH 2 and GDH 2-1 reported by King and Cook (1981), were observed at the p Is between pH 6.56–6.76 on the gel. No phenotypes with GDH 3 component were detected so far. The allele frequency of GDH3may be very low among Japanese. The results of family study suggest that these phenotypes are inherited in the autosomal codominant trait. The allele frequencies were GDH1=0.522 and GDH2=0.478.

SummaryPlasma AT-III exhibits a microheterogeneous form with pIs distributed over a narrow pH range by analytical agarose gel isoelectric focusing followed by immunofixation. Three new variants, each of which was the heterozygous state between the common and each variant components, were observed in a total 370 samples from unrelated healthy donors. These variants have at least normal activities of the thrombin inactivation in the presence of heparin, and their immunological antigen concentrations in plasma are in the normal range.