Emiko Okuma

Simultaneous determination of d- and l-amino acids in the nervous tissues of crustaceans using precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate and reversed-phase ion-pair high-performance liquid chromatography

After the derivatization of D- and L-amino acids with (+)-1-(9-fluorenyl)ethyl chloroformate, nineteen amino acids were separated into their D- and L-enantiomers and from other physiological amino compounds by reversed-phase ion-pair high-performance liquid chromatography. The separation was performed by three separate runs differing in mobile phase compositions and gradient profiles. Tyrosine, tryptophan and cysteine could not be detected because of their weak reactions with the derivatization reagent. Of seven D-amino acids found in the crustacean nervous tissues and eyes, D-alanine, D-arginine and D-aspartate were the most abundant and widely distributed.

Role of free d- and l-alanine in the Japanese mitten crab Eriocheir japonicus to intracellular osmoregulation during downstream spawning migration

Abstract Changes of d - and l -alanine and other osmolytes were examined in the tissues and hemolymph of the Japanese mitten crab Eriocheir japonicus captured during the maturation in the river and during the course of spawning migration downstream. Only d - and l -alanine and inorganic ions increased significantly in muscle during the maturation in freshwater from July to October and during downstream migration to estuaries in October. Inorganic ions in muscle was the highest in estuaries. In the crabs captured in the sea, glycine and d - and l -alanine were largely elevated in place of the inorganic ions declined. No other free amino acid increased in the muscle of the sea specimens. In hemolymph, total amino acids comprised only 4-10% of total osmolytes and decreased with maturation and during migration. Hemolymph inorganic ions were the highest also in estuaries. These data suggest that, in addition to a role as osmolyte, d - and l- alanine in the tissues of this strong hyperosmoregulator play an important role in the adjustment of salinity tolerance prior to and during downstream migration toward the sea.

Occurrence of Alanine Racemase in Crustaceans and the Changes of the Properties During Seawater Acclimation of Crayfish

Alanine racemase activity was detected in the muscle and hepatopancreas of six macruran species. Optimal pH was around 8.5 irrespective of species and tissues. Apparent Michaelis constants ranged from 48 to 157 mM for muscle enzyme and 35 to 239 mM for hepatopancreas enzyme. The enzyme specifically catalyzed the racemization of d- and l-alanine. The enzyme did not require pyridoxal 5′-phosphate as well as FAD as a cofactor. Pyruvate and l-alanine showed strong inhibition in the direction of d to l. During seawater acclimation of crayfish Procambarus clarkii, alanine racemase activity increased about twice in muscle and 1.5 times in hepatopancreas. Michaelis constant, on the other hand, decreased 33% for muscle enzyme and 65% for hepatopancreas enzyme, suggesting the increase of substrate affinity during seawater acclimation. The activity in the physiological pH range (6.5–7.5) also increased with increasing salinity.