METABOLIC ENZYMES AND REGULATION OF FREE D-AMINO ACIDS IN INVERTEBRATES

从黑虎对虾肌肉中分离出丙氨酸消旋酶。从纯化酶的多肽片段中获得了部分氨基酸序列。这些序列与一些细菌的酶有相似性，但相似性在40%以下。对虾酶在结构和性质上与细菌酶有很大的不同。根据这些部分序列制备抗体，用于检测酶的亚细胞分布和筛选cDNA文库。D-氨基酸和D-天冬氨酸氧化酶在鱼类中的分布具有物种特异性，并被认为与食物习性有关。添加D-丙氨酸可提高鲤鱼肠道、肝胰腺和肾脏中D-氨基酸氧化酶的活性，表明该酶是一种诱导型酶。D-天冬氨酸或D-谷氨酸对D-天冬氨酸氧化酶无诱导作用。两种酶的活性均为亚细胞…。从鲤鱼肝胰腺组织中克隆D-氨基酸氧化酶基因，得到801个碱基的扩增产物，编码267个氨基酸残基。在小龙虾、对虾和硬壳中，D-丙氨酸、L-丙氨酸随环境盐度的升高而显著增加，表明D-丙氨酸是这些无脊椎动物细胞内等渗调节的主要渗透调节物质。低盐习服后，小龙虾肌肉和肝胰腺中D-、L-丙氨酸含量升高，缺氧后恢复过程中D-、L-丙氨酸含量下降。根据这些数据，D-丙氨酸、L-丙氨酸与乳酸一起被认为是小龙虾的厌氧终产物。本研究阐明了D-丙氨酸在无脊椎动物中的生理作用，并首次测定了丙氨酸消旋酶的部分氨基酸序列。不含D-丙氨酸的鱼类将肝脏和肾脏中的D-氨基酸与D-氨基酸和D-天冬氨酸氧化酶代谢成相应的α-酮酸和解毒的D-氨基酸。较少

Alanine racemase was isolated from the muscle of black-tiger prawn. Partial amino acid sequences were obtained from the peptide fragments of purified enzyme. These sequences showed similarity with the enzyme from some bacteria but the similarities were below 40%. The prawn enzyme was considered to be rather different from bacterial counterpart in its structure and properties. Antibody was prepared on the basis of these partial sequences and used for the examination of subcellular distribution of the enzyme and the screening of cDNA library.Distribution of D-amino acid and D-aspartate oxidases in fish was species specific and considered to be related to food habits. The activity of D-amino acid oxidase increased in intestine, hepatopancreas, and kidney of carp with the administration of D-alanine, suggesting the enzyme is an inducible one. No enzyme induction occurred in D-aspartate oxidase with the administration of D-aspartate or D-glutamate. Both enzyme activities were subcellularly … More distributed in peroxisomes as is the case in mammals.cDNA cloning of D-amino acid oxidase from carp hepatopancreas gave a PCR product of 801 bp encoding 267 amino acid residues. The amino acid sequence showed similarities of 58-59% to human and porcine one.In the tissues of crayfish, kuruma prawn, and hard clam, D-, L-alanine increased significantly with increasing environmental salinity, indicating that D-alanine is a major osmolyte responsible for intracellular isosmotic regulation in these invertebrates. After hypersalinity acclimation, crayfish received severe anoxia increased D-, L-alanine in muscle and hepatopancreas which declined during recovery after anoxia. From these data, D-, L-alanine were considered to be anaerobic end-products together with lactate in crayfish.In the present study, physiological roles of D-alanine in invertebrates were clarified and the partial amino acid sequences of alanine racemase were determined for the first time. It was also clarified that fish species having no D-alanine metabolized D-amino acids to corresponding α-keto acid with D-amino acid and D-aspartate oxidases in liver and kidney and detoxified D-amino acids. Less