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

无脊椎动物中的代谢酶和游离 D-氨基酸的调节

基本信息

批准号：
12460091
负责人：
ABE Hiroki
金额：
$ 10.43万
依托单位：
THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

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

从黑虎虾的肌肉中分离得到丙氨酸消旋酶，从纯化酶的肽片段中获得部分氨基酸序列，这些序列与一些细菌的酶相似，但相似度低于40％。根据这些部分序列制备了与细菌对应物有很大不同的抗体，并用于检查酶的亚细胞分布和cDNA文库的筛选。D-氨基酸和D-氨基酸的分布。鱼类中的 D-天冬氨酸氧化酶具有物种特异性，并且被认为与饮食习惯有关。施用 D-丙氨酸后，鲤鱼的肠道、肝胰腺和肾脏中的 D-氨基酸氧化酶活性增加，表明该酶是一种诱导型酶。一。给予 D-天冬氨酸或 D-谷氨酸时，D-天冬氨酸氧化酶中没有发生酶诱导。这两种酶活性均分布在亚细胞内。与哺乳动物中的情况一样。从鲤鱼肝胰腺中克隆 D-氨基酸氧化酶的 PCR 产物，得到编码 267 个氨基酸残基的 801 bp 的 PCR 产物，其氨基酸序列与人和猪的氨基酸序列有 58-59% 的相似性。小龙虾、车虾和硬蛤的组织中，D-、L-丙氨酸随着环境盐度的增加而显着增加，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剂，在高盐度驯化后，小龙虾的肌肉和肝胰腺中的 D-、L-丙氨酸增加，从这些数据来看，D-、L-丙氨酸在缺氧后的恢复过程中下降。 -丙氨酸被认为是小龙虾中的厌氧终产物。在本研究中，丙氨酸的生理作用阐明了无脊椎动物中的D-丙氨酸，并首次测定了丙氨酸消旋酶的部分氨基酸序列，还阐明了不含D-丙氨酸的鱼类将D-氨基酸代谢为具有D-氨基酸的相应α-酮酸。肝脏和肾脏中的酸性和D-天冬氨酸氧化酶以及解毒的D-氨基酸较少。