Nucleotide Sequence of Lactate Dehydrogenase from the Cephlochordate Branchiostoma belchiri and the Properties of the Gene Product by Escherichia coli

头索类文昌鱼乳酸脱氢酶的核苷酸序列及大肠杆菌基因产物的特性

• 批准号: 13640702
• 负责人: IMAI Toshio
• 金额: $ 1.79万
• 依托单位: Toho University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13640702/
• 关键词: Amphioxus; Branchiostoma belchiri; LDH gene; Nucleotide sequence; Expression; Enzymatic properties; LDH; LDHアミノ酸配列; 比較酵素学

A part of a study on molecular evolution and comparative enzymology of lactate dehydrogenase (LDH), we analyzed the LDH gene of the protochordate Amphioxus (B.belcheri), which, it is believed, is the phylogenetic predecessor of vertebrates and possesses their ancestral characteristics including enzymatic properties of vertebrates. The complete sequence of 999 bases in the LDH gene of Amphioxus was revealed, with 67%-86% being homologous with the LDH gene of other organisms. The amino acid sequence corresponding to the nucleotide sequence of the LDH protein was determined from this sequence, and the secondary structure of this protein in comparison with that of other organisms was estimated. Although the region involved in the enzymatic function of LDH was completely conserved, some regions around the active site varied in different organisms. The molecular phylogenetic tree, the preparation of which was based on the amino acid sequence, revealed that the LDH of Amphioxus may correspond to the phylogenetic predecessor to the vertebrate, i.e., the stage prior to the branching of LDH into LDH-A and LDH-B. in addition, we attempted to synthesize the LDH protein of B.belcheri by using an expression system obtained from Escherichia coli (E. coli) and to determine its characterization. The LDH protein was purified from an E. coli suspension, and the purified enzyme was demonstrated to be homogeneous by 2D-electrophoresis. The LDH was enzymologically characterized as having a molecular weight of 140 kDa, and isoelectric point of 7.5, and optimum pHs of 9.3-9.6 and 7.4-7.6 in L-P and P-L reactions, respectively. Thermal stability was examined at various temperatures during 30 minutes of incubation, and an 50% enzyme activity was observed even at 50℃. The apparent Km of LDH for pyruvate, lactate, NADH and NAD^+ were 1.7 × 10^<-4>M, 1.6 ×10^<-2>M, 3.7 × 10^<-4>M and 8.2 × 10^<-4>M, respectively.

作为乳酸脱氢酶（LDH）分子进化和比较酶学研究的一部分，我们对原脊索动物文昌鱼（B.belcheri）的LDH基因进行了分析。结果表明，文昌鱼LDH基因全长999个碱基，与其它生物LDH基因的同源性为67%~ 86%。从该序列确定对应于LDH蛋白质的核苷酸序列的氨基酸序列，并估计该蛋白质与其它生物的二级结构相比的二级结构。虽然参与LDH的酶功能的区域是完全保守的，但活性位点周围的一些区域在不同的生物体中是不同的。根据氨基酸序列构建的分子系统树显示文昌鱼LDH可能对应于脊椎动物的系统发育前身，LDH分支为LDH-A和LDH-B之前的阶段。另外，我们尝试通过使用从大肠杆菌（Escherichia coli）获得的表达系统来合成B.belcheri的LDH蛋白。coli）并确定其特性。从大肠杆菌中纯化LDH蛋白。大肠杆菌悬浮液中，纯化后的酶经双向电泳证明是均一的。LDH的酶学性质为分子量为140 kDa，等电点为7.5，L-P和P-L反应的最适pH分别为9.3-9.6和7.4-7.6。在不同温度下孵育30分钟期间检查热稳定性，并且即使在50℃下也观察到50%的酶活性。LDH对丙酮酸、乳酸、NADH和NAD^+的表观Km分别为1.7 × 10^<-4>M、1.6 ×10^<-2>M、3.7 × 10^<-4>M和8.2 × 10^<-4>M。