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

作为乳酸脱氢酶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）的乳酸脱氢酶基因进行了分析，认为文昌鱼是脊椎动物的系统发育祖先，具有脊椎动物的酶学特性等祖先特征。文昌鱼LDH基因的完整序列为999个碱基，其中67% ~ 86%与其他生物的LDH基因同源。根据该序列确定了LDH蛋白核苷酸序列对应的氨基酸序列，并估计了该蛋白与其他生物的二级结构。虽然参与LDH酶促功能的区域是完全保守的，但在不同的生物体中，活性位点周围的一些区域是不同的。基于氨基酸序列构建的文昌鱼分子系统发育树显示，文昌鱼的LDH可能对应于脊椎动物的系统发育前身，即LDH分化为LDH- a和LDH- b之前的阶段。此外，我们尝试利用大肠杆菌（e.c oli）的表达系统合成B.belcheri的LDH蛋白，并确定其特性。从大肠杆菌悬浮液中纯化LDH蛋白，通过2d电泳证明纯化酶是均匀的。经酶学鉴定，该LDH在L-P和P-L反应中的分子量为140 kDa，等电点为7.5，最适ph值分别为9.3 ~ 9.6和7.4 ~ 7.6。在30分钟的培养过程中，在不同温度下测试了热稳定性，即使在50℃下也观察到50%的酶活性。丙酮酸、乳酸、NADH和NAD^+的LDH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。

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.