|Budget Amount *help
¥2,800,000 (Direct Cost : ¥2,800,000)
Fiscal Year 2002 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 2001 : ¥2,100,000 (Direct Cost : ¥2,100,000)
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.