|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1991 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1990 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Role of malic enzyme in Bradyrhizobium japonicum energy metabolism were surveyed, and the data suggested the enzyme was a key enzyme to improve the energy metabolism.
An NADP-malic enzyme, a possible key enzyme for the energy metabolism of Bradyrhizobium japonicum A1017, was purified to homogeneity by ammonium sulfate precipitation, DEAE-cellulose column chromatography, preparative isoelectric-focusing, Sephacryl S-200 column, 2', 5'-ADP-Sepharose 4B column and NAD-agarose column chromatographies.
The purified enzyme showed its optimun pH at 7.4, and the pI value was 6.4. The reaction was dependent on NADP, with the Km values of 4.9 muM and 61 muM against NADP and malate respectively. The molecular weight of the enzyme subunit was calculated to be 59, 000 on SDS-PAGE, and the result of Sephacryl S-200 gel chromatography suggested that the native enzyme was present as dimer. Among metabolites present in the nodule cell extract, malonate, succinate, oxalacetate, ATP, ADP and NADPH in the enzyme reaction system showed strong regulatory effect on the activity under physiological concentrations.
From the purified enzyme, the N-terminal amino acid sequence was analysed. From the data 4 DNA probes were synthesized. A genomic DNA library was also prepared after selecting ca. 2 kbp DNA fragment by sucrose density gradient centrifugation. Following ligating and packaging of the DNA to lamda phage, the plaque hybridization was performed and selected the positive clone.