PURIFICATION AND CHARACTERIZATION OF NADH DEHYDROGENASE FROM BACILLUS-MEGATERIUM

NADH dehydrogenase of Bacillus megaterium was isolated from the sonica te soluble fraction. The enzyme was purified approximately 61-fold by a combination of ammonium sulfate fractionation and column chromatogra phy on DEAE-Sephadex and hydroxyapatite. The purified enzyme has an ap parent molecular weight of 42 000 as determined by SDS-polyacrylamide gel electrophoresis and activity staining for NADH-MTT (4,5-dimethylth iazol-2-yl)-2,5-diphenyltetrazolium bromide) oxidoreductase. The enzym e is specific for NADH and has a pH optimum of 7.5-7.8. The apparent K (m) values for NADH are 15.7, 34.8, and 69.2 muM as determined for NAD H-DCIP (dichlorophenol-indophenol), NADH-ferricyanide, and NADH-MTT ox idoreductases. FAD is the prosthetic group of the enzyme. NAD+ acts as a competitive inhibitor. The inhibition studies suggest that NADH deh ydrogenase is the primary electron donor of the NADH oxidase system. L ocalization studies and inhibition studies together indicate that the NADH oxidase is a complex of membrane-bound enzymes and coenzymes.