title: Purification and characterization of NADH dehydrogenase from Bacillus subtilis.
abstract: NADH dehydrogenase from Bacillus subtilis W23 has been isolated from membrane vesicles solubilized with 0.1% Triton X-100 by hydrophobic interaction chromatography on an octyl-Sepharose CL-4B column. A 70-fold purification is achieved. No other components could be detected with sodium dodecyl sulphate polyacrylamide gel electrophoresis. Ferguson plots of the purified protein indicated no anomalous binding of sodium dodecyl sulphate and an accurate molecular weight of 63 000 could be determined. From the amino acid composition a polarity of 43.8% was calculated indicating that the protein is not very hydrophobic. Optical absorption spectra and acid extraction of the enzyme chromophore followed by thin-layer chromatography showed that the enzyme contains 1 molecule FAD/molecule. The enzyme was found to be specific for NADH. NADPH is oxidized at a rate which is less than 6% of the rate of NADH oxidation. The activity of the enzyme as determined by NADH:3-(4'-5'-dimethyl-thiazol-2-yl)2,4-diphenyltetrazolium bromide oxidoreduction is optimal at 37 C and pH 7.5-8.0. The purified enzyme has a Kapp for NADH of 60 microM and a V of 23.5 mumol NADH/min X mg protein. These parameters are not influenced by phospholipids. The enzyme activity is hardly or not at all affected by NADH-related compounds such as ATP, ADP, AMP, adenosine, deoxyadenosine, adenine and nicotinic amide indicating the high binding specificity of the enzyme for NADH.
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000302:isolation & purification; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography; D003579:Cytochrome Reductases / Q000302:isolation & purification*; D007122:Immunoelectrophoresis; D008970:Molecular Weight; D009245:NADH Dehydrogenase / Q000302:isolation & purification*; D012995:Solubility; D013053:Spectrophotometry
chemical_list: D001426:Bacterial Proteins; D003579:Cytochrome Reductases; D009245:NADH Dehydrogenase