title: Isolation and characterization of a FAD-dependent NADH diaphorase from Methanospirillum hungatei strain GP1.
abstract: Crude extracts of Methanospirillum hungatei strain GP1 contained NADH and NADPH diaphorase activities. After a 483-fold purification of the NADH diaphorase the enzyme was further separated from contaminating proteins by polyacrylamide disc gel electrophoresis. Two distinct activity bands were extracted from the acrylamide, each one having oxygen, 2,6-dichlorophenolindophenol, and cytochrome c linked activities. In these preparations NADPH could not replace NADH as electron donor. During the initial purification steps all activity was lost due to the removal of a readily released cofactor. Enzyme activity was restored by either FAD or a FAD fraction isolated from M. hungatei. Oxidase activity exhibited a broad pH optimum from 7.0 to 8.5 and apparent Km values of 26 microM for NADH and 0.2 microM for FAD. Superoxide anion, formed in the presence of oxygen, accounted for all of the NADH consumed in the reaction. The molecular weight of the diaphorase was about 117 500 by sodium dodecyl sulfate gel electrophoresis. Sulfhydryl reagents and chelating agents were inhibitory. Inactivation, which occurred during storage in phosphate buffer at 4 degrees C, was delayed by dithiothreitol. The isolated NADH diaphorase lacked NADPH:NAD transhydrogenase and NAD reductase activities.
mesh_terms: D008058:Dihydrolipoamide Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D004583:Electrons; D019605:Euryarchaeota / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013481:Superoxides
chemical_list: D013439:Sulfhydryl Reagents; D013481:Superoxides; D005182:Flavin-Adenine Dinucleotide; D008058:Dihydrolipoamide Dehydrogenase