Some properties of a detergent-solubilized NADPH-cytochrome c(cytochrome P-450) reductase purified by biospecific affinity chromatography.
NADPH-cytochrome c (cytochrome P-450) reductase (EC 1.6.2.4) has been purified to homogeneity, as judged by sodium dodecyl sulfate disc gel electrophoresis, from detergent-solubilized rat and pig liver microsomes using an affinity chromatography procedure. Treatment of microsomes with a polyethoxynonylphenyl ether plus either cholate or deoxycholate and subsequent batch-wise DEAE-cellulose chromatography followed by biospecific affinity chromatography on Sepharose 4B-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (2'5'-ADP-Sepharose 4B) result in a greater than 30% yield of purified reductase from microsomes. The enzyme contains 1 mol each of FAD and FMN and exhibits a molecular weight of 78,000 g mol-1 estimated by comparison with protein standards on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The turnover numbers calculated on the basis of flavin are 1360 min-1 and 1490 min-1 at 25 degrees for the pig and rat liver enzymes, respectively. Titration of these purified preparations aerobically with both NADPH and potassium ferricyanide demonstrated unequivocally that the air-stable, reduced form of NADPH-cytochrome c (P-450) reductase contains 2 electron equivalents, confirming recent results obtained by Masters et al. (Masters, B. S. S., Prough, R. A., and Kamin, H. (1975) Biochemistry 14, 607-613) for the proteolytically solubilized enzyme. In addition, these preparations are capable of reconstituting benzphetamine N-demethylation activity in the presence of partially purified cytochrome P-450 and dilauroylphosphatidylcholine, as measured by formaldehyde formation from benzphetamine.
D000332:Aerobiosis; D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods; D003579:Cytochrome Reductases / Q000302:isolation & purification*; D005292:Ferricyanides; D005486:Flavin Mononucleotide / Q000032:analysis; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D012995:Solubility; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
D005292:Ferricyanides; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D003579:Cytochrome Reductases; D009251:NADPH-Ferrihemoprotein Reductase