Physicochemical properties of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase from bovine adrenocortical microsomes.
Adrenocortical NADPH-cytochrome P-450 reductase (EC. 1.6.2.4) was purified from bovine adrenocortical microsomes by detergent solubilization and affinity chromatography. The purified cytochrome P-450 reductase was a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being electrophoretically homogeneous and pure. The cytochrome P-450 reductase was optically a typical flavoprotein. The absorption peaks were at 274, 380 and 45 nm with shoulders at 290, 360 and 480 nm. The NADPH-cytochrome P-450 reductase was capable of reconstituting the 21-hydroxylase activity of 17 alpha-hydroxyprogesterone in the presence of cytochrome P-45021 of adrenocortical microsomes. The specific activity of the 21-hydroxylase of 17 alpha-hydroxyprogesterone in the reconstituted system using the excess concentration of the cytochrome P-450 reductase, was 15.8 nmol/min per nmol of cytochrome P-45021 at 37 degrees C. The NADPH-cytochrome P-450 reductase, like hepatic microsomal NADPH-cytochrome P-450 reductase, could directly reduce the cytochrome P-45021. The physicochemical properties of the NADPH-cytochrome P-450 reductase were investigated. Its molecular weight was estimated to be 80 000 +/- 1000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical ultracentrifugation. The cytochrome P-450 reductase contained 1 mol each FAD and FMN as coenzymes. Iron, manganese, molybdenum and copper were not detected. The Km values of NADPH and NADH for the NADPH-cytochrome c reductase activity and those of cytochrome c for the activity of NADPH-cytochrome P-450 reductase were determined kinetically. They were 5.3 microM for NADPH, 1.1 mM for NADH, and 9-24 microM for cytochrome c. Chemical modification of the amino acid residues showed that a histidyl and cysteinyl residue are essential for the binding site of NADPH of NADPH-cytochrome P-450 reductase.
D000302:Adrenal Cortex / Q000201:enzymology*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D012995:Solubility; D013050:Spectrometry, Fluorescence
D009251:NADPH-Ferrihemoprotein Reductase