Kinetic Properties of Nicotinamide Adenine Dinucleotide Phosphate-Cytochrome P-450 Reductase from Porcine Adrenal Microsomes

Two forms of nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase, reductase I (MW 78000) and reductase II (MW 72000), were purified as a single component on SDS-polyacrylamide gel electrophoresis from the porcine adrenal microsomes. When cytochrome c was used as an electron acceptor, specific activities of these reductases were 51.8μmol/min/mg for reductase I and 53.1μmol/min/mg for reductase II, respectively. These reductases were able to reduce artificial electron acceptors, such as ferricyanide, dichlorophenol indophenol, nitroblue tetrazolium and neotetrazolium. Two forms of reductase were optically flavoprotein. On the kinetic analysis, the apparent Km values of cytochrome c for reductase I and reductase II were 40.8 and 21.3μM ; those of NADPH for reductase I and reductase II were 35.7 and 22.7μM, respectively. Reductase I was able to support cytochrome P-450 monooxygenase reactions on the reconstituted system, such as 21-hydroxylation, 17α-hydroxylation and cleavage reaction of C17-C20 bond by highly purified cytochrome P-450 (21-hydroxylase), P-450 (17α-hydroxylase-C17, 20 lyase). The apparent Km values were 0.33μM for P-450 (21-hydroxylase) and 0.58μM for P-450 (17α-hydroxylase-C17, 20 lyase), when progesterone was used as substrate. However, reductase II was unable to support the monooxygenase reaction. From these observations, it is discussed that reductase II which retains the flavin prosthetic group is a cytochrome P-450 reductase lacked a small hydrophobic peptide (MW 6000) which is specific binding site to cytochrome P-450 for the electron transfer.