Interaction between cytochrome P-450 (P-450C21) and NADPH-cytochrome P-450 reductase from adrenocortical microsomes in a reconstituted system.
The interaction between P-450C21 and NADPH-cytochrome P-450 reductase, both purified from bovine adrenocortical microsomes, has been investigated in a reconstituted system with a nonionic detergent, Emulgen 913, by kinetic analysis and gel filtrations. Steady state kinetic data in progesterone 21-hydroxylation showed formation of an equimolar complex between the two enzyme proteins at low Emulgen concentration. Steady state kinetic studies on the electron transfer from NADPH to P-450C21 via the reductase showed that a stable complex formation between the two enzyme proteins was not involved in the steady state electron transfer at high Emulgen concentration. In stopped flow experiments, a time course of the P-450C21 reduction showed biphasic kinetics composed of fast and slow phases. The dependence of kinetic parameters on Emulgen concentration indicates that the fast phase corresponds to the electron transfer within the complex and the slow phase to the electron transfer through a random collision between P-450C21 and the reductase. The stable complex formation between P-450C21 and the reductase has been clearly demonstrated by gel filtration. The stable complex was composed of several molecules of the two enzyme proteins at an equimolar ratio, which was active for progesterone 21-hydroxylation and had a tendency to dissociate at high Emulgen concentration.
D000302:Adrenal Cortex / Q000201:enzymology*; D000818:Animals; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D017137:Nonoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011485:Protein Binding; D013255:Steroid 21-Hydroxylase / Q000378:metabolism
D003902:Detergents; D017137:Nonoxynol; D011092:Polyethylene Glycols; D003577:Cytochrome P-450 Enzyme System; D013255:Steroid 21-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase