Purified NADPH cytochrome P-450 reductase. Interaction with hepatic microsomes and phospholipid vesicles.

Homogeneousdetergent-solubilized NADPH-cytochrome P-450 reductase but not steapsin-solubilized cytochrome c reductase was incorporated spontaneously into microsomes, microsomes treated with steapsin-protease to remove endogenous reductase, and syn thetic phospholipid vesicles.This incorporated reductase was active in electron transfer to cytochrome P-450, as shown by its participation in the NADPH-dependent demethylation of benzphetamine.Microsomeand vesicle-bound reductase was not removed by washing in high salt buffers, indicating hydrophobic binding forces.Detergent-solubilized reductase was stabilized against heat denaturation by Renex 690 and phospholipid vesicles, and appeared to aggregate in the absence of detergent;however, no such protective effects or aggregation was observed with the steapsin-solubilized reductase.Treatment of detergent-solubilized reductase *vesicle complexes with steapsin protease released cytochrome c reductase activity from the vesicles and converted the protein to a form with the electrophoretic mobility of the steapsin protease-solubilized enzyme and a 12,500-dalton fragment.These results and the ability of phospholipid vesicles to protect the 12,500dalton fragment against further proteolysis indicate that detergent-solubilized reductase contains a separable membrane binding domain, at least part of which is intercalated directly into the hydrophobic core of the phospholipid bilayer.The amino acid composition of the membrane binding domain, calculated as the difference between detergent-solubihzed reductase and steapsin-solubilized reductase, was not especially hydrophobic, but did contain 32.9% Asx and Glx, which may indicate that only a portion of the membrane binding domain is enmeshed in the hydrophobic core of the membrane.The cytochrome P-450.dependentmixed function oxidase system which catalyzes the hydroxylation of a wide variety of drugs, fatty acids, alkanes, steroids, polycyclic hydrocarbons,