Studies on the interaction of steroid substrates with adrenal microsomal cytochrome P-450 (P-450C21) in liposome membranes.
Cytochrome P-450 (P-450C21), purified from bovine adrenocortical microsomes, was incorporated into the single bilayer liposomes of egg yolk phosphatidylcholine by gel filtration, using a high pressure liquid chromatography system. Interaction of the steroid substrates, 17 alpha-hydroxyprogesterone and progesterone, with P-450C21 in the liposomes was studied in the equilibrium state by measuring substrate-induced spectral change. The apparent dissociation constant of the P-450C21-substrate complex increased with phosphatidylcholine concentration in the system, showing the substrate to be partitioned between the aqueous and lipid phases. Partition coefficients, determined by equilibrium dialysis and the Hummel-Dreyer method, were 3500 for progesterone and 2000 for 17 alpha-hydroxyprogesterone at 25 degrees C. The binding process of the substrates to P-450C21 in the liposomes and their dissociation were measured by a stopped flow method. The apparent rate of substrate binding to P-450C21 in the liposomes was not effected by substrate partitioning, indicating partitioning to occur much more quickly than substrate binding to P-450C21. Absorption changes observed in the stopped flow experiments were analyzed at a rapid equilibrium of partitioning. Based on these results, the substrate binding site of P-450C21 was concluded to face the lipid phase of the liposome membranes.
D019326:17-alpha-Hydroxyprogesterone; D000302:Adrenal Cortex / Q000201:enzymology*; D000368:Aged; D000818:Animals; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006801:Humans; D006908:Hydroxyprogesterones / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology*; D008954:Models, Biological; D010713:Phosphatidylcholines / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism*
D006908:Hydroxyprogesterones; D008081:Liposomes; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D011374:Progesterone; D019326:17-alpha-Hydroxyprogesterone; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol