Interaction of steroids with adrenal cytochrome P-450 (P-450(17)alpha,lyase) in liposome membranes.
Purified cytochrome P-450(17)alpha,lyase from guinea-pig adrenal microsomes, which catalyzes progesterone 17 alpha-hydroxylation and sequentially C17-C20 bond cleavage of the 17 alpha-hydroxyprogesterone, was successfully incorporated into liposomal membranes composed of only phosphatidylcholine or of a phospholipid mixture of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine at a molar ratio of 5:3:1. Although the purified P-450(17)alpha,lyase was readily converted into P-420 in the detergent-solubilized system without substrates, the P-450 embedded in the liposomal membranes was found to be quite stable without the substrates. Using the P-450(17)alpha,lyase-proteoliposomes, the interaction of steroids with P-450(17)alpha,lyase was studied for progesterone, 17 alpha-hydroxyprogesterone and androstenedione in the liposomal system by optical difference spectroscopy and by equilibrium dialysis. The partition coefficients of steroids between the aqueous phase and the liposomal membranes were determined by the equilibrium dialysis. They were about 1.4-1.6-times higher in phosphatidylcholine liposomes than in the liposomes of the lipid mixture. The dissociation constants of the P-450-steroid complexes were calculated from the apparent dissociation constants using the partition coefficients for the situation where the substrate-binding site faces the lipid phase of the membranes or where it faces the aqueous phase. The dissociation constant in the former case was not affected by the lipid composition. These results suggest that P-450(17)alpha,lyase might interact only with the substrates in the lipid phase of the liposomal membranes.
Kominami
S
S
Faculty of Integrated Arts and Sciences, Hiroshima University, Japan.
Higuchi
A
A
Takemori
S
S
D000311:Adrenal Glands / Q000201:enzymology*; D000818:Animals; D006168:Guinea Pigs; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism*; D008566:Membranes / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D013254:Steroid 17-alpha-Hydroxylase / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013256:Steroids / Q000378:metabolism*
D008081:Liposomes; D013256:Steroids; D013250:Steroid Hydroxylases; D013254:Steroid 17-alpha-Hydroxylase