Catalytic properties of the liver microsomal hydroxylase system in reconstituted phospholipid vesicles.
Two types of cytochrome P-450, P-450LM2 and P-450LM3, have been purified from rabbit liver microsomes and incorporated into phospholipid vesicles by a cholate gel filtration technique together with purified preparations of NADPH-cytochrome P-450 reductase. The catalytic properties of the vesicles have been compared with a system reconstituted with small amounts of dilauroylphosphatidylcholine (DLPC). 6 beta-Hydroxylation of androstenedione proceeded at a rate 10 times higher in the vesicles compared to the DLPC-system. The kinetics for the reaction were the same in the vesicles as in intact microsomes i.e. sigmoidal substrate curves were obtained and Hill-coefficients of about 1.4 were calculated in these systems. In contrast, Michaelis-Menten kinetics were obtained for 6 beta-hydroxylation in the DLPC-system. The results could indicate cooperativity between different P-450 molecules in the intact membrane but not in the DLPC-system. P-450LM2-catalyzed 16-hydroxylation of androstenedione was in contrast to the situation with P-450LM3 inhibited in the vesicles as compared to the DLPC system. It is suggested that for evaluation of substrate specificity and other properties of different types of liver microsomal P-450, phospholipid vesicles may be a more relevant integration level than the DLPC-system.
D000735:Androstenedione; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D006900:Hydroxylation; D007700:Kinetics; D008081:Liposomes; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism; D010084:Oxidation-Reduction; D010634:Phenobarbital / Q000494:pharmacology; D010713:Phosphatidylcholines; D011817:Rabbits; D013250:Steroid Hydroxylases / Q000378:metabolism*
D008081:Liposomes; D010713:Phosphatidylcholines; D000735:Androstenedione; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D009251:NADPH-Ferrihemoprotein Reductase; D010634:Phenobarbital