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.