Incorporation of purified components of the rabbit liver microsomal hydroxylase system into phospholipid vesicles

Abstract

Methods are described for incorporation of purified forms of rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450 LM 2 , P-450 LM 3 and P-450 LM 4 (LM, liver microsomes) into phospholipid vesicles. It was found that each cytochrome could individually be incorporated into preformed phospholipid vesicles in the absence of cholate. However, NADPH-cytochrome P-450 reductase prevented incorporation of P-450 by this method, a phenomenon possibly inherent in the formation of complexes between P-450 and the reductase in solution. Using the cholate-gel filtration technique it was possible to prepare monolamellar phosphatidylcholine vesicles containing any of the cytochromes and P-450 reductase in good yields. It was found that P-450 LM 3 -containing vesicles had a mean diameter of 47 nm, whereas vesicles formed under the same conditions but containing P-450 LM 4 were much smaller (mean diameter 33 nm). Vesicles formed with P-450 LM 2 were homogeneous in density (1.04 g/cm 3) according to isopycnic centrifugation in Ficoll but not in size (44–72 nm). These findings, taken together with results obtained from treatment of the cytochromes in soluble form and in reconstituted vesicles with the nonpenetrating reagent, p- diazobenzene sulphonate, indicate a unidirectional, relatively peripheral orientation of P-450 LM 4 with the major part localized on the outside of the vesicles. Experiments with trypsin and cytochrome c- reduction demonstrated a unidirectional orientation of P-450 reductase towards the outside of the vesicles.