Abstract
Abstract Partially purified cytochromes P-450 and P-448 were obtained from hepatic microsomes of rats treated with either phenobarbital or 3-methylcholanthrene, using the ionic detergent sodium cholate and the nonionic detergent emulgen 911. The preparations contained 9 to 11 nmoles of cytochrome P-450 or P-448 per mg of protein and were free of NADPH-cytochrome c reductase and cytochrome b5. The total phospholipid content of the partially purified cytochrome P-450, per mg of protein, was reduced by more than 98% relative to microsomes. A partially purified NADPH-cytochrome c reductase preparation, essentially free of cytochrome b5 and phospholipid, is also described. Reconstitution of the cytochrome P-450 and reductase fractions with phospholipid resulted in restoration of enzymatic activity for the oxidative metabolism of a variety of substrates. Since the ratio of cytochrome P-450 to cytochrome b5 in this reconstituted system was greater than 200 to 1, it is suggested that cytochrome b5 is not an obligatory component of the mixed function oxidase system.
Highlights
Cytochrome P-450 (O.D. 418) eluted as two peaks, one which eluted in the column volume, and another which eluted at 0.1 Y KCl
The peak which eluted in the column volume contained most of the phosphatidylcholine and epoxide hydrase and accounted for approximately 25y0 of the total cytochrome P-450 eluted
The peak which eluted at 0.1 M KC1 contained the remainder of the cytochrome P-450 and was free of cytochrome bs and NADPH-cytochrome c reductase which eluted at higher ionic strength
Summary
Purified cytochromes P-450 and P-448 were obtained from hepatic microsomes of rats treated with either phenobarbital or 3-methylcholanthrene, using the ionic detergent sodium cholate and the nonionic detergent emulgen 911. The mixed function oxidase system of rat and rabbit liver microsomes has been resolved into three fractions consisting of cytochrome P-450, NADPH-cytochrome c reductase and a lipid component later identified as phosphatidylcholine (l-4). When these three components are recombined, the resulting reconstituted system catalyzes the hydrosylation of a wide variety of substrates, including drugs, other xenobiotics, steroids, and fatty acids [2,3,4]. Cytochrome P-450 and lipid are reconstituted, the system metabolizes a variety of substrates in the presence of NADPH and molecular oxygen and still has the same substrate specificity as our less purified preparations, but the system shows essentially an absolute requirement for all three components
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