Multiple Forms of Cytochrome P-450 Purified from Liver Microsomes of Phenobarbital- and 3-Methylcholanthrene-Pretreated Rabbits

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Abstract
A method is described for the separation and purification of different forms of cytochrome P-450 from liver microsomes of phenobarbital (PB)- and 3-methylcholanthrene (MC)-pre-treated rabbits. It consists of solubilization of microsomes with cholate, followed by successive chromatography on ω-aminooctyl Sepharose 4B, hydroxylapatite and CM-Sephadex C-50 columns. Separation of different forms of cytochrome P-450 is achieved in the amino-octyl Sepharose and hydroxylapatite chromatography steps. This method permits the separation of three forms of cytochrome P-450, i.e. “P-4501,” “P-4502,” and “P-4481,” from PB-induced microsomes; P-4501, the main cytochrome P-450 component in these microsomes, and P-4481 can each be obtained in a gel-electrophoretically homogeneous state, whereas P-4502 can be obtained in a partially purified state. Application of the same method to MC-induced microsomes led to the purification of P-4481, the main component in these microsomes, to homogeneity and to partial purification of a fourth form, i.e. “P-4502.” P-4481 from MC-induced microsomes seems to be identical with P-4481 from PB-induced microsomes in monomeric molecular weight (54,000), amino acid composition and chromatographic behavior. However, P-4481 from MC-induced microsomes, but not P-4481 from PB-induced microsomes, contains 0.18 to 0.88 mol of tightly bound MC per mol of protein. P-4501 has a molecular weight of 49,000 and its amino acid composition is clearly different from that of P-4481. Although P-4502 is similar to P-4501 in molecular weight, they differ from each other in chromatographic behavior. P-4502 seems to be different from P-4501 in molecular weight, though they are similar to each other in chromatographic behavior. All the cytochrome P-450 preparations can be freed from the detergents used in the purification procedure by CM-Sephadex C-50 chromatography. Detergent-free P-4501, P-4502, and P-4481 exist in aqueous solution as oligomeric aggregates.