Multiple forms of cytochrome P-450 purified from liver microsomes of phenobarbital- and 3-methylcholanthrene-pretreated rabbits. I. Resolution, purificaton, and molecular properties.

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)-pretreated rabbits. It consists of solubilization of microsomes with cholate, followed by successive chromatography on omega-aminooctyl Sepharose 4B, hydroxylapatite and CM-Sephadex C-50 columns. Separation of different forms of cytochrome P-450 is achieved in the aminooctyl Sepharose and hydroxylapatite chromatograhy steps. This method permits the separation of three forms of cytochrome P-450, i.e. "P-450(1)," "P-450(2)," and "P-488(1)," from PB-induced microsomes; P-450(1), the main cytochrome P-450 component in these microsomes, and P-448(1) can each be obtained in a gel-electrophoretically homogeneous state, whereas P-450(2) can be obtained in a partially purified state. Application of the same method to MC-induced microsomes led to the purification of P-448(1), the main component in these microsomes, to homogeneity and to partial purification of a fourth form, i.e. "P-450(3)." P-448(1) from MC-induced microsomes seems to be identical with P-448(1) from PB-induced microsomes in monomeric molecular weight (54,000), amino acid composition and chromatographic behavior. However, P-448(1) from MC-induced microsomes, but not P-448(1) from PB-induced microsomes, contains 0.18 to 0.88 mol of tightly bound MC per mol of protein. P-450(1) has a molecular weight of 49,000 and its amino acid composition is clearly different from that of P-448(1). Although P-450(2) is similar in molecular weight, they differ from each other in chromatographic behavior. P-450(3) seems to be different from P-450(1) 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-450(1), P-450(2), and P-448(1) exist in aqueous solution as oligomeric aggregates.