Existence and Separation of Three Forms of Cytochrome P-450 from Rat Liver Microsomes

Abstract

Abstract Rat liver microsomes contain three spectrally distinguishable forms of cytochrome P-450. Each form is identified qualitatively and quantitatively by visible spectral changes that occur when the cytochromes P-450 combine with various ligands, such as cyanide. In addition to cytochrome P-450, rat liver microsomes also contain cytochrome b5, which may be removed from the microsomes by digestion with Subtilisin VII. The relative amounts and spectral properties of the three forms of cytochrome P-450 remaining in treated particles are not altered by the partial proteolytic digestion. With difference spectral assays, methods have been developed for the chromatographic separation of the three forms of cytochrome P-450 in Subtilisin-treated particles. The particles are first treated with deoxycholate, and the clarified suspension is chromatographed on diethylaminoethylcellulose; a discontinuous gradient of KCl is used. Analysis of the affinities of the three separated forms (Forms I, II, and III) for various ligands yields the following binding constants for cyanide: Form I, K = 0.5 mm; Form II, K = 1.5 mm; Form III, K = 5.0 mm; and for octylamine: Form I, K1 = 0.025 mm and K2 = 0.12 mm; Form II, K1 = 0.016 mm and K2 = 0.056 mm; Form III, K = 0.30 mm. The binding constants are essentially identical with those observed for the three forms of cytochrome P-450 in microsomes and Subtilisin-treated particles. Thus, Subtilisin treatment, solubilization, or chromatography does not alter either the relative amounts of the three forms or their difference spectral characteristics. Assays of the three forms after various pretreatments of rats have been undertaken. The relative amounts of the three forms of cytochrome P-450 are altered by pretreatment of the rats. Form III is increased by pretreatment with 3-methylcholanthrene; Form II is preferentially induced by phenobarbital; and dietary ethyl alcohol preferentially induces Form I, the form of cytochrome P-450 that exhibits the highest affinity for cyanide. Thus, this work has provided not only an assay for the various forms of cytochrome P-450 and a chromatographic separation for future work on recombination of the multienzymic oxidases, but a functional relationship for the three forms is suggested by the induction experiments. These are only spectrally identifiable forms that may be separated by these procedures, and, to date, there is no information on whether or not these forms differ structurally.