Purification and characterization of the rat liver microsomal cytochrome P-450 involved in the 4-hydroxylation of debrisoquine, a prototype for genetic variation in oxidative drug metabolism.

Abstract

Genetic polymorphism in oxidative drug metabolism is perhaps best exemplified in the case of debrisoquine 4-hydroxylase activity, where the incidence of deficient metabolism ranges from 1% to 30% in various populations and this defect is also linked to an impaired ability to metabolize a number of other drugs effectively. Sprague-Dawley (SD) rats possess this activity, but females of the DA strain do not, although total cytochrome P-450 (P-450) levels are similar. We have purified, by using debrisoquine 4-hydroxylase activity as an assay, a minor P-450 to electrophoretic homogeneity from male SD rats and designate this as P-450UT-H. P-450UT-H differs from eight other purified rat liver P-450s as judged by peptide mapping and immunochemical analysis and thus appears to be isozymic with these other P-450s. P-450UT-H exhibited considerably more debrisoquine 4-hydroxylase activity than any of the other purified P-450s and, on a total P-450 basis, more than total microsomal P-450. Antibodies raised against P-450UT-H specifically recognized P-450UT-H and inhibited more than 90% of the debrisoquine hydroxylase activity present in SD rat liver microsomes. The level of P-450UT-H in SD rat liver microsomes accounted for less than 10% of the total P-450, as judged by immunochemical quantitation. These assays also indicated that the level of P-450UT-H in female DA rat liver microsomes is only about 5% of that in male or female SD rat liver microsomes, consonant with the view that deficiency of this form of P-450 is responsible for the defective debrisoquine 4-hydroxylase activity in the former animals.(ABSTRACT TRUNCATED AT 250 WORDS)