Purification and Characterization of Two Functionally Different Human Liver Substrate Bufuralol* Cytochrome P-450 Isozymes Involved in Impaired Hydroxylation of the Prototype

The debrisoquine/sparteine-type polymorphism of drug oxidation presumably is caused by the absence or deficiency of cytochrome P-450 (P-450) isozyme(s). Using bufuralol 1‘-hydroxylation as a prototype reaction of this polymorphism, two functionally distinct forms, P-450 buf I and P-450 buf 11, with identical apparent M, of 50,000 were purified from liver microsomes of three different human livers. P-450 buf I exhibited a marked selectivity for the (+)-enantiomer of bufuralol ((-)/(+) ratio = 0.15), P-450 buf I1 was nonstereoselective ((-)/(+) ratio = 1.03). The K , values for (-)and (+)-bufuralol were 31 and 54 p~ with P450 buf I and 314 and 245 MM with P-450 buf 11. P450 buf I1 generated two other metabolites in addition to 1’-OH-bufuralol which were not observed with P450 buf I. Using the inhibitor quinidine, a Ki of 0.06 PM was observed with P-450 buf 1 as opposed to 80 p~ with P-450 buf I1 for bufuralol 1’-hydroxylation. A strong immunochemical relatedness of P-450 buf I and P-450 buf I1 was found since polyclonal antibodies against either form recognized the heterologous antigen to the same extent as the homologous antigen on Western blots and in immunoinhibition and in immunoprecipitation experiments. Cross-reactivity of these antibodies with a microsomal nonheme protein of unknown function (apparent M, 50,000) also was noted. Western blots of microsomes of in vivo and in vitro phenotyped extensive and poor metabolizer individuals revealed no correlation of in vivo-determined metabolic ratio, microsomal ctivity, and amount of immunoreactive material. Antibodies against P-450 buf I and P-450 buf I1 inhibited bufuralol 1’-hydroxylation in microsomes of in vivo and in vitro phenotyped poor metabolizer individuals demonstrating that the residual activities are immunochemically related to the activities in extensive metabolizers.