Abstract
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 II, with identical apparent Mr 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 II was nonstereoselective((-)/(+) ratio = 1.03). The Km values for (-)- and (+)-bufuralol were 31 and 54 microM with P-450 buf I and 314 and 245 microM with P-450 buf II. P-450 buf II generated two other metabolites in addition to 1'-OH-bufuralol which were not observed with P-450 buf I. Using the inhibitor quinidine, a Ki of 0.06 microM was observed with P-450 buf I as opposed to 80 microM with P-450 buf II for bufuralol 1'-hydroxylation. A strong immunochemical relatedness of P-450 buf I and P-450 buf II 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 Mr 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 activity, and amount of immunoreactive material. Antibodies against P-450 buf I and P-450 buf II 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.
Highlights
PURIFICATION AND CHARACTERIZATION OF TWO FUNCTIONALLY DIFFERENT HUMAN LIVER CYTOCHROME P-450 ISOZYMES INVOLVED IN IMPAIRED HYDROXYLATION OF THE PROTOTYPE SUBSTRATE BUFURALOL*
The debrisoquine/sparteine-typepolymorphism of variety of clinically important drugs.Severalgeneticpolydrug oxidation presumably is caused by the absencemorrphismsin oxidative drug metabolismhavebeen recogdeficiency of cytochrome P-450 (P-4i5s0oz)yme(s). nized, and theirclinical relevance has been established [1,2,3]
Using bufuralol 1‘-hydroxylationas a prototype reac- One of the best studied examples of a genetic variation of tion of this polymorphism, two functionally distinct drug oxidation is the debrisoquine/sparteine-typepolymorforms, P-450buf I and P-450buf 11, with identical phism which occurs in 3-10% of Caucasian populations and apparent M, of 50,000were purified from livmericro- causes impaired biotransformation of drugs such as debrisosomes of three different human livers
Summary
Dept. of Anesthesia, Stanford UniversitySchool were strictlydesignated according tothesubstrates usedfor the of Medicine, Stanford, CA 94305. According to the same principle and extending this study, we report on the purification from the same humlaivner preparation of two functionally polymorphic cytochrome P450 isozymes highly active in the 1'-hydroxylation of bufuralol. Polyclonal antibodies against cytochromes P-450 buf I and P-450 buf I1 have been raised and used to immunochemically characterize the bufuralol1'-hydroxylationactivityin microsomesfrom in vivo and in vitro phenotyped extensive andpoor metabolizer individuals. These datallow the formulationof several alternative hypotheses which could explain the molecular mechanism causing thedebrisoquine/sparteine-typepolymorphism. HAP pool I and HAP pool I I indicate the fractions pooled on the basis of characteristic properties of bufuralol 1'-hydroxylation (see text)
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