Debrisoquine/sparteine-type polymorphism of drug oxidation. 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-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.
D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D003647:Debrisoquin / Q000378:metabolism*; D004983:Ethanolamines / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007546:Isoquinolines / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010084:Oxidation-Reduction; D013034:Sparteine / Q000378:metabolism*; D013329:Structure-Activity Relationship
D004983:Ethanolamines; D007527:Isoenzymes; D007546:Isoquinolines; D013034:Sparteine; C010831:bufuralol; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C043872:bufuralol 1'-hydroxylase; D003647:Debrisoquin