Purification and characterization of the human liver cytochromes P-450 involved in debrisoquine 4-hydroxylation and phenacetin O-deethylation, two prototypes for genetic polymorphism in oxidative drug metabolism.
Two forms of cytochrome P-450 were purified to apparent homogeneity from several different preparations of human liver microsomes. One form, designated P-450DB, had relatively high catalytic activity towards the drugs debrisoquine, sparteine, bufuralol (both the (+)- and (-)-isomers), encainide, and propranolol and appears to be the enzyme involved in the polymorphic distribution of oxidative activities towards these substrates in humans. The other form, designated P-450PA, had relatively high phenacetin O-deethylase activity and appears to be involved in the variation of this activity among humans. Polyclonal antibodies raised to the two enzymes were specific for the antigens as judged by immunoelectrophoresis and immuno-inhibition studies. The two enzymes and their activities were distinguished by chromatographic separation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid composition, immuno-inhibition studies, and steady-state kinetic assays. Immunochemical studies suggest that each form represents only a small fraction of the total cytochrome P-450 in human liver microsomes. These biochemical studies provide a basis for better understanding the mechanisms which underlie genetic polymorphisms involving P-450 cytochromes in humans.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D019388:Cytochrome P-450 CYP1A2; D019389:Cytochrome P-450 CYP2D6; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D003647:Debrisoquin / Q000378:metabolism; D004983:Ethanolamines / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D007122:Immunoelectrophoresis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D006899:Mixed Function Oxygenases / Q000032:analysis*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000032:analysis*; D010615:Phenacetin / Q000378:metabolism; D011110:Polymorphism, Genetic; D051381:Rats; D011919:Rats, Inbred Strains; D013237:Stereoisomerism
D000596:Amino Acids; D004983:Ethanolamines; C010831:bufuralol; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2; D019389:Cytochrome P-450 CYP2D6; D010615:Phenacetin; D003647:Debrisoquin