Polymorphism of human cytochrome P-450

Abstract

1. The cytochrome P-450 forms involved in debrisoquine 4-hydroxylation (P-450_DB), phenacetin O-deethylation (P-450_PA), S-mephenytoin 4-hydroxylation (P-450_MP), and nifedipine 1, 4-oxidation (P-450_NF) have been purified to electrophoretic homogeneity from human liver microsomes. All of these reactions show in vivo polymorphism in humans. Evidence for the roles of the purified proteins in these processes comes from in vitro reconstitution and immunoinhibition studies.

2. The rat orthologs of these enzymes are as follows—P-450_DB: P-450_UT-H; P-450_PA: P-450_ISF-G; P-450_MP: P-450_UT-I; P-450_NF; P-450_NF: P-450_PCN-E. Only in the case of P-450_UT-H is the primary rat ortholog the same cytochrome P-450 which catalyses the catalytic reaction under consideration.

3. Reconstitution and immunochemical studies establish that the following reactions are catalysed by the individual P-450_s—P-450_DB: debrisoquine 4-hydroxylation, sparteine Δ⁵-oxidation, bufuralol 1′-hydroxylation, encainide O-demethylation, and propanolol 4-hydroxylation; P-450_PA: phenacetin O-deethylation; P-450_MP: S-mephenytoin 4-hydroxylation and tolbutamide methyl hydroxylation; P-450_NF: oxidation of nifedipine and 16 other substituted dihydropyridines, estradiol 2- and 4-hydroxylation, aldrin epoxidation, benzphetamine N-demethylation and 6β-hydroxylation of testosterone, androstenedione and cortisol.

4. A cDNA clone has been isolated that corresponds to rat P-450_UT-H, as shown by a number of criteria. Studies with this probe establish that the sex and strain variation in debrisoquine 4-hydroxylase and related activities is related to differences in the levels of a 2-0 kb length mRNA present. The same probe also hybridizes to a human mRNA of the same size.

5. Polyclonal and monoclonal antibodies have been utilized to isolate several cDNA clones related to P-450_NF from a bacteriophage λgt11 expression library; the largest of these is 2·3 kb in length.

6. Several cDNA clones related to P-450_MP have also been isolated from a λgt11 library. One of these (2·5 kb) contains all of the coding sequence except for the last 14 nucleotides at the 5′ end. The entire coding region has been sequenced; all unambiguously defined N-terminal amino acid residues of P-450_MP are coded for by the nucleotide sequence.