Microheterogeneity in the major phenobarbital-inducible forms of rabbit liver microsomal cytochrome P-450 as revealed by nucleotide sequencing of cloned cDNAs

Abstract

We have isolated one full-length cDNA clone, termed pHP1, and a number of clones of shorter insert lengths, tentatively called b14, b46, etc., all encoding phenobarbital- (PB-) inducible forms of rabbit liver microsomal cytochrome P-450, and determined their nucleotide sequences. The polypeptides encoded by these cDNAs can be classified into five types, represented by HP1, b14, b46, b52, and b54, the deduced amino acid sequences of which are more than 95% similar to one another. Amino acid differences among them total 24 positions, which are distributed over the entire sequence, in contrast to the microheterogeneity observed in two PB-inducible rat liver microsomal cytochromes P-450 (P-450b and P-450e). The primary structure deduced for the HP1 protein is 97% similar to that determined for rabbit P-450 LM2 (form 2), which has been purified by Coon and co-workers [van der Hoeven, T. A., Haugen, D. A., and Coon, M. J. (1974) Biochem. Biophys. Res. Commun. 60, 569-675; Haugen, D. A., and Coon, M. J. (1976) J. Biol. Chem. 251, 7929-7939] as the major PB-inducible form of rabbit liver microsomal cytochrome P-450. The amino acid sequence of P-4501, which we have purified as the major PB-inducible rabbit liver cytochrome P-450, was partially determined with the sequence reported for P-450 LM2 as a reference. The two sequences are closely similar to each other, but at least two amino acid differences can be detected between them. The primary structure predicted from the nucleotide sequence of b14 cDNA, which covers only the carboxyl-terminal 40% of the entire sequence, is identical with that determined for P-4501. It is concluded that the liver of PB-treated rabbits expresses at least five distinct but closely similar forms of cytochrome P-450, all of which are encoded by genes belonging to the rabbit P450IIB (major PB-inducible) subfamily.