Molecular cloning and expression of rabbit sterol 12 alpha-hydroxylase

Sterol 12 alpha-hydroxylase is an important enzyme in bile acid biosynthesis, responsible for the balance between formation of cholic acid and chenodeoxycholic acid, The enzyme has been purified to apparent homogeneity from rabbit liver (Ishida, H., Noshiro, M., Okuda, R., and Goon, M. J. (1992) J. Biol. Chem. 267, 21519-21323), and we here describe the cloning and sequencing of a cDNA coding for this enzyme, After tryptic digestion of purified protein in a polyacrylamide gel, eight different peptides were isolated and sequenced, Using oligonucleotides deduced from the amino acid sequences, clones were isolated from a rabbit liver cDNA library, In addition to several overlapping clones, one full-length clone was obtained that coded for a polypeptide of 500 amino acids, corresponding to a molecular mass of 57 kDa. All of the eight peptides and the reported NH2-terminal amino acid sequence were matched against the sequence. The peptide sequence showed a 39% similarity with human prostacyclin synthase (CYP8) and 31% similarity with the rate-limiting enzyme in over-all synthesis of bile acids, the cholesterol 7 alpha-hydroxylase (CYP7) of the rabbit, The similarity with most other sterol cytochrome P-450 hydroxylases was less, Thus, this species of cytochrome P-450 should belong to a group of its own, here denoted CYP12, Transfection of COS cells with the coding part of the cDNA resulted in a significant expression of sterol 12 alpha-hydroxylase activity toward 7 alpha-hydroxy-4-cholesten-3-one. Northern blotting showed that the enzyme was exclusively expressed in the liver, The major mRNA fraction in rabbit liver had a size of approximately 2.9 kilobases, and those found in rat and human liver were about 2.5 and 4.5 kilobases, respectively, Fasting of rats and mice led to a severalfold increase in both enzyme activity and mRNA levels, In contrast, starvation of rabbits had little or nostimulatory effect on enzyme activity and mRNA levels.