MOLECULAR-CLONING AND EXPRESSION OF RABBIT STEROL 12-ALPHA-HYDROXYLASE

Sterol 12 alpha-hydroxylase is an important enzyme in bile acid biosyn thesis, responsible for the balance between formation of cholic acid a nd chenodeoxycholic acid, The enzyme has been purified to apparent hom ogeneity from rabbit liver (Ishida, H., Noshiro, M., Okuda, R., and Go on, M. J. (1992) J. Biol. Chem. 267, 21519-21323), and we here describ e the cloning and sequencing of a cDNA coding for this enzyme, After t ryptic digestion of purified protein in a polyacrylamide gel, eight di fferent peptides were isolated and sequenced, Using oligonucleotides d educed from the amino acid sequences, clones were isolated from a rabb it liver cDNA library, In addition to several overlapping clones, one full-length clone was obtained that coded for a polypeptide of 500 ami no acids, corresponding to a molecular mass of 57 kDa. All of the eigh t peptides and the reported NH2-terminal amino acid sequence were matc hed against the sequence. The peptide sequence showed a 39% similarity with human prostacyclin synthase (CYP8) and 31% similarity with the r ate-limiting enzyme in over-all synthesis of bile acids, the cholester ol 7 alpha-hydroxylase (CYP7) of the rabbit, The similarity with most other sterol cytochrome P-450 hydroxylases was less, Thus, this specie s of cytochrome P-450 should belong to a group of its own, here denote d CYP12, Transfection of COS cells with the coding part of the cDNA re sulted in a significant expression of sterol 12 alpha-hydroxylase acti vity toward 7 alpha-hydroxy-4-cholesten-3-one. Northern blotting showe d that the enzyme was exclusively expressed in the liver, The major mR NA 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 kilobas es, respectively, Fasting of rats and mice led to a severalfold increa se in both enzyme activity and mRNA levels, In contrast, starvation of rabbits had little or nostimulatory effect on enzyme activity and mRN A levels.