ISOLATION OF A BOVINE FULL-LENGTH CYTOCHROME-P450 (CYP3A) CDNA SEQUENCE AND ITS FUNCTIONAL EXPRESSION IN V79 CELLS

From a bovine liver cDNA library in lambda Max1 a 1870 bp cDNA was iso lated using the human CYP3A4 cDNA as a probe. The cDNA-deduced amino a cid sequence encoded a protein of 507 amino acids and exhibited homolo gies of 76, 72 and 64% with canine CYP3A12, human CYP3A4 and rat CYP3A 1, respectively. Furthermore, a very high homology of 91.7% was observ ed with the deduced amino acid sequence of a partial CYP3A cDNA from d warf goat. A striking observation was that both the bovine and the goa t cDNA exhibit a 4 amino acid extension at the C-terminus, which is du e to a frame-shifting insertion of 2 nt. The bovine CYP3A cDNA was clo ned in a retroviral vector, transfected to V79 cells and cells were se lected for cytochrome P450 expression. The expressed enzyme was shown to catalyze the 6 beta-hydroxylation of testosterone, which could also be observed in a V79 cell line expressing human CYP3A4. In the bovine CYP3A cell line, however, 6 beta-hydroxytestosterone was not found to be the major metabolite. This cell line additionally showed high leve ls of hydroxylase activity at the 2 beta and 12 beta position of testo sterone. The cDNA-expressed testosterone hydroxylase activity could be inhibited with the specific CYP3A inhibitors, tiamulin and ketoconazo le. (C) 1997 Elsevier Science B.V.