Department of Veterinary Pharmacology, Utrecht University, P.O. Box 80176, NL-3508 TD Utrecht, The Netherlands.
Environ Toxicol Pharmacol. 1997 Feb 15;3(1):17-24. doi: 10.1016/s1382-6689(96)00133-0.
From a bovine liver cDNA library in λMaxl a 1870 bp cDNA was isolated using the human CYP3A4 cDNA as a probe. The cDNA-deduced amino acid sequence encoded a protein of 507 amino acids and exhibited homologies of 76, 72 and 64% with canine CYP3A12, human CYP3A4 and rat CYP3A1, respectively. Furthermore, a very high homology of 91.7% was observed with the deduced amino acid sequence of a partial CYP3A cDNA from dwarf goat. A striking observation was that both the bovine and the goat cDNA exhibit a 4 amino acid extension at the C-terminus, which is due to a frame-shifting insertion of 2 nt. The bovine CYP3A cDNA was cloned in a retroviral vector, transfected to V79 cells and cells were selected for cytochrome P450 expression. The expressed enzyme was shown to catalyze the 6β-hydroxylation of testosterone, which could also be observed in a V79 cell line expressing human CYP3A4. In the bovine CYP3A cell line, however, 6β-hydroxytestosterone was not found to be the major metabolite. This cell line additionally showed high levels of hydroxylase activity at the 2β and 12β position of testosterone. The cDNA-expressed testosterone hydroxylase activity could be inhibited with the specific CYP3A inhibitors, tiamulin and ketoconazole.
从牛肝 cDNA 文库中，使用人 CYP3A4 cDNA 作为探针，分离出 1870bp 的 cDNA。该 cDNA 推导的氨基酸序列编码一个由 507 个氨基酸组成的蛋白质，分别与犬 CYP3A12、人 CYP3A4 和大鼠 CYP3A1 的同源性为 76%、72%和 64%。此外，与来自矮山羊的部分 CYP3A cDNA 的推导氨基酸序列观察到非常高的同源性为 91.7%。一个显著的观察结果是，牛和山羊 cDNA 在 C 末端都有 4 个氨基酸的延伸，这是由于 2 个核苷酸的移码插入所致。牛 CYP3A cDNA 被克隆到逆转录病毒载体中，转染到 V79 细胞中，并选择细胞进行细胞色素 P450 表达。表达的酶被证明能够催化睾酮的 6β-羟化，这也可以在表达人 CYP3A4 的 V79 细胞系中观察到。然而，在牛 CYP3A 细胞系中，未发现 6β-羟睾酮是主要代谢物。该细胞系还显示出睾酮 2β 和 12β 位置的羟化酶活性的高水平。cDNA 表达的睾酮羟化酶活性可以被特异性 CYP3A 抑制剂替米考星和酮康唑抑制。
