Jo Corbin C, Mapes S M, Lee Young M, Conley Alan J
Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
Mol Cell Endocrinol. 2003 Aug 29;206(1-2):147-57. doi: 10.1016/s0303-7207(02)00422-7.
Recombinant porcine gonadal and placental, and the human and bovine, isozymes of aromatase cytochrome P450 (P450arom) were over-expressed in insect cells, purified and quantified by difference spectroscopy. Human and bovine P450arom exhibited greater apparent molecular size than either porcine isozyme prompting an examination of N-linked glycosylation and amino-terminal peptide sequence. Comparisons of substrate affinities and turnover were also made. In contrast to human and bovine P450arom which are N-linked glycoproteins, neither isozyme of porcine P450arom is glycosylated, explaining in part their lower molecular size. Differences found in N-terminal peptide sequences were unlikely to influence apparent molecular size or enzyme function. Human and bovine P450arom had similar affinities and turnovers for androstenedione (approximately 200 nM, 3/min) and testosterone (approximately 350 nM, 2/min). The porcine isozymes had 10-fold higher affinities but correspondingly lower turnovers, particularly the gonadal P450arom. Overall, the catalytic efficiency (Vmax/Km) was similar for all but porcine gonadal P450arom which was much lower. These data emphasize the structural and functional variability of even the most conserved of proteins among diverse species wherein such differences have previously remained unexpected.
重组猪性腺和胎盘以及人和牛的芳香化酶细胞色素P450（P450arom）同工酶在昆虫细胞中过表达，通过差示光谱法进行纯化和定量。人和牛的P450arom表现出比任何一种猪同工酶更大的表观分子大小，这促使人们对N-连接糖基化和氨基末端肽序列进行研究。还对底物亲和力和周转率进行了比较。与作为N-连接糖蛋白的人和牛P450arom不同，猪P450arom的两种同工酶都没有糖基化，这部分解释了它们较小的分子大小。在氨基末端肽序列中发现的差异不太可能影响表观分子大小或酶功能。人和牛的P450arom对雄烯二酮（约200 nM，3/分钟）和睾酮（约350 nM，2/分钟）具有相似的亲和力和周转率。猪同工酶的亲和力高10倍，但相应的周转率较低，尤其是性腺P450arom。总体而言，除猪性腺P450arom低得多外，所有酶的催化效率（Vmax/Km）相似。这些数据强调了即使是不同物种中最保守的蛋白质在结构和功能上的变异性，而这种差异以前一直出乎意料。
