FUNCTIONAL OVARIAN AND PLACENTAL ISOFORMS OF PORCINE AROMATASE
FUNCTIONAL OVARIAN AND PLACENTAL ISOFORMS OF PORCINE AROMATASE
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DOI：
10.1016/0303-7207(95)03607-9
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发表时间：
1995-08-30
影响因子：
4.1
通讯作者：
CONLEY, AJ
中科院分区：
文献类型：
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作者：
CORBIN, CJ;KHALIL, MW;CONLEY, AJ
从猪胎盘、卵巢内膜和颗粒层组织中克隆了猪芳香化酶细胞色素P-450的功能亚型，并在体外表达了全长cDNA。猪卵泡膜和颗粒膜表达相同形式的P-450 arom。该卵巢cDNA编码501个氨基酸的蛋白质，在N-末端比胎盘P-450 arom同种型（503个残基）短两个氨基酸。总的来说，这两种异构体表现出93%的核苷酸和87%的氨基酸的同一性，彼此，都是高度同源的，在核苷酸和氨基酸水平上，人和牛的P-450 arom，也503个氨基酸的蛋白质。预测的氨基酸序列的分析进一步表明，假定与人P-450 arom基因的内含子-外显子边界保守，对应于假定的外显子III、V和IX的cDNA区域在猪胎盘和卵巢酶中是保守的，而序列变异发生在所有其他假定的外显子中。在体外表达表明，编码猪胎盘P-450 arom的cDNA是几乎10倍以上的活性在雌酮的合成雄烯二酮比卵巢亚型，合成更多的19 OH-雄烯二酮比雌酮。转染的Cos 1细胞的Western分析表明，活性的差异不是由于cDNA的表达水平，因为在用每个构建体转染的细胞中观察到类似水平的免疫可检测蛋白。这两种异构体是敏感的抑制活性的特定芳香化酶抑制剂，4 OH-雄烯二酮和CGS 16949 A。此外，由卵巢P-450 arom cDNA编码的酶的活性被细胞色素P-450 11 β-羟化酶抑制剂依托咪酯抑制，但胎盘P-450 arom亚型没有。这些功能差异与在涉及新鲜匀浆组织中P-450 arom活性的类似实验中所做的观察结果一致。这些数据提供了证据，存在不同的，种内亚型的P-450 arom，第一次在任何物种中描述，并表明猪具有独特的机制，调节雄激素代谢。
Functional isoforms of porcine aromatase cytochrome P-450 were cloned from placenta, and ovarian theca interna and granulosa tissues, and full length cDNAs were expressed in vitro. Porcine theca and granulosa expressed an identical form of P-450arom. This ovarian cDNA encoded for a protein of 501 amino acids, two amino acids shorter at the N-terminal end than placental P-450arom isoform (503 residues). Overall, the two isoforms exhibited 93% nucleotide and 87% amino acid identity with each other, and both were highly homologous, at the nucleotide and amino acid levels, to human and bovine P-450arom, also 503 amino acid proteins. Analysis of the predicted amino acid sequence further suggested that the regions of the cDNAs, corresponding to presumed exons III, V and IX, assuming conservation of intron-exon boundaries with the human P-450arom gene, were conserved in the porcine placental and ovarian enzymes, while sequence variance occurred in all other putative exons. In vitro expression indicated that the cDNA encoding porcine placental P-450arom was almost 10-fold more active in the synthesis of estrone from androstenedione than was the-ovarian isoform which synthesized more 19OH-androstenedione than estrone. Western analysis of transfected Cos1 cells suggested that the differences in activity were not due to levels of expression of the cDNAs since similar levels of immunodetectable protein were observed in cells transfected with each construct. Both isoforms were sensitive to inhibition of activity by the specific aromatase inhibitors, 4OH-androstenedione and CGS16949A. In addition, activity of the enzyme encoded by the ovarian P-450arom cDNA was suppressed by etomidate, an inhibitor of cytochrome P-450 11 beta-hydroxylase, but the placental P-450arom isoform was not. These functional differences were consistent with observations made in similar experiments involving P-450arom activity in freshly homogenized tissues. These data provide evidence of the existence of distinct, intraspecies isoforms of P-450arom, the first described in any species and suggest that pigs possess a unique mechanism for regulating androgen metabolism.