Tremblay Y, Fleury A, Beaudoin C, Vallée M, Bélanger A
Laboratory of Molecular Endocrinology, CHUL Research Center, Québec, Canada.
DNA Cell Biol. 1994 Dec;13(12):1199-212. doi: 10.1089/dna.1994.13.1199.
In mammalian and fish species, P450c17 mediates both 17 alpha-hydroxylase and 17,20-lyase activities in the synthesis of steroid hormones. Previous results have shown that among the adrenal steroid hydroxylase enzymes involved in adrenal C19 steroid and glucocorticoid synthesis, regulation of cytochrome P450c17 is of primary importance because it is localized at the key branch between glucocorticoid and C19 steroid synthesis. A cDNA library from guinea pig adrenal was constructed, and the complete 17 alpha-hydroxylase cytochrome P450 cDNA was isolated. The guinea pig P450c17 cDNA includes the full-length coding region (1,524 nucleotide), the complete 3' untranslated region (169 nucleotide), and 39 bases of the 5' untranslated region. Our clone shares most of the features of the other P450c17 cDNAs; however, in addition, we identified a novel conserved region of 18 amino acids located in exon I between residues 80 and 97. This region presents the highest percentage of identity among the other P450c17 enzymes and is positioned one helixturn upstream of the important Ser106 on the corresponding human form. On Northern blot, the cDNA hybridizes with a major 1.8-kb mRNA and with two other related P450c17 mRNA of about 3 and 4 kb. P450c17 mRNA is equally distributed in male and female gonads and adrenals. Characterization of the enzymatic activity shows that 17 alpha-hydroxylase and 17,20-lyase are carried by a single protein, but in homogenates 17,20-lyase activity is barely detectable. Moreover, we demonstrate in vitro and in vivo that the guinea pig enzyme preferentially has very high levels of 17 alpha-hydroxylase and 17,20-lyase activities only toward delta 4 steroids. Second-messenger cyclic adenosine monophosphate and adrenocorticotropin specifically increased the abundance of P450c17 mRNA levels in guinea pig adrenal cells.
在哺乳动物和鱼类中，P450c17在类固醇激素合成过程中介导17α-羟化酶和17,20-裂解酶的活性。先前的研究结果表明，在参与肾上腺C19类固醇和糖皮质激素合成的肾上腺类固醇羟化酶中，细胞色素P450c17的调节至关重要，因为它位于糖皮质激素和C19类固醇合成的关键分支处。构建了豚鼠肾上腺的cDNA文库，并分离出完整的17α-羟化酶细胞色素P450 cDNA。豚鼠P450c17 cDNA包括全长编码区（1524个核苷酸）、完整的3'非翻译区（169个核苷酸）和5'非翻译区的39个碱基。我们的克隆具有其他P450c17 cDNA的大多数特征；然而，此外，我们还鉴定出一个位于外显子I中第80至97位残基之间的18个氨基酸的新保守区域。该区域在其他P450c17酶中具有最高的同一性百分比，并且在相应的人类形式中位于重要的Ser106上游一个螺旋圈处。在Northern印迹分析中，该cDNA与一条主要的1.8 kb mRNA以及另外两条约3 kb和4 kb的相关P450c17 mRNA杂交。P450c17 mRNA在雄性和雌性性腺及肾上腺中分布均匀。酶活性的表征表明，17α-羟化酶和17,20-裂解酶由单一蛋白质承担，但在匀浆中几乎检测不到17,20-裂解酶活性。此外，我们在体外和体内证明，豚鼠酶仅对δ4类固醇优先具有非常高的17α-羟化酶和17,20-裂解酶活性水平。第二信使环磷酸腺苷和促肾上腺皮质激素特异性增加了豚鼠肾上腺细胞中P450c17 mRNA水平的丰度。
