Miyata M, Nagata K, Shimada M, Yamazoe Y, Kato R
Department of Pharmacology, School of Medicine, Keio University, Tokyo, Japan.
Arch Biochem Biophys. 1994 Nov 1;314(2):351-9. doi: 10.1006/abbi.1994.1453.
P450/6 beta A gene was isolated from a rat genomic library and its encoding protein was characterized by the expression of the corresponding cDNA in COS-1 cells. Between exon regions of P450/6 beta A gene and P450PCN2 cDNA (F. J. Gonzalez, B.-J. Song, and J. P. Hardwick (1986) Mol. Cell. Biol. 6, 2969-2976), 12 nucleotide differences were observed, involving two amino acid changes, His-->Asp [429] and Asp-->Gly [445], respectively. A cDNA (6 beta-A cDNA), whose nucleotide sequence was completely identical with the corresponding exon of P450/6 beta A gene, was isolated from a rat cDNA library. Northern blotting using specific oligonucleotide probes showed that 6 beta-A mRNA, but not P450PCN2 mRNA, was a major form in livers of the male rats. 6 beta-A protein expressed in COS-1 cells (at about 0.1 to 0.3% of total microsomal protein) catalyzed testosterone 6 beta-hydroxylation. The reaction was enhanced by the addition of NADPH-P450 reductase (2.5-fold) and by the simultaneous addition of cytochrome b5 and NADPH-P450 reductase (13.7-fold). Catalytic properties of 6 beta-A for typical CYP3A substrates were consistent with those of purified rat P450(6 beta-1) and P450(6 beta-3), corresponding, respectively, to CYP3A2 or the variant form (K. Nagata, F. J. Gonzalez, Y. Yamazoe, and R. Kato (1990) J. Biochem. 107, 718-725). Apparent Michaelis constants (Km) of 6 beta-A for testosterone 6 beta-hydroxylation and a O-dealkylation ratio of propoxycoumarin to pentoxycoumarin, however, showed higher degrees of similarity to those of purified P450(6 beta-3) than those of P450(6 beta-1).
从大鼠基因组文库中分离出P450/6βA基因，并通过在COS-1细胞中表达相应的cDNA对其编码蛋白进行了表征。在P450/6βA基因和P450PCN2 cDNA（F. J. 冈萨雷斯、B.-J. 宋和J. P. 哈德威克（1986年）《分子与细胞生物学》6，2969 - 2976）的外显子区域之间，观察到12个核苷酸差异，分别涉及两个氨基酸变化，即His→Asp [429]和Asp→Gly [445]。从大鼠cDNA文库中分离出一个cDNA（6β - A cDNA），其核苷酸序列与P450/6βA基因的相应外显子完全相同。使用特异性寡核苷酸探针进行的Northern印迹分析表明，6β - A mRNA而非P450PCN2 mRNA是雄性大鼠肝脏中的主要形式。在COS-1细胞中表达的6β - A蛋白（约占总微粒体蛋白的0.1%至0.3%）催化睾酮6β - 羟基化反应。通过添加NADPH - P450还原酶（2.5倍）以及同时添加细胞色素b5和NADPH - P450还原酶（13.7倍），该反应得到增强。6β - A对典型CYP3A底物的催化特性与纯化的大鼠P450(6β - 1)和P450(6β - 3)一致，分别对应于CYP3A2或变体形式（K. 永田、F. J. 冈萨雷斯、Y. 山添和R. 加藤（1990年）《生物化学杂志》107，718 - 725）。然而，6β - A对睾酮6β - 羟基化的表观米氏常数（Km）以及丙氧基香豆素与戊氧基香豆素的O - 脱烷基化比率，与纯化的P450(6β - 3)的相似程度高于与P450(6β - 1)的相似程度。
