Human placenta estrogen synthetase (aromatase) purified by affinity chromatography.
Human placenta estrogen synthetase (aromatase) purified by affinity chromatography.
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通过亲和层析纯化人胎盘雌激素合成酶（芳香酶）。
DOI：
10.1016/s0021-9258(18)61668-0
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
D. Hagerman
中科院分区：
文献类型：
--
作者：
D. Hagerman
用DEAE-Trisacryl和睾酮-琼脂糖层析法从新鲜人胎盘微粒体中纯化微粒体雌激素合成酶（细胞色素P-450 ES），又称芳香化酶。以雄烯二酮为底物，NADPH为电子供体，部分纯化的人胎盘P-450还原酶为电子载体，进行雌激素合成酶测定。纯化的P-450的比细胞色素P-450含量为0.67 nmol mg-1蛋白质，并且制剂不含细胞色素P-420。最大吸光度为448.5 nm。纯化的P-450 ES组分的比雌激素合成酶活性为35 nmol min-1 nmol-1的细胞色素P-450或23.3 nmol min-1 mg-1的蛋白质。后一个值显示了179倍的纯化，在两步程序中产率大于1%。以雄烯二酮为芳香化底物测定了反应动力学常数。P-450的Km为1.4 nM，Vmax为37 nmol min-1 nmol-1。纯化的酶以相同的速率芳香化雄烯二酮和睾酮;雄烯二酮只产生雌酮，睾酮只产生雌二醇-17 β。脱氢表雄酮未检测到芳香化或以其他方式代谢。16 α-羟基睾酮和16 α-羟基雄烯二酮均未芳香化。在直接测定中未检测到羟基类固醇脱氢酶或还原酶。在芳构化测定孵育混合物中未检测到游离反应中间体。该产品的纯度和制备的简单性推荐其用于酶的进一步研究。
Microsomal estrogen synthetase (cytochrome P-450ES), also known as aromatase, was purified from fresh human placenta microsomes by DEAE-Trisacryl and testosterone-agarose chromatography. Estrogen synthetase assays were done with androstenedione as substrate, NADPH as electron donor, and a partially purified P-450 reductase from human placenta as the electron carrier. The specific cytochrome P-450 content of the purified P-450 was 0.67 nmol mg-1 of protein, and the preparation contained no cytochrome P-420. The absorbance maximum was 448.5 nm. The specific estrogen synthetase activity of the purified P-450ES fraction was 35 nmol min-1 nmol-1 of cytochrome P-450 or 23.3 nmol min-1 mg-1 of protein. The latter value shows a 179-fold purification with a yield greater than 1% in the two-step procedure. Kinetic constants for the reaction were measured with androstenedione as the aromatizable substrate. The Km was 1.4 nM and the Vmax was 37 nmol min-1 nmol-1 of P-450. The purified enzyme aromatized androstenedione and testosterone at identical rates; androstenedione gave only estrone, and testosterone gave only estradiol-17 beta. Dehydroepiandrosterone was not detectably aromatized or otherwise metabolized. Neither 16 alpha-hydroxytestosterone nor 16 alpha-hydroxyandrostenedione was aromatized. No hydroxysteroid dehydrogenase or reductase was detected in direct assays. No free reaction intermediates were detected in aromatization assay incubation mixtures. The purity of the product and the simplicity of the preparation recommend it for use in further studies of the enzyme.