A simple and rapid procedure for the purification of phenobarbitalinducible cytochrome P-450 from rat liver microsomes
A simple and rapid procedure for the purification of phenobarbitalinducible cytochrome P-450 from rat liver microsomes
复制标题
从大鼠肝微粒体中纯化苯巴比妥诱导的细胞色素 P-450 的简单快速方法
DOI：
10.1016/0003-9861(79)90006-7
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发表时间：
1979
影响因子：
3.9
通讯作者：
A. Y. Lu
中科院分区：
文献类型：
--
作者：
S. B. West;Mou;G. Miwa;A. Y. Lu
已经开发出一种快速而简单的程序，用于从苯巴比妥预处理的大鼠的肝微粒体中纯化苯巴比妥诱导形式的细胞色素P-450。 2 天内，可以从 4 g 微粒体蛋白中回收约 1000–1500 nmol 高度纯化的细胞色素 P-450，具体含量为 16 nmol/mg 蛋白。该程序包括用胆酸钠溶解微粒体蛋白，用聚乙二醇分级，以及室温下在DEAE-纤维素上进行柱层析。所得 DEAE-纤维素组分在十二烷基硫酸钠存在下在聚丙烯酰胺凝胶上进行电泳，作为最小分子量为 52,000 的主要蛋白质条带和一些微弱的条带。在 QAE Sephadex A-25 上进一步层析基本上消除了这些微弱的条带，并将比含量略微增加至 17 nmol/mg 蛋白质。这种形式的细胞色素P-450的含量相对较低，似乎存在于未经处理的大鼠的微粒体中，因为通过该程序可以以DEAE-纤维素部分的形式回收不到1%。苯巴比妥可在不同年龄和性别的大鼠中诱导出相同的形式。在最佳测定条件下的重构系统中，这种形式的细胞色素P-450 催化苯异丙胺的 N-去甲基化，周转数大于 100，并在 16α 位置但不在 6β 或 7α 位置羟基化睾酮。
A rapid and simple procedure has been developed for the purification of a phenobarbital-inducible form of cytochromeP-450 from the liver microsomes of phenobarbitalpretreated rats. Within 2 days approximately 1000–1500 nmol of highly purified cytochromeP-450 with a specific content of 16 nmol/mg protein can be recovered from 4 g of microsomal protein. The procedure consists of solubilization of microsomal protein with sodium cholate, fractionation with polyethylene glycol, and column chromatography at room temperature on DEAE-cellulose. The resulting DEAE-cellulose fraction electrophoreses on polyacrylamide gels in the presence of sodium dodecyl sulfate as a major protein band with a minimum molecular weight of 52,000 and a few faint bands. Further chromatography on QAE Sephadex A-25 essentially removes these faint bands and increases the specific content slightly to 17 nmol/mg protein. Relatively low amounts of this form of cytochromeP-450 appear to be present in microsomes of untreated rats since less than 1% can be recovered as the DEAE-cellulose fraction by this procedure. An identical form is inducible by phenobarbital in rats of different ages and sex. In a reconstituted system under optimal assay conditions, this form of cytochromeP-450 catalyses the N-demethylation of benzphetamine with a turnover number greater than 100 and hydroxylates testosterone at the 16α position but not at the 6β or 7α position.