Tukey R H, Robinson R, Holm B, Falany C N, Tephly T R
Drug Metab Dispos. 1982 Mar-Apr;10(2):97-101.
A technique has been developed which rapidly separates and purifies UDP-glucuronosyltransferases from liver microsomes of untreated rabbits. by use of this method, highly purified estrone and p-nitrophenol UDP-glucuronosyltransferases can be obtained in good yield in about 48 hr. Microsomes were solubilized with the nonionic detergent Emulgen 911 in low ionic strength buffers and applied to a DEAE-cellulose column equilibrated with low ionic strength buffers. UDP-glucuronosyltransferase activities were then eluted in a stepwise fashion with increasing concentration of KCl. Three fractions were studied. The first two fractions contained only estrone UDP-glucuronosyltransferase activity while a third contained p-nitrophenol UDP-glucuronosyltransferase activity. Each fraction was directly applied to a UDP-hexanolamine Sepharose-4B column, which was then washed extensively with KCl, and the transferases were eluted with UDP-glucuronic acid. A method for separating the transferases on the affinity column is presented. Testosterone and morphine could not be conjugated by any of the purified enzymes.
已开发出一种技术，可从未经处理的兔肝脏微粒体中快速分离和纯化UDP-葡萄糖醛酸基转移酶。使用该方法，可在约48小时内以高收率获得高度纯化的雌酮和对硝基苯酚UDP-葡萄糖醛酸基转移酶。微粒体在低离子强度缓冲液中用非离子洗涤剂乳化剂911溶解，并应用于用低离子强度缓冲液平衡的DEAE-纤维素柱。然后用浓度递增的KCl逐步洗脱UDP-葡萄糖醛酸基转移酶活性。研究了三个级分。前两个级分仅含有雌酮UDP-葡萄糖醛酸基转移酶活性，而第三个级分含有对硝基苯酚UDP-葡萄糖醛酸基转移酶活性。每个级分直接应用于UDP-己醇胺琼脂糖-4B柱，然后用KCl广泛洗涤，并用UDP-葡萄糖醛酸洗脱转移酶。提出了一种在亲和柱上分离转移酶的方法。睾酮和吗啡不能被任何纯化的酶结合。
