Preparation of a High Performance Affinity Gel for Purification of UDP-Glucuronyltransferase

该项目旨在开发和准备一种新的高性能凝胶，用于UDP-葡萄糖基转移（UDP-GT）的分离和净化。在第一年，从6-succinylmorphine-omega-氨基氯胺衍生出的Sepharose 4 B凝胶被发现，可以分离出一种良好的UDP-葡萄糖转移的同位素酶。由于终端吗啡是容易水解的，可释放的，omega-氨基琼脂糖4 B凝胶的成功酸衍生物被认为是高性能凝胶的基本体和一种准备好的凝胶，omega-碳丙基氨基琼脂糖4 B，成功分离UDP-GTs。在第一年，UDP-GT在大鼠的liver micromes中被纯化是通过使用凝胶后续纯化的UDP-六苯胺Sepharose 4 B列，其中是一个新的专门用于吗啡的酶。在下一年，另一种同位素酶在几乎纯净的形式中以4-羟基联苯和4-硝基酚的特定活性形式纯化。凝胶是另一种，应用于纯化的liver微UDP-GTs在几内亚猪和beagle狗中。没有人被报告说在几内亚猪和狗中的纯净UDP-GT。一种同位素酶，其中苯羟基基亚群的活性很高，从几内亚猪中净化。单体分子重量约为54，000，N-末端氨基酸序列是以均匀的方式对大鼠和雌激素UDP-GT进行大鼠序列的睾丸激素UDP-GT。两个UDP-GTs在纯形式的男性和女性狗中被孤立。一种同工酶净化剂被命名为吗啡UDP-GT，到吗啡和单体分子重量的高活性为50，000。另一种分子重量的酶为52，000展示的广泛基底特性，并命名为UDP-GT_<Dog-1>。UDP-GT_<Dog-1>的N-终止氨基酸序列与大鼠4-甲基甲苯UDP-GT的序列相同。Thus， 4种新的UDP-GT的同位素已经成功地孤立和净化，并在这个项目中使用了新的亲和力凝胶。

This project was intended to develop and prepare a new high performance gel for separation and purification of UDP-Glucuronyltransferases (UDP-GT). In the first year, a Sepharose 4B gel derivative of 6-succinylmorphine-omegaーaminooctylamine was found to separate well isoenzymes of UDP-glucuronyltransferases. Since the terminal morphine was easily hydrolyzable and released entirely, succinic acid derivative of omegaーaminooctyl Sepharose 4B gel was assumed to be an essential body of the high performance gel and a prepared gel, omega-carboxypropionylaminooctyl Sepharose 4B, worked successfully for separation of UDP-GTs. In the first year, an isoenzyme of UDP-GT in liver microsomes of rats was purified by use of the gel following purification with UDP-hexanolamine Sepharose 4B column, Which was a new enzyme specific to morphine. Next year, another isoenzyme was purified from rats in almost pure form with specific activities to 4-hydroxybiphenyl and 4-nitrophenol. The gel was, further, applied for use in the purification of liver microsomal UDP-GTs in guinea pigs and beagle dogs. No attempt has been reported to purify UDP-GT in guinea pigs and dogs. An isoenzyme which has high activity to phenolic hydroxyl group of substrates was purified from guinea pigs. The monomer molecular weight was about 54,000 and N-terminus amino acid sequence was homologous to sequences of testosterone UDP-GT of rats and estrone UDP-GT in rabbits. Two UDP-GTs were isolated in pure form from male and female dogs. An isoenzyme purified was named morphine UDP-GT due to the high activity to morphine and the monomer molecular weight was 50,000. Another enzyme of molecular weight 52,000 Showed broad substrate specificity and was named UDP-GT_<Dog-1>. Nーterminus amino acid sequence of UDP-GT_<Dog-1> was homologous to the sequence of 4-methylumbelliferone UDP-GT in rats. Thus, 4 new isoenzymes of UDP-GT were successfully isolated and purified by use of the new affinity gel prepared in this project.