权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

用于纯化 UDP-葡萄糖醛酸转移酶的高性能亲和凝胶的制备

基本信息

批准号：
01571213
负责人：
OGURI Kazuta
金额：
$ 1.34万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

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.

本项目旨在开发和制备一种新型高效凝胶，用于分离纯化尿苷二磷酸葡萄糖醛酸转移酶（UDP-GT）。在第一年，发现6-琥珀酰吗啡-ω-氨基辛胺的Sepharose 4 B凝胶衍生物能够很好地分离UDP-葡萄糖醛酸转移酶的同工酶。由于末端吗啡易于水解并完全释放，因此假定ω-羧基丙酰氨基辛基Sepharose 4 B凝胶的琥珀酸衍生物是高性能凝胶的基本主体，并且所制备的凝胶ω-羧基丙酰氨基辛基Sepharose 4 B成功地用于分离UDP-GT。第一年，用UDP-己醇胺Sepharose 4 B柱纯化后，用凝胶纯化了大鼠肝微粒体中的UDP-GT同工酶，这是一种新的吗啡特异性酶。第二年，从大鼠中纯化出几乎纯的另一种同工酶，具有对4-羟基联苯和4-硝基苯酚的特异性活性。该凝胶进一步用于纯化豚鼠和比格犬的肝微粒体UDP-GT。没有尝试在豚鼠和狗中纯化UDP-GT的报道。从豚鼠体内分离纯化了一种对底物酚羟基具有高活性的同工酶。其N端氨基酸序列与大鼠睾酮UDP-GT和家兔雌酮UDP-GT序列同源。从雄性和雌性犬中分离出两种纯形式的UDP-GT。纯化的同工酶对吗啡有很高的活性，命名为吗啡UDP-GT，单体分子量为50，000。另一种分子量为52，000的酶具有广泛的底物特异性，命名为UDP-GT_<Dog-1>。其N末端氨基酸序列<Dog-1>与大鼠4-甲基伞形酮UDP-GT序列同源。因此，利用本项目制备的新型亲和凝胶成功地分离纯化了4种新的UDP-GT同工酶。