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

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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.