Studies on Narcotic UDP-Glucuronosyltransferases for effective and safty use in clinical application

UDP葡萄糖醛酸转移酶（UGTs）是一种将核苷酸葡萄糖酸盐转移到小疏水分子上的超家族酶。吗啡的一种活性葡糖苷，吗啡6-葡糖苷（M6G），一直被认为对人类的镇痛作用有兴趣。除人类外，豚鼠的葡萄糖醛酸吗啡对M6G具有高容量。吗啡UGT的纯化为UGT55K和UGT59K可能的异聚物的制备提供了条件。用金黄色葡萄球菌V8蛋白酶原位酶切后测定其内部氨基酸序列，设计降解引物。从豚鼠肝脏cDNA文库中筛选编码UGT55K的pGUGT2基因，用PCR探针克隆得到该基因。然而，它没有覆盖开放阅读框。UGT55K编码523个氨基酸的序列经5- race和3-RACE测定，并进一步对1821个碱基的全长PCR产物进行分析。以UGT55K cDNA为探针克隆UGT59K cDNA。经5-RACE分析，获得了编码529个氨基酸的UGT59K cDNA 2504个长度超过5个的序列。推断的氨基酸序列相似性为76.5%，推测这两个基因都属于豚鼠UGT2B簇的成员。UGT55K和UGT59K最近被UGT命名委员会命名为gpiUGT2B21和gpiUGT2B22。在pSVL载体上构建了UGT2B21和UGT2B22 cdna表达质粒。每个载体用多胺转染试剂包裹在脂质体中，单次和双次转染COS-1细胞。采用高效液相色谱法和薄层色谱法测定微体中表达的UGT活性。ugt2b21葡萄糖醛酸吗啡3位，4-羟基联苯，冰片，睾酮，雄酮，雌三醇。M6G活性和氯霉素糖醛酸化仅在UGT2B22共转染的UGT2B21细胞中可见。我们还没有得知UGT2B22的底物。目前的研究结果表明，UGT异构体可以作为异聚物，比同聚物获得更广泛的底物。少

UDP Glucuronosyltransferases (UGTs) represent a superfamily of enzymes that transfer a nucleotide glucuronate to small hydrophobic molecules. An active glucuronide of morphine, morphine 6-glucuronide (M6G), has been interested in to be responsible for the analgesia in humans. Besides humans, guinea pigs glucuronidate morphine to M6G with high capacity. The purification of morphine UGT in guinea pig gave us a preparation of possible hetero-oligomer of UGT55K and UGT59K.Their internal amino acid sequences were determined after in situ digestion with S.aureus V8 protease and then degenerated primers were designed. pGUGT2 that encodes UGT55K was cloned with the PCR probe by the screening from guinea pig liver cDNA library. However it didn't cover the open reading frame. The sequence of UGT55K encoding 523 amino acids was determined after 5- and 3-RACE, and further analysis of the full length PCR product of 1821 bases. The UGT59K cDNA was cloned with the UGT55K cDNA as a probe. The 2504 bas … More e length sequences of UGT59K cDNA that encodes 529 amino acids was determined after 5-RACE.The similarity of deduced amino acid sequences was 76.5%, and both genes were expected to be members of UGT2B cluster in guinea pigs. UGT55K and UGT59K were recently named gpiUGT2B21 and gpiUGT2B22 by UGT Nomenclature Committee.The expression plasmids of UGT2B21 and UGT2B22 cDNAs were constructed in pSVL vector. Each vector was enclosed in liposomes with polyamine transfection reagent, and subjected to single and dual transfection to COS-1 cells. Expressed UGT activities in the microsomes were determined by HPLC and radioluminogram on TLC plates. UGT2B21glucuronidated morphine 3-position, 4-hydroxybiphenyl, borneol, testosterone, androsterone and estriol. M6G activity and chloramphenicol glucuronidation were only seen in UGT2B22 co-transfected UGT2B21 cell. We have learned no substrate yet for UGT2B22. The present results indicates that UGT isomers could act as hetero-oligomers by accessing more broad substrates than homo-oligomers. Less