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

麻醉性UDP-葡萄糖醛酸基转移酶在临床应用中有效和安全使用的研究

基本信息

批准号：
08557088
负责人：
OGURI Kazuta
金额：
$ 7.74万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

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

UDP葡萄糖醛酸转移酶（UGT）代表将核苷酸葡萄糖醛酸转移到小疏水分子的酶的超家族。吗啡6-葡糖苷酸（M6 G）是吗啡的一种活性葡糖苷酸，具有镇痛作用。除人类外，豚鼠将吗啡葡萄糖醛酸苷高容量地转化为M6 G。通过纯化豚鼠吗啡UGT，制备了UGT 55 K和UGT 59 K可能的异源寡聚体，经金黄色葡萄球菌V8蛋白酶原位酶切后测定其内部氨基酸序列，并设计简并引物。从豚鼠肝cDNA文库中筛选UGT 55 K基因，用PCR探针克隆到pGUGT 2。然而，它没有覆盖开放的阅读框架。通过5-RACE和3-RACE，对UGT 55 K基因的全长1821个碱基的PCR产物进行分析，确定了UGT 55 K基因编码523个氨基酸。以UGT 55 K cDNA为探针克隆UGT 59 K cDNA。2504低音 ...更多信息 5-RACE法测定了UGT 59 K cDNA全长序列，编码529个氨基酸，推导的氨基酸序列相似性为76.5%，推测两个基因均属于UGT 2B簇。UGT命名委员会将UGT 55 K和UGT 59 K分别命名为gpiUGT 2B 21和gpiUGT 2B 22。将每个载体用多胺转染试剂包封在脂质体中，并对COS-1细胞进行单转染和双转染。通过HPLC和TLC板上的放射发光图测定微粒体中表达的UGT活性。UGT 2B 21葡萄糖醛酸化吗啡3位，4-羟基联苯，睾酮，雄酮和雌三醇。仅在UGT 2B 22共转染的UGT 2B 21细胞中观察到M6 G活性和氯霉素葡萄糖醛酸化。我们还没有发现UGT 2B 22的底物。目前的结果表明，UGT异构体可以作为异源寡聚体，通过访问更广泛的底物比同源寡聚体。少