| Project/Area Number |
08557088
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Anesthesiology/Resuscitation studies
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| Research Institution | KYUSHU UNIVERSITY |
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Principal Investigator |
OGURI Kazuta KYUSHU UNIV., FAC.PHARM.SCI., PROFESSOR, 薬学部, 教授 (70037589)
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| Co-Investigator(Kenkyū-buntansha) |
ISHII Yuji KYUSHU UNIV., FAC.PHARM.SCI., INSTRUCTOR, 薬学部, 助手 (90253468)
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| Project Period (FY) |
1996 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥12,100,000 (Direct Cost: ¥12,100,000)
Fiscal Year 1998: ¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 1997: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥3,700,000 (Direct Cost: ¥3,700,000)
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| Keywords | UDP-glucuronosyltransferase / guinea pig / oligomer / protein interaction / morphine / active metabolite / morphine-6-glucuronide / morphine-3-glucuronide / グルクロン酸転移酵素 / クローニング / 遺伝子発現 / ヒト / 肝臓 / グルクロン酸抱合 / 薬物代謝 |
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| Research Abstract |
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
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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
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