The structure of complex of sulfonamide antiglaucomatous agent with carbonic anhydrase as studied by NMR spectroscopy
| Project/Area Number |
04454443
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Ophthalmology
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| Research Institution | Osaka Prefectural College of Nursing (1994)
Osaka University (1992-1993) |
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Principal Investigator |
KISHIDA Kenichi Osaka Prefectural college of Nursing Faculty of Nursing Professor, 看護学部, 教授 (80028563)
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| Co-Investigator(Kenkyū-buntansha) |
KOBAYASHI Yuji Osaka University Institute for Protein Research Associate Professor, 蛋白質研究所, 助教授 (20127228)
西川 淳一 大阪大学, 薬学部, 助手 (90218131)
松尾 武清 大阪大学, 教養部, 教授 (50029691)
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| Project Period (FY) |
1992 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥5,700,000 (Direct Cost: ¥5,700,000)
Fiscal Year 1994: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1993: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1992: ¥3,200,000 (Direct Cost: ¥3,200,000)
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| Keywords | Carbonic anhydrase II / Sulfonamide / Acetazolamide / Nuclear magnetic resonance / Histidine / ^<13>C-edited NOESY / 多次元NMR法 / 二重標識法 / His64 / ^<13>Cや^<15>Nで標識 / HSQC法 / 安定同位元素標識 / His-64 / 炭酸脱水酵素 / 変異源処理 / グリシン / 質量分析 |
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| Research Abstract |
In order to obtain information on the structure of the complex of acetazolamide, a potent antiglaucomatous agent, with carbonic anhydrase II,^1H,^<13>C and ^<15>N-nuclear magnetic resonance study was conducted. As for the preparation of isotope-labeled carbonic anhydrase II,cDNA encoding human carbonic anhydrase II was transduced into the host cells (BL21 strain of E.coli) which were auxotrophic for histidine and glycine. The cDNA was supplied from another laboratory. The cells were isolated in our own laboratory. The enzyme was separated from cells grown in a medium containing ^<13>C and ^<15>N-labeled histidine and glycine, and purified by both affinity and ion-exchange chromatography. [acetamide-2-^<13>C, ^<15>NJ-acetazolamide was synthesized according to a reported procedure. The following NMR measurements were carried out. They are CO-filtered ^<15>N-HSQC,3D-HNCA,2D-HCalphaCbetaH, (H) Cbeta (CalphaCdelta) H.Through these procedures, we distinguishd NMR signals from His64 residue. Then, we measured NMR of the complex of human carbonic anhydrase II with acetazolamide by means of ^<13>C-edited NOESY.We did not detect NOE (nuclear Overhauser effect) interaction between acetamide moiety of acetazolamide and His64 residue of the enzyme. but we did detect a reaction between the moiety and a residue which is suspected to be phenylalanine. The present results do not co-incide with our hypothesis that acetamide moiety may interact with His64 residue of the enzyme, although further study is required before coming to a final conclusion.
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Report
(4 results)
Research Products
(7 results)
