| Project/Area Number |
16590021
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Chemical pharmacy
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| Research Institution | Nigata University of Phermacy and Applied Life Sciences |
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Principal Investigator |
KITAGAWA Kouki Nigata University of Phermacy and Applied Life Sciences, Faculty of Pharmaceutical Sciences, Professor (60093853)
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| Co-Investigator(Kenkyū-buntansha) |
SEKIGAWA Yumi Niigata University of Pharmacy and Applied Life Sciences, Faculty of Pharmaceutical Sciences Research, Assistant (50329330)
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| Project Period (FY) |
2004 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2005: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2004: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | protein sulfation / chemical synthesis / solid-phase peptide synthesis / sulfated tyrosine / thioester segment condensation / MALDI-TOF-MS / α-conotoxin / cholecystokinin / 質量分析 / MALDI-TOFMS / α-コノトキシン / チオエステルセグメント縮合 |
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| Research Abstract |
1. We examined the disulfide-bond forming reaction which does not cause the loss of sulfate ester on Tyr(SO3H) residue. α-Conotoxins (PnIA and PnIB), which contain two disulfide-bonds, were prepared by two approaches; a simultaneous oxidation approach and a two-step selective oxidation approach. Both approaches did not produce the desulfated peptide throughout the synthesis. Another sulfated a-conotoxin, EpI, was also synthesized by the side-chain anchoring strategy in which β-carboxyl group of Asp residue was anchored with polymer-support. These three sulfated α-conotoxins were proved to be almost equipotent on biological activity compared with their non-sulfate counterparts. This result implies that the sulfate ester on these peptides does not govern their biological activities.
2. Preparation of peptide thioester which contains the Tyr(SO3H) residue(s) is critical for the chemical synthesis of the large-molecular-form of sulfated peptide. After assembly of the Tyr(SO3H)-containing peptide by Fmoc-SPPS, the protected peptide was subjected to the thioesterification followed by the removal of protecting groups with TFA (4℃). The Tyr(SO3H)-containing peptide thioester was used for the Ag+-mediated segment condensation to prepare the objective sulfated peptide.
3. Various sulfated peptides prepared by us were subjected to MALDI-TOF-MS analyses to obtain information on MS of sulfated peptides and proteins. Also we examined the novel approach to determine the sulfated site(s) of peptides and proteins using a combination of chymotrypsin digestion and MS analysis.
4. Rat cholecystokinin (CCK)-peptides varying the molecular size, were synthesized and used as authentic markers on HPLC to determine the proteolytic products from the precursor protein. From these studies, it is suggested that various proteases and prohormone convertases are involved in CCK processing and their actions are cell and tissue specific.
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