1997 Fiscal Year Final Research Report Summary
Molecular biological and biochemical study of plasms platelet-activating factor-metabolic enzymes for new anti-PAF drugs
| Project/Area Number |
07557302
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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 |
Biological pharmacy
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| Research Institution | Teikyo University |
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Principal Investigator |
SETAKA Morio Teikyo University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (70012630)
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| Co-Investigator(Kenkyū-buntansha) |
FUJITA Masamichi Nisshin Shokuhin, Tokyo Laboratory, Researcher, 研究員
SHIMIZU Fuzuki Teikyo University, Faculty of Pharmaceutical Sciences, Research Assistant, 薬学部, 教務職員
SATO Noriko Teikyo University, Faculty of Pharmaceutical Sciences, Research Associate, 薬学部, 助手 (80162460)
YOKOYAMA Kazuaki Teikyo University, Faculty of Pharmaceutical Sciences, Research Associate, 薬学部, 助手 (50246021)
KARASAWA Ken Teikyo University, Faculty of Pharmaceutical Sciences, Lecturer, 薬学部, 講師 (50186029)
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| Project Period (FY) |
1995 – 1997
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| Keywords | PAF-Acetylhydrolase / PAF (Platelet-Activating Factor) / cDNA Cloning / Genomic DNA Cloning / lysoPAF-acetyltransferase / 1ysoPAFアセチルトランスフェラーゼ |
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| Research Abstract |
1.cDNA cloning of plasma platelet-activating factor-acetylhydrolase and gene expression
Internal amino acid seauences of purified guinea pig plasma platelet-activating factor-acetylhydrolase (Mw 63 kDa) were determined using a gas-phase amino acid sequencer, and PCR was performed using first-strand cDNA derived from guinea pig liver as a template. Four positive clones were screened from 300,000 pfu of a guinea pig liver cDNA library using the 600-bp PCR fragment as a probe, and 5' RACE was performed. The recombinant protein expressed in E.coli showed the enzymatic activity. The cDNA encoded 436 amino acids (predicted Mw 49 kDa) and contained consensus sequences for an active site of serine-esterases and three putative N-linked glycosylation sites.
2.Analysis using plasma platelet-activating factor-acetylhydrolase-specific antibody
The recombinant protein was used to immunize a rabbit, and a mono specific antibody was obtained. This antibody recognized a 63-kDa protein contained in guinea pig plasma, suggesting that the enzyme was modified by glycosylation. This enzyme was found to be secreted from mouse mast cells upon stimulation using this antibody.
3.Genomic cloning of platelet-activating factor-acetylhydrolase
Genomic DNA containing the 5' -flanking region of platelet-activating factor-acetylhydrolase was cloned, and about 6 kbp was sequenced. The promotor region is investigated by reporter gene assay. Analysis of the expression mechanism of this enzyme and its contribution to diseases at the molecular level is planned in combination with immunochemical methods.
4.Purification of acetyltransferase
Solubilization of this enzyme by various detergents was found to be quite difficult, and a trial to purify the enzyme is performed.
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[Publications] Toru Sasaki, Takayuki Tohyama, Kennichi Oda, Hinako Toyama, Shin-ichi Ishii, Michio Senda, Ken Karasawa, Noriko Sato, Morio Setaka, Shoshichi Nojima, Tadashi Nozaki and Pierre Braquet.: "A distribution study of ^<11>C platelet-activating factor (PAF) analogs in normal and tumor-bearing mice." Nuclear Medicine and Biology. 23. 309-314 (1996)
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