Molecular biological and biochemical study of plasms platelet-activating factor-metabolic enzymes for new anti-PAF drugs

抗PAF新药血浆血小板活化因子-代谢酶的分子生物学和生化研究

• 批准号: 07557302
• 负责人: SETAKA Morio
• 金额: $ 1.66万
• 依托单位: Teikyo University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557302/
• 关键词: 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; lysoPAFアセチルトランスフェラーゼ; PAFアセチルトランスフェラーゼ; タンパク精製; 遺伝子クローニング

1.cDNA cloning of plasma platelet-activating factor-acetylhydrolase and gene expressionInternal 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 antibodyThe 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-acetylhydrolaseGenomic 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 acetyltransferaseSolubilization of this enzyme by various detergents was found to be quite difficult, and a trial to purify the enzyme is performed.

1.血浆血小板活化因子-乙酰水解酶的cDNA克隆及基因表达采用气相氨基酸测序仪测定纯化的豚鼠血浆血小板活化因子-乙酰水解酶(Mw 63 kDa)的内部氨基酸序列，并以豚鼠肝脏来源的第一链cDNA为模板进行PCR。以600bp的PCR片段为探针，从300,000pfu的豚鼠肝脏cDNA文库中筛选出4个阳性克隆，并进行5'RACE。在大肠杆菌中表达的重组蛋白显示出酶活性。该cDNA编码436个氨基酸（预测Mw 49 kDa），包含丝氨酸酯酶活性位点和三个推定的N联糖基化位点的共有序列。2.血浆血小板活化因子-乙酰水解酶特异性抗体分析将重组蛋白用于免疫兔子，获得单特异性抗体。该抗体可识别豚鼠血浆中含有的 63 kDa 蛋白质，表明该酶经过糖基化修饰。发现该抗体刺激后小鼠肥大细胞会分泌该酶。3.血小板活化因子-乙酰水解酶基因组克隆克隆了含有血小板活化因子-乙酰水解酶5'侧翼区的基因组DNA，并测序了约6kbp。通过报告基因测定来研究启动子区域。计划结合免疫化学方法在分子水平上分析该酶的表达机制及其对疾病的贡献。4.乙酰转移酶的纯化发现用各种去污剂溶解该酶是相当困难的，因此进行了纯化该酶的试验。

项目成果

Karasawa,K.,et al.: "Cloning,Expression and Characterization of Plasma Platelet-Activating Factor-Acetylhydrolase from Guinea Pig" J.Biochem.120. 838-844 (1996)

Karasawa,K.,et al.：“豚鼠血浆血小板激活因子乙酰水解酶的克隆、表达和表征”J.Biochem.120。

Ken-ichi Nakajima, Makoto Murakami, Ryohei Yanoshita Yuji Samejima, Ken Karasawa, Morio Setaka, Shoshichi Nojima and Ichiro Kudo: "Activated mast cells release extracellular type platelet-activating factor acetylhydrolase that contribute to autocrine inac

Ken-ichi Nakajima、Makoto Murakami、Ryohei Yanoshita Yuji Samejima、Ken Karasawa、Morio Setaka、Shoshichi Nojima 和 Ichiro Kudo：“激活的肥大细胞释放细胞外型血小板激活因子乙酰水解酶，有助于自分泌 inac

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) anal

Toru Sasaki、Takayuki Tohyama、Kennichi Oda、Hinako Toyama、Shin-ichi Ishii、Michio Senda、Ken Karasawa、Noriko Sato、Morio Setaka、Shoshichi Nojima、Tadashi Nozaki 和 Pierre Braquet。：“^<11>C 血小板的分布研究

Karasawa, K., et al.: "Cloning,Expression and Characterization of Plasma Platelet-Activating Factor-Acetylhydrolase from Guinea Pig" J.Biochem.120. 838-844 (1996)

Karasawa, K. 等人：“豚鼠血浆血小板激活因子乙酰水解酶的克隆、表达和表征”J.Biochem.120。

Ken Karasawa, Osamu Kuge, Kiyoshi Kawasaki, Masahiro Nishijima, Yasuko Nakano, Motowo Tomita, Kazuaki Yokoyama, Morio Setaka and Shoshichi Nojima.: "Cloning, expression and characterization of plasma platelet-activating factor-acetylhydrolase from Guinea

Ken Karasawa、Osamu Kuge、Kiyoshi Kawasaki、Masahiro Nishijima、Yasuko Nakano、Motowo Tomita、Kazuaki Yokoyama、Morio Setaka 和 Shoshichi Nojima.：“来自几内亚的血浆血小板激活因子乙酰水解酶的克隆、表达和表征