Molecular biological and biochemical study of plasma Platelet-activating factor-acetylhydrolase ; its regulation and function

血浆血小板激活因子-乙酰水解酶的分子生物学和生化研究；

• 批准号: 07672386
• 负责人: KARASAWA Ken
• 金额: $ 1.15万
• 依托单位: Teikyo University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07672386/
• 关键词: PAF (Platelet-Activating Factor); PAF-Acetylhydrolase; Molecular Cloning; PAT

1.Molecular cloning of the cDNA of plasma platelet-activating factor-acetylhydrolase Purified guinea pig plasma platelet-activating factor-acetylhydrolase (Mw 63 kDa) was digested with lysylendopeptidase. The 9 resulting peptides were isolated by reverse-phase HPLC,and their amino acid sequences were determined using a gas-phase amino acid sequencer. The deduced oligonucleotide primers were then syntesized on the basis of the petide sequences, and PCR was performed using first-strand cDNA derived from guinea pig liver poly (A)^+ RNA as a template. A specific fragment of about 600 bp was amplified. Four positive clones were screened from 300,000 pfu of a guinea pig liver cDNA library using the PCR fragment as a probe. None of these contained the N-terminal region of the protein, and 5' RACE was performed. 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.Gene expression and antibody preparationThe full-length cDNA was ligated in an E.coli expression vector with a His tag. The recombinant protein showed platelet-activating factor-acetylhydrolase activity, and was purified using a Ni^+ column and SDS-PAGE.The purified protein was used to immunize a rabbit, and a mono-specific antibody was obtained. This antibody recognized a 63-kDa protain contained in guinea pig plasma, suggesting that the enzyme was modified by blycosylation. The significance of this post-translational modification remaines to be elucidated.Cloning of the genomic DNA is performed. Recently, the expression of this enzyme was reported to be regulated by estradiol in the liver and macrophages and by interleukins in mast cells. Analysis of the regulation and function of this enzyme at the molecular level is planned using promoter region analysis and immunochemical methods.

1.血浆血小板活化因子乙酰水解酶cDNA的分子克隆用赖氨酰内肽酶消化纯化的豚鼠血浆血小板活化因子乙酰水解酶（Mw 63 kDa）。通过反相HPLC分离得到的9种肽，并使用气相氨基酸测序仪测定其氨基酸序列。根据肽段序列合成寡核苷酸引物，以豚鼠肝poly（A）^+ RNA第一链cDNA为模板进行PCR。扩增出约600 bp的特异性片段。以PCR片段为探针，从30万pfu的豚鼠肝cDNA文库中筛选出4个阳性克隆。这些都不含蛋白质的N-末端区域，并进行5' RACE。该cDNA编码436个氨基酸（预测分子量为49 kDa），含有丝氨酸酯酶活性位点和3个推测的N-连接糖基化位点的共有序列。2.基因表达和抗体纯化将全长cDNA连接到带有His标签的大肠杆菌表达载体中。重组蛋白经Ni^+柱和SDS-PAGE纯化后，免疫家兔，获得单特异性抗体。该抗体识别豚鼠血浆中含有的63-kDa蛋白质，表明该酶被blycosylation修饰。这种翻译后修饰的意义有待阐明。最近，据报道，这种酶的表达在肝脏和巨噬细胞中受到雌二醇的调节，在肥大细胞中受到白细胞介素的调节。计划使用启动子区域分析和免疫化学方法在分子水平上分析这种酶的调节和功能。

项目成果

Ken Karasawa et al.: "Cloning,expression and characterization of plasma platelet activating factor acetylhydrolase from Guinea Pig" Journal of Biochemistry. 120. 838-844 (1996)

Ken Karasawa 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”生物化学杂志。

唐沢 健: "動脈硬化症と血小板活性化因子(PAF) -PAF分解酵素の分子生物学的研究-" 杉山産業化学研究所年報. 55-68 (1995)

Ken Karasawa：“动脉硬化和血小板活化因子（PAF）-PAF降解酶的分子生物学研究-”杉山工业化学研究所年度报告55-68（1995）。

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 P

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

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

Toru Sasaki et al.: "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)

Toru Sasaki等人：“11C血小板激活因子(PAF)类似物在正常和荷瘤小鼠中的分布研究”核医学和生物学。