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Study of platelet-activating factor (PAF) carrier protein in human serum.

人血清中血小板活化因子（PAF）载体蛋白的研究。

基本信息

批准号：
02807204
负责人：
MIWA Masao
金额：
$ 1.09万
依托单位：
University of Shizuoka
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

The behavior of platelet-activating factor(PAF)produced in stimulated human polymorphonuclear leukocytes(PMN)was investigated in the presence of serum under conditions close to those existing in vivo. When the cells were stimulated in the presence of the serum obtained from a PAF acetylhydrolase(PAF-AH)-deficient Japanese subject, over 60% of synthesized PAF was detected in the extracellular medium by bioassay, scintillation proximity RIA and selected ion monitoring/gas chromatography/mass spectrography analysis. The release of PAF from PMN after stimulation with FMLP and A23187 was also observed in the presence of normal serum treat ed with acid to inactivate PAF-AH. The heterogeneity of the molecular species of extracellu. 1ar PAF was similar to that of intracellular PAF produced in- stimulated PMN in the presence of PAF-Aft-deficient serum, ruling out the possibility that a specific molecular species of PAF was preferentially released from the cells in the presence of the serum. As … More these data suggested the occurrence of PAF-releasing factor(s)in the serum, an attempt was made to partially purify this factor from PAF-AH-deficient serum and acid-treated normal serum by ammonium sulfate fractionation and column chromatography with DEAE-Cellulofine and Sepharose CL-6B. The molecular mass of PAF-releasing factor revealed on a TSK gel G3000 SW HPLC column was 24OkDa, which was differnt from that of albumin. The binding assay, newly developed for this study, revealed that the PAF-binding activity of PAF-releasing factor is stronger than that of albumin, and that the PAF-releasing factor forms a complex with PAF at low concentration (10^9 M). PAF bound to this factor was difficult to be hydrolyzed by serum PAF-AH. On the other hand, the PAF/PAF-releasing factor complex had aggregatory activity toward washed rabbit platelets. These observations suggest that certain protein(s)releases and carries the PAF newly synthesized by PMN in blood plasma/serum. Thus it appears that PAF functions as an autacoid in vivo, along with other mediators. Less

在血清存在的情况下，在接近体内存在的条件下，研究了受刺激的人多形核白细胞（PMN）中产生的血小板激活因子（PAF）的行为。当在从 PAF 乙酰水解酶 (PAF-AH) 缺陷的日本受试者获得的血清存在下刺激细胞时，通过生物测定、闪烁邻近 RIA 和选择离子监测/气相色谱/质谱分析，在细胞外培养基中检测到超过 60% 的合成 PAF。在用酸处理以灭活 PAF-AH 的正常血清存在的情况下，还观察到用 FMLP 和 A23187 刺激后 PAF 从 PMN 中释放。细胞外分子种类的异质性。 1ar PAF 与在 PAF-Aft 缺乏的血清存在下产生的刺激 PMN 的细胞内 PAF 相似，排除了在存在血清的情况下 PAF 的特定分子种类优先从细胞中释放的可能性。由于这些数据表明血清中存在 PAF 释放因子，因此尝试通过硫酸铵分级分离和使用 DEAE-Cellulofine 和 Sepharose CL-6B 的柱色谱从 PAF-AH 缺陷血清和酸处理的正常血清中部分纯化该因子。 TSK gel G3000 SW HPLC柱显示PAF释放因子的分子量为24OkDa，与白蛋白的分子量不同。本研究新开发的结合测定表明，PAF 释放因子的 PAF 结合活性强于白蛋白，并且 PAF 释放因子在低浓度（10^9 M）下与 PAF 形成复合物。与该因子结合的PAF很难被血清PAF-AH水解。另一方面，PAF/PAF释放因子复合物对洗涤的兔血小板具有聚集活性。这些观察结果表明，某些蛋白质释放并携带血浆/血清中 PMN 新合成的 PAF。因此，PAF 与其他介质一起在体内发挥自体激素的作用。较少的