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Studies on the biosynthetic mechanism of platelet-activating factor and its physiological significance in alveolar macrophages

肺泡巨噬细胞血小板激活因子生物合成机制及其生理意义的研究

基本信息

批准号：
62480425
负责人：
WAKU Keizo
金额：
$ 4.16万
依托单位：
Faculty of Pharmaceutical Sciences, Teikyo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

1. Studies on the acylation mechanism of alkylether phospholipids. We studied the acylation mechanism of 1-alkyl-glycerophosphocholine (GPC), using 1-alkyl-GPC as an acceptor and diacyl-GPC, including ^<14>C-arachidonic acid at the 2-position, as an acyl donor, by rabbit alveolar macrophage microsomes. This transacylation reaction does not need CoA, ATP or Mg^<2+>. C-20 fatty acids, especially arachidonic acid (20:4) and docosahexaenoic acid (22:6) are the good substrates for this enzymatic reaction.2. Release of arachidonic acid from alkylether phospholipids and incorporation of acetyl residue into 1-alkyl-GPC. Lyso platelet-activating factor (lyso PAF, 1-alkyl-GPC) produced by phospholipase A_2 action on ether choline glycerophospholipid (CGP) was acetylated or acylated by acetyltransferase or transacylase, competitively. The specific activity of acetyltransferase was 474 pmole/min/10^6cell lysate (acetylCoA; 100 m) and increased by 2-3 times on the cell stimulation with A23187. That of transacylase was 98 pmole/min/10^6cell lysate and significant increase of the activity was not observed on cell stimulation. on the other hand, the acetyltransferase activity was quite dependent on the concentration of acetyl-CoA in the case of no addition of exogenous acetyl-CoA. Therefore, in this case, lyso PAF seemed to be acylated mainly by transacylase with 20:4, rather than by acetyltransferase. The localization of these enzymes or substrates in cells shoud be studied in the next step.

1.烷基醚磷脂的酰化反应机理研究。用兔肺泡巨噬细胞微粒体研究了1-烷基-甘油磷胆碱(GPC)的酰化反应机理，以1-烷基-GPC为受体，2-位含~&lt；14&gt；C-花生四烯酸的双酰基-GPC为酰基供体。这种转酰化反应不需要辅酶A、三磷酸腺苷或镁离子。C-20脂肪酸，特别是花生四烯酸(20：4)和二十二碳六烯酸(22：6)是该酶反应的良好底物。烷基醚磷脂中花生四烯酸的释放和乙酰基残基在1-烷基-GPC中的掺入。溶血型血小板激活因子(lyso PAF，1-烷基-GPC)由磷脂酶A_2作用于乙基胆碱甘油磷脂(CGP)而产生，乙酰基转移酶或转酰基酶竞争性地乙酰化或酰化。乙酰基转移酶比活力为474pmole/min/10~6细胞裂解物(乙酰辅酶A；100m)，经A23187刺激后比活力提高2~3倍。转酰基酶的活力为98pmole/min/10~6细胞裂解物，在细胞刺激下未见明显的活性增加。另一方面，在不添加外源乙酰辅酶A的情况下，乙酰转移酶的活性与乙酰辅酶A的浓度有很大的依赖关系。因此，在这种情况下，lyso PAF似乎主要是通过20：4的转酰基酶而不是乙酰转移酶来酰化的。这些酶或底物在细胞中的定位应在下一步进行研究。