Hattori M, Arai H, Inoue K
Department of Health Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
J Biol Chem. 1993 Sep 5;268(25):18748-53.
Platelet-activating factor (PAF) acetylhydrolase, which removes the acetyl moiety at the sn-2 position, has been found in plasma and tissue cytosol. PAF acetylhydrolase in bovine brain cytosol was chromatographically separated into three distinct fractions, all of which exhibited pH optima in the neutral to mild alkaline region and were unaffected by EDTA. We have purified the major fraction of the enzyme to near homogeneity. The purified enzyme had a molecular mass of about 100 kDa, as estimated by gel filtration chromatography, and gave three distinct bands of 45, 30, and 29 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides exclusively co-migrated with the activity throughout the purification steps. These data suggest that this set of polypeptides corresponds to the subunits of bovine brain PAF acetylhydrolase. Diisopropyl fluorophosphate completely inhibited the activity at 0.1 mM. [3H]Diisopropyl fluorophosphate labeled only the 29-kDa polypeptide, suggesting that this polypeptide possesses an active serine residue(s). The purified enzyme displayed similar activity against PAF and oxidatively modified phosphatidylcholine, but did not hydrolyze phosphatidylcholine or phosphatidylethanolamine with two long chain acyl groups. Thus, the intracellular PAF acetylhydrolase is likely to be a new member of the calcium-independent phospholipases A2 in mammalian tissues.
血小板活化因子（PAF）乙酰水解酶可去除sn-2位的乙酰基部分，已在血浆和组织胞质溶胶中被发现。牛脑胞质溶胶中的PAF乙酰水解酶经色谱分离为三个不同的组分，所有组分在中性至弱碱性区域均表现出最适pH值，且不受乙二胺四乙酸（EDTA）影响。我们已将该酶的主要组分纯化至接近均一。通过凝胶过滤色谱法估计，纯化后的酶分子量约为100 kDa，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上分别呈现出45 kDa、30 kDa和29 kDa的三条明显条带。在整个纯化步骤中，这些多肽与活性完全共迁移。这些数据表明，这组多肽对应于牛脑PAF乙酰水解酶的亚基。氟磷酸二异丙酯在0.1 mM时完全抑制活性。[3H]氟磷酸二异丙酯仅标记29 kDa的多肽，表明该多肽具有活性丝氨酸残基。纯化后的酶对PAF和氧化修饰的磷脂酰胆碱表现出相似的活性，但不水解含有两个长链酰基的磷脂酰胆碱或磷脂酰乙醇胺。因此，细胞内PAF乙酰水解酶可能是哺乳动物组织中钙非依赖性磷脂酶A2的一个新成员。
