Schalkwijk C G, Märki F, Wiesenberg I, van den Bosch H
Centre for Biomembranes and Lipid Enzymology, University of Utrecht, The Netherlands.
J Lipid Mediat. 1991 Jul-Aug;4(1):83-95.
Monoclonal antibodies against rat liver mitochondrial phospholipase A2 were found to be cross-reactive with the phospholipase A2 present in caseinate-induced rat peritoneal exudate, both in dot-blot and in monoclonal antibody-Sepharose binding experiments. Immunoaffinity purification of the exudate enzyme yielded enzyme preparations with an estimated enrichment of about 54000-fold in a single chromatographic step. Sodium dodecylsulfate-polyacrylamide gelectrophoresis showed, apart from the presence of a 14 KDa phospholipase A2 band, several other bands at higher molecular weights. After Western blotting and immunostaining only a 14 kDa phospholipase A2 was detected, but upon storage of the sample in dodecylsulfate and dithiotreitol associated forms of this phospholipase A2 appeared. Similar tendencies to form multimers were observed starting from immunopurified and homogeneous phospholipase A2 preparations obtained from rat platelets and from rat liver mitochondria. This phenomenon is likely to be of general importance for the interpretation of results obtained in Western blots using antiphospholipase A2 antibodies.
在斑点印迹和单克隆抗体 - 琼脂糖结合实验中，发现抗大鼠肝脏线粒体磷脂酶A2的单克隆抗体与酪蛋白诱导的大鼠腹腔渗出液中的磷脂酶A2发生交叉反应。对渗出液酶进行免疫亲和纯化，在单个色谱步骤中得到的酶制剂估计富集约54000倍。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳显示，除了存在一条14 kDa的磷脂酶A2条带外，还有几条分子量更高的条带。蛋白质免疫印迹和免疫染色后，仅检测到一条14 kDa的磷脂酶A2，但当样品在十二烷基硫酸盐和二硫苏糖醇中储存时，该磷脂酶A2的相关形式出现。从大鼠血小板和大鼠肝脏线粒体获得的免疫纯化且均一的磷脂酶A2制剂开始，也观察到形成多聚体的类似趋势。这种现象可能对于解释使用抗磷脂酶A2抗体在蛋白质免疫印迹中获得的结果具有普遍重要性。
