Kim D K, Bonventre J V
Department of Life Science, Pohang Institute of Science and Technology, Korea.
Biochem J. 1993 Aug 15;294 ( Pt 1)(Pt 1):261-70. doi: 10.1042/bj2940261.
Phospholipase A2 (PLA2) plays a key role in the production of intracellular and extracellular chemical mediators such as arachidonic acid, eicosanoids and platelet-activating factor, which modulate membrane channel activity, signal transduction, are vasoactive and chemotactic, and are implicated in many pathophysiological mechanisms of inflammation and tissue injury. We previously identified, purified and characterized an arachidonic acid-selective cytosolic 100-110 kDa PLA2 from bovine platelets and rat kidney that is activated during cell stimulation. The purification schemes previously published resulted in low yields of enzyme, insufficient for extensive biochemical characterization. We report the purification of a large-molecular-mass (100 kDa) PLA2 from pig spleen, bovine kidney and bovine lung, using a novel large-scale purification scheme. The enzyme was purified to near homogeneity from an acidified extract obtained from 4.8 kg of pig spleen by sequential use of DEAE-cellulose anionic exchange, Butyl-Toyopearl hydrophobic chromatography and DEAE-5PW h.p.l.c., and further purified by non-denaturing PAGE. This purification scheme will permit the preparation of quantities of purified native enzyme sufficient to study its properties and regulation. To generate antiserum against the PLA2 enzyme, the 100 kDa protein was excised and electroeluted from SDS/PAGE gels of the active fractions after DEAE-5PW h.p.l.c., and this was used as antigen. This polyclonal antibody against pig spleen 100 kDa PLA2 protein reacted with 100 kDa bands in preparations partially purified from bovine platelets, kidney and lung as well as pig spleen, and immunoprecipitated PLA2 activity from these sources. The antibody also immunoprecipitated a 100 kDa protein from cytosolic fractions of cultured renal mesangial cells, human erythroleukaemia cells and human monocytic U937 cells. Considerable PLA2 activity was present in the immunoprecipitates. To our knowledge this antibody is unique in its ability to permit measurement of PLA2 activity in the immunoprecipitate itself, and will be a useful tool for the study of the regulation and the activation mechanisms of the native PLA2 enzyme.
磷脂酶A2（PLA2）在细胞内和细胞外化学介质的产生中起关键作用，如花生四烯酸、类二十烷酸和血小板活化因子，这些介质可调节膜通道活性、信号转导，具有血管活性和趋化性，并参与炎症和组织损伤的许多病理生理机制。我们之前从牛血小板和大鼠肾脏中鉴定、纯化并表征了一种花生四烯酸选择性胞质100 - 110 kDa的PLA2，其在细胞刺激过程中被激活。先前发表的纯化方案酶产量低，不足以进行广泛的生化表征。我们报告了使用一种新型大规模纯化方案从猪脾脏、牛肾脏和牛肺中纯化出一种大分子质量（100 kDa）的PLA2。通过依次使用DEAE - 纤维素阴离子交换、丁基 - Toyopearl疏水色谱和DEAE - 5PW高效液相色谱，从4.8千克猪脾脏获得的酸化提取物中，将该酶纯化至接近均一性，然后通过非变性聚丙烯酰胺凝胶电泳进一步纯化。这种纯化方案将允许制备足够数量的纯化天然酶，以研究其性质和调节。为了产生针对PLA2酶的抗血清，从DEAE - 5PW高效液相色谱后活性组分的SDS / PAGE凝胶中切下100 kDa蛋白质并进行电洗脱，并将其用作抗原。这种针对猪脾脏100 kDa PLA2蛋白的多克隆抗体与从牛血小板、肾脏和肺以及猪脾脏部分纯化的制剂中的100 kDa条带发生反应，并从这些来源免疫沉淀PLA2活性。该抗体还从培养的肾系膜细胞、人红白血病细胞和人单核细胞U937细胞的胞质组分中免疫沉淀出一种100 kDa蛋白质。免疫沉淀物中存在相当数量的PLA2活性。据我们所知，这种抗体在允许测量免疫沉淀物本身中的PLA2活性方面具有独特能力，并且将成为研究天然PLA2酶的调节和激活机制的有用工具。
