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首页> 美国卫生研究院文献>Biochemical Journal >Purification of a 100 kDa phospholipase A2 from spleen lung and kidney: antiserum raised to pig spleen phospholipase A2 recognizes a similar form in bovine lung kidney and platelets and immunoprecipitates phospholipase A2 activity.
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Purification of a 100 kDa phospholipase A2 from spleen lung and kidney: antiserum raised to pig spleen phospholipase A2 recognizes a similar form in bovine lung kidney and platelets and immunoprecipitates phospholipase A2 activity.

机译：从脾肺和肾中纯化100 kDa磷脂酶A2：针对猪脾磷脂酶A2产生的抗血清在牛肺肾和血小板中识别出相似的形式并免疫沉淀磷脂酶A2活性。

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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.
机译：磷脂酶A2（PLA2）在细胞内和细胞外化学介质（如花生四烯酸，类花生酸和血小板活化因子）的产生中起关键作用，它们调节膜通道活性，信号转导，具有血管活性和趋化性，并涉及许多病理生理学炎症和组织损伤的机制。我们先前从牛血小板和大鼠肾脏中鉴定，纯化和鉴定了花生四烯酸选择性胞质100-110 kDa PLA2，并在细胞刺激过程中被激活。先前发表的纯化方案导致酶的产量低，不足以进行广泛的生化表征。我们报告使用新型大规模纯化方案从猪脾，牛肾和牛肺中纯化大分子（100 kDa）PLA2。通过依次使用DEAE-纤维素阴离子交换，丁基-Toyopearl疏水色谱法和DEAE-5PW h.p.c.从4.8公斤猪脾中获得的酸化提取物中将酶纯化至接近均一，然后通过非变性PAGE进一步纯化。该纯化方案将允许制备足以研究其性质和调节的纯化的天然酶。为了产生针对PLA2酶的抗血清，在DEAE-5PW h.p.c.之后，从活性级分的SDS / PAGE凝胶中切出100kDa的蛋白质并对其进行电洗脱，并将其用作抗原。这种针对猪脾的100 kDa PLA2蛋白多克隆抗体与从牛血小板，肾脏和肺以及猪脾中部分纯化的制剂中的100 kDa条带反应，并通过这些来源免疫沉淀了PLA2活性。该抗体还从培养的肾小球膜细胞，人红白血病细胞和人单核U937细胞的胞质级分中免疫沉淀出100 kDa的蛋白质。免疫沉淀物中存在相当大的PLA2活性。据我们所知，该抗体在允许测量免疫沉淀本身中PLA2活性方面具有独特的能力，并且将是研究天然PLA2酶的调控和激活机制的有用工具。

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