Smith D M, Waite M
Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina.
J Leukoc Biol. 1992 Dec;52(6):670-8. doi: 10.1002/jlb.52.6.670.
We describe here and partially characterize a Ca(2+)-independent phospholipase A2 that acts on phosphatidylinositol in normal human peripheral blood neutrophils. Neutrophils incubated with myo-[3H]inositol to form [3H]phosphatidylinositol and then stimulated with the calcium ionophore A23187 produced [3H]lysophosphatidylinositol. This deacylation was further characterized in cell sonicates by the specific release of [3H]arachidonic acid from exogenous [1-14C]stearoyl-2-[3H]arachidonyl-phosphatidylinositol. This phospholipase A2 is Ca2+ independent, retaining full activity in the presence of 10 mM EDTA, and is optimally active at alkaline pH (pH 9). A phosphatidylinositol-hydrolyzing phospholipase C activity was characterized by the production of [3H]-/[14C]-diglycerides. This phospholipase C activity is dependent on the presence of exogenous Ca2+ and is optimally active at neutral pH (pH 7.5). The lipoxygenase/cyclooxygenase inhibitors eicosatetraenoic acid and nordihydroguaiaretic acid and the calmodulin antagonist trifluoperazine were the only compounds tested that showed significant inhibition of phospholipase A2 activity. However, none of these phosphatidylinositol-hydrolyzing phospholipase A2 inhibitory compounds resulted in the accumulation of any radiolabeled diglyceride, monoglyceride, or phosphatidic acid intermediates. Following subcellular fractionation on sucrose density gradients, it was found that the plasma membrane-enriched fractions contained the highest specific activity for phospholipase A2; however, the cytosolic fraction contained a large part of the total phospholipase A2 activity. Furthermore, when neutrophils were first exposed to several agents, including lipopolysaccharide, phorbol myristate acetate, or N-formyl-methionyl-leucyl- phenylalanine, and then subfractionated, there was a significant translocation of the enzyme activity from the cytosolic fraction to the membrane-enriched fractions. These data suggest that this Ca(2+)-independent, phosphatidylinositol-hydrolyzing phospholipase A2 may play an important role in early cell activation, providing free arachidonic acid for subsequent metabolism into biologically active eicosanoids.
我们在此描述并部分表征了一种不依赖钙离子的磷脂酶A2，它作用于正常人外周血中性粒细胞中的磷脂酰肌醇。用肌醇-[3H]孵育中性粒细胞以形成[3H]磷脂酰肌醇，然后用钙离子载体A23187刺激，产生了[3H]溶血磷脂酰肌醇。通过从外源性[1-14C]硬脂酰-2-[3H]花生四烯酰-磷脂酰肌醇中特异性释放[3H]花生四烯酸，在细胞超声裂解物中对这种脱酰基作用进行了进一步表征。这种磷脂酶A2不依赖钙离子，在10 mM EDTA存在下仍保持全部活性，并且在碱性pH（pH 9）下活性最佳。一种水解磷脂酰肌醇的磷脂酶C活性通过产生[3H]-/[14C]-甘油二酯来表征。这种磷脂酶C活性依赖于外源性钙离子的存在，并且在中性pH（pH 7.5）下活性最佳。脂氧合酶/环氧化酶抑制剂二十碳四烯酸和去甲二氢愈创木酸以及钙调蛋白拮抗剂三氟拉嗪是所测试的仅有的显示出对磷脂酶A2活性有显著抑制作用的化合物。然而，这些水解磷脂酰肌醇的磷脂酶A2抑制性化合物均未导致任何放射性标记的甘油二酯、甘油单酯或磷脂酸中间体的积累。在蔗糖密度梯度上进行亚细胞分级分离后，发现富含质膜的级分中磷脂酶A2的比活性最高；然而，胞质级分中含有大部分的总磷脂酶A2活性。此外，当中性粒细胞首先暴露于几种试剂，包括脂多糖、佛波酯或N-甲酰甲硫氨酰亮氨酰苯丙氨酸，然后进行亚分级分离时，酶活性从胞质级分向富含膜的级分有显著的转位。这些数据表明，这种不依赖钙离子、水解磷脂酰肌醇的磷脂酶A2可能在早期细胞活化中起重要作用，为随后代谢为生物活性类花生酸提供游离花生四烯酸。
