McCord M, Chabot-Fletcher M, Breton J, Marshall L A
Department of Inflammation and Respiratory Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
J Invest Dermatol. 1994 Jun;102(6):980-6. doi: 10.1111/1523-1747.ep12384234.
The phospholipase A2 (PLA2) activities that are localized in the keratinocyte cytosolic and microsomal fractions were biochemically and pharmacologically characterized. The cytosol and to a lesser extent the microsome were sensitive to heat treatment and stable in the presence of sulfhydryl reducing agents. Both fractions were almost totally inactivated by reduction of pH to 2. The cytosolic activity demonstrated a sevenfold preference for arachidonic acid over oleic acid in the sn-2 position of substrate phospholipid and the microsome exhibited a fourfold preference. Neither the cytosol nor the microsome was inactivated by a neutralizing mouse monoclonal antibody 3F10 generated against recombinant human (rh) type II 14-kDa PLA2. Western immunoblot analysis of both fractions identified a high-molecular-mass protein in keratinocyte cytosol but not the microsome that migrated with rh 85-kDa PLA2. Neither the cytosol nor the microsome possessed immunoreactive bands that migrated with rh type II 14-kDa PLA2 when probed with monoclonal antibody 3F10. Further analysis of the cytosolic activity showed that it was activated by submicromolar concentrations of Ca2+, reduced by arachidonyl trifloromethylketone, a selective 85-kDa PLA2 inhibitor, but was unaffected by C-7 phosphonate phospholipid, a selective 14-kDa PLA2 transition state inhibitor. Taken together, the data supports the existence of a PLA2 activity in the cytosol that displays characteristics that are indistinguishable from those exhibited by the 85-kDa PLA2. Alternatively, both the cytosol and microsome were devoid of type II 14-kDa-like PLA2 activity. The failure of 12-epi scalaradial, a 14-kDa PLA2 inhibitor, to modify A23187-stimulated keratinocyte prostaglandin E2 release, was consistent with the biochemistry and suggests that the 85-kDa PLA2 may play an important role in keratinocyte prostaglandin E2 formation.
对定位于角质形成细胞胞质溶胶和微粒体部分的磷脂酶A2（PLA2）活性进行了生化和药理学特性分析。胞质溶胶以及程度稍轻的微粒体对热处理敏感，在巯基还原剂存在的情况下稳定。将pH降至2时，两个部分几乎完全失活。胞质溶胶活性在底物磷脂的sn-2位置对花生四烯酸的偏好是油酸的七倍，微粒体则表现出四倍的偏好。针对重组人（rh）II型14-kDa PLA2产生的中和性小鼠单克隆抗体3F10，既不会使胞质溶胶也不会使微粒体失活。对两个部分的蛋白质免疫印迹分析表明，角质形成细胞胞质溶胶中有一种高分子量蛋白质，但微粒体中没有，该蛋白质与rh 85-kDa PLA2迁移情况相同。当用单克隆抗体3F10探测时，胞质溶胶和微粒体均没有与rh II型14-kDa PLA2迁移情况相同的免疫反应条带。对胞质溶胶活性的进一步分析表明，它被亚微摩尔浓度的Ca2+激活，被选择性85-kDa PLA2抑制剂花生四烯酰三氟甲基酮抑制，但不受选择性14-kDa PLA2过渡态抑制剂C-7膦酸磷脂的影响。综上所述，数据支持胞质溶胶中存在一种PLA2活性，其表现出的特性与85-kDa PLA2无法区分。或者说，胞质溶胶和微粒体均缺乏II型14-kDa样PLA2活性。14-kDa PLA2抑制剂12-表斯卡拉瑞尔未能改变A23187刺激的角质形成细胞前列腺素E2释放，这与生化分析结果一致，表明85-kDa PLA2可能在角质形成细胞前列腺素E2形成中起重要作用。
