Murakami M, Kudo I, Inoue K
Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Adv Exp Med Biol. 1992;318:27-34. doi: 10.1007/978-1-4615-3426-6_3.
Phospholipase A2 activity in lysates of mast cells and their related cells [mouse bone marrow-derived IL-3 dependent mast cells (BMMC), rat connective tissue mast cells (CTMC), and rat mastocytoma RBL-2H3 cells] was measured using phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC) as exogenous substrates. Both BMMC and RBL cells showed rather high phospholipase A2 activity, whereas CTMC showed only weak activity. These cells contained at least three types of phospholipase A2. Type 1 enzyme showed no appreciable affinity to heparin, and preferentially hydrolyzed either PC or PE, both of which have an arachidonic acid at the sn-2 position. The activity was absorbed by monoclonal antibody against rabbit platelet cytosolic 85-kDa phospholipase A2. Type 2 enzyme had an affinity to heparin, and was completely inhibited by anti-rat platelet 14-kDa secretory phospholipase A2. This enzyme could be expressed as an "ecto-type" enzyme on the cell surface and might be secreted from cells when mast cells are activated. Type 3 enzyme also had an affinity to heparin, but was separated from type 2 enzyme on reverse-phase HPLC. This enzyme did not interact with anti-14-kDa secretory enzyme antibody. Purified type 3 enzyme (30-kDa) specifically hydrolyzed PS. p-Bromophenacylbromide inhibited all types of phospholipase A2, whereas mepacrine inhibited type 2 and type 3 enzymes, but not type 1 enzyme. Type 2 enzyme was also inhibited by the specific antibody, complement degradation product, and a small-molecular-weight inhibitor. Histamine release was inhibited by all these inhibitors, whereas PGD2 production was inhibited only by p-bromophenacylbromide. Possible roles for these phospholipases A2 in mast cell function are proposed.
使用磷脂酰乙醇胺（PE）、磷脂酰丝氨酸（PS）和磷脂酰胆碱（PC）作为外源性底物，测定肥大细胞及其相关细胞（小鼠骨髓来源的白细胞介素3依赖性肥大细胞（BMMC）、大鼠结缔组织肥大细胞（CTMC）和大鼠肥大细胞瘤RBL - 2H3细胞）裂解物中的磷脂酶A2活性。BMMC和RBL细胞均表现出相当高的磷脂酶A2活性，而CTMC仅表现出较弱的活性。这些细胞含有至少三种类型的磷脂酶A2。1型酶对肝素没有明显亲和力，优先水解在sn - 2位含有花生四烯酸的PC或PE。该活性被抗兔血小板胞质85 kDa磷脂酶A2的单克隆抗体吸收。2型酶对肝素有亲和力，并被抗大鼠血小板14 kDa分泌型磷脂酶A2完全抑制。这种酶可以在细胞表面表达为“外切型”酶，并且在肥大细胞被激活时可能从细胞中分泌出来。3型酶也对肝素有亲和力，但在反相高效液相色谱上与2型酶分离。该酶不与抗14 kDa分泌型酶抗体相互作用。纯化的3型酶（30 kDa）特异性水解PS。对溴苯甲酰溴抑制所有类型的磷脂酶A2，而米帕林抑制2型和3型酶，但不抑制1型酶。2型酶也被特异性抗体、补体降解产物和小分子抑制剂抑制。组胺释放被所有这些抑制剂抑制，而前列腺素D2的产生仅被对溴苯甲酰溴抑制。提出了这些磷脂酶A2在肥大细胞功能中的可能作用。
