Biochemical Studies on Platelet Phospholipase A_2

(1)用超声法制备兔血小板可溶组分，经肝素-琼脂糖柱层析后，在肝素结合组分和非结合组分中均检测到磷脂酶A2活性。在肝素结合组分中检测到的活性似乎属于分泌型14-KDa II型磷脂酶A2，因为它与抗人14-KDa II型磷脂酶A2单抗结合。在肝素非结合部分中发现的活性与相同的抗体没有明显的反应。当用氮空化法轻微破坏血小板时，肝素非结合磷脂酶A2主要恢复到血小板胞浆部分。肝素非结合组分比含有亚油酰基残基的磷脂更有效地水解sn-2位上含有花生四烯酰基残基的磷脂。因此，兔血小板含有两种磷脂酶A2：一种是颗粒…中分泌型14-kDa II型磷脂酶A2一种是胞质磷脂酶A2，偏爱花生四烯酰基残基。(2)从兔和人血小板中纯化的非肝素结合型磷脂酶A2活性接近均一。它们的相对分子质量分别为88 kDa和90 kDa。纯化的酶表现出对脂肪酸的选择性；它比含有亚油酰残基的sn-2位含有花生四烯基残基的磷脂更有效地水解酶。纯化的酶与磷脂酰胆碱或磷脂酰乙醇胺的催化活性在10^~(-7)~10^~(-6)~10~(-6)钙离子存在下急剧增加。这些特点与那些不同。因此，这些高相对分子质量的酶被归类为一种新的磷脂酶A2，可能参与刺激依赖的花生四烯酸残基在兔血小板中的释放。(3)制备了兔血小板88-kDa磷脂酶A2的单抗。这些抗体与在兔肺、肝和脑中检测到的磷脂酶A2活性发生反应，表明存在具有相似特性的酶。较少

(1) When a rabbit platelet soluble fraction was prepared by sonication and subjected to heparin--Sepharose column chromatography, phospholipase A2 activity was detected in both heparin-binding and non-binding fractions. The activity detected in the heparin-binding fraction appeared to belong to the secretory 14-KDa group II phospholipase A2, because it bound to anti- human 14-KDa group II phospholipase A2 monoclonal antibody. The activity found in the heparin- non-binding fraction did not appreciably react with the same antibody. When platelets were gently disrupted by the nitrogen cavitation method, the heparin-non-binding phospholipase A2 was mainly recovered in the platelet cytosolic fraction. The heparin-non-binding fraction hydrolyzed a phospholipid bearing an arachidonoyl residue at the sn-2 position more effectively than one with a linoleoyl residue. Thus, rabbit platelets contain two kinds of phospholipase A2 ; one is the secretory 14--kDa group II phospholipase A2 in the granu … More le fraction and the other is a cytoslic phospholipase A2 with a preference for arachidonoyl residue.(2) The heparin-non-binding phospholipase A2 activities were purified to near homogeneity from rabbit and human platelets. Their molecular weights were about 88 kDa and 90 kDa, respectively. The purified enzymes exhibited a fatty acid preference ; it hydrolyzed phospholipid bearing an arachidonoyl residue at the sn-2 position more effectively than that with a linoleoyl residue. The catalytic activity of the purified enzyme with phosphatidylcholine or phosphatidylethanolamine increased sharply in the presence of between 10^<-7> and 10^<-6> calcium ion. These characteristics differ from those. Therefore, these high-molecular weight enzymes are classified to a novel phospholipase A2 and may participate in the stimulus-dependent release of arachidonoyl residues in rabbit platelets.(3) Monoclonal antibodies were raised against rabbit platelet 88-kDa phospholipase A2. These antibodies reacted with phospholipase A2 activities detected in rabbit lung, liver and brain, indicating the exisetence of enzymes with similar characteristics. Less