Biochemical Studies on Platelet Phospholipase A_2

(1)超声法制备兔血小板可溶性组分，经肝素-Sepharose柱层析，检测肝素结合和非结合组分中磷脂酶A2活性。在肝素结合部分检测到的活性似乎属于分泌型14-KDa II组磷脂酶A2，因为它与抗人14-KDa II组磷脂酶A2单克隆抗体结合。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。当氮空化法轻微破坏血小板时，肝素非结合磷脂酶A2主要在血小板胞浆部分被回收。肝素非结合部分水解在sn-2位置具有花生四烯醇基残基的磷脂比具有亚麻油基残基的磷脂更有效。因此，兔血小板含有两种磷脂酶A2；一种是分泌14- kDa II型磷脂酶A2的颗粒，另一种是偏爱花生四烯酰基残基的细胞降解磷脂酶A2。(2)兔和人血小板中肝素非结合磷脂酶A2的活性纯化接近同源性。它们的分子量分别约为88 kDa和90 kDa。纯化酶表现出对脂肪酸的偏好；它水解sn-2位含有花生四烯酰基残基的磷脂比含有亚麻油基残基的磷脂更有效。在10^<-7> ~ 10^<-6>钙离子存在时，纯化酶对磷脂酰胆碱或磷脂酰乙醇胺的催化活性急剧提高。这些特征与那些不同。因此，这些高分子量酶被归类为一种新型磷脂酶A2，并可能参与兔血小板中花生四烯醇残基的刺激依赖性释放。(3)制备兔血小板88-kDa磷脂酶A2单克隆抗体。这些抗体与兔肺、肝和脑中检测到的磷脂酶A2活性反应，表明存在具有相似特征的酶。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