Muszbek L, Laposata M
Department of Pathology and Laboratory Medicine, University of Pennsylvania, School of Medicine, Philadelphia 19104.
Blood. 1989 Sep;74(4):1339-47.
Covalent attachment of fatty acid to proteins plays an important role in association of certain proteins with hydrophobic membrane structures. In platelets, the structure of many membrane glycoproteins (GPs) has been examined in detail, but the question of fatty acid acylation of platelet proteins has not been addressed. In this study, we wished to determine (a) whether platelet proteins could be fatty acid acylated; and, if so, (b) whether these modified proteins were present in isolated platelet membranes and cytoskeletal fractions; and (c) if the pattern of fatty acid acylated proteins changed on stimulation of the platelets with the agonist thrombin. We observed that in platelets allowed to incorporate 3H-palmitate, a small percentage (1.37%) of radioactivity incorporated into the cells became covalently bound to protein. Selective cleavage of thioester, thioester plus O-ester, and amide-linked 3H-fatty acids from proteins, and their subsequent analysis by high-performance liquid chromatography (HPLC) indicated that the greatest part of 3H-fatty acid covalently bound to protein was thioester-linked 3H-palmitate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography, at least ten major radiolabeled proteins were detected. Activation of platelets by thrombin greatly increased the quantity of 3H-palmitoylated proteins associated with the cytoskeleton. Nearly all radiolabeled proteins were recovered in the membrane fraction, indicating that these proteins are either integral or peripheral membrane proteins or proteins tightly associated to membrane constituents. Components of the GPIIb-IIIa complex were not palmitoylated. Thus, platelet proteins are significantly modified posttranslationally by 3H-palmitate, and incorporation of palmitoylated proteins into the cytoskeleton is a prominent component of the platelet response to thrombin stimulation.
脂肪酸与蛋白质的共价连接在某些蛋白质与疏水膜结构的结合中起着重要作用。在血小板中，许多膜糖蛋白（GPs）的结构已被详细研究，但血小板蛋白的脂肪酸酰化问题尚未得到解决。在本研究中，我们希望确定：（a）血小板蛋白是否可以被脂肪酸酰化；如果可以，（b）这些修饰后的蛋白是否存在于分离的血小板膜和细胞骨架组分中；以及（c）在用激动剂凝血酶刺激血小板后，脂肪酸酰化蛋白的模式是否发生变化。我们观察到，在允许掺入3H-棕榈酸的血小板中，掺入细胞的放射性的一小部分（1.37%）与蛋白质共价结合。从蛋白质中选择性切割硫酯、硫酯加O-酯以及酰胺连接的3H-脂肪酸，并随后通过高效液相色谱（HPLC）进行分析，结果表明与蛋白质共价结合的3H-脂肪酸的最大部分是硫酯连接的3H-棕榈酸。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和荧光自显影，检测到至少十种主要的放射性标记蛋白。凝血酶激活血小板大大增加了与细胞骨架相关的3H-棕榈酰化蛋白的数量。几乎所有放射性标记蛋白都在膜组分中回收，这表明这些蛋白要么是整合膜蛋白或外周膜蛋白，要么是与膜成分紧密相关的蛋白。GPIIb-IIIa复合物的组分未被棕榈酰化。因此，血小板蛋白在翻译后被3H-棕榈酸显著修饰，并且棕榈酰化蛋白掺入细胞骨架是血小板对凝血酶刺激反应的一个突出组成部分。
