Beurling-Harbury C, Harbury P B
Department of Medicine, University of Illinois, College of Medicine, Chicago 60680.
Thromb Haemost. 1990 Apr 12;63(2):286-90.
Actin is the major ATP and ADP binding protein in platelets, 0.9-1.3 nmol/10(8) cells, 50-70% in the unpolymerized state. The goal of these experiments was to develop a method for extracting all protein-bound ATP and ADP from undisturbed platelets in plasma. Extraction of actin-bound ADP is routine while extraction of actin-bound ATP from platelets in buffer has been unsuccessful. Prior to extraction the platelets were exposed to 14-C adenine, to label the metabolic and actin pools of ATP and ADP. The specific activity was determined from the actin-bound ADP in the 43% ethanol precipitate. Sequential ethanol and perchlorate extractions of platelet rich plasma, and the derived supernatants and precipitates were performed. ATP concentrations were determined with the luciferase assay, and radioactive nucleotides separated by TLC. A total of 1.18 nmol/10(8) cells of protein-bound ATP and ADP was recovered, 52% ATP (0.61 nmol). The recovery of protein-bound ADP was increased from 0.3 to 0.57 nmol/10(8) cells. This approach for the first time successfully recovered protein bound ATP and ADP from platelets in a concentration expected for actin.
肌动蛋白是血小板中主要的ATP和ADP结合蛋白，含量为0.9 - 1.3 nmol/10⁸个细胞，其中50 - 70%处于未聚合状态。这些实验的目的是开发一种从血浆中未受干扰的血小板中提取所有与蛋白质结合的ATP和ADP的方法。提取与肌动蛋白结合的ADP是常规操作，但从缓冲液中的血小板中提取与肌动蛋白结合的ATP一直未成功。在提取之前，将血小板暴露于¹⁴C腺嘌呤，以标记ATP和ADP的代谢池和肌动蛋白池。从43%乙醇沉淀物中与肌动蛋白结合的ADP测定比活性。对富含血小板的血浆进行乙醇和高氯酸盐的顺序提取，并对所得上清液和沉淀物进行处理。用荧光素酶测定法测定ATP浓度，并用薄层层析法分离放射性核苷酸。共回收了1.18 nmol/10⁸个细胞的与蛋白质结合的ATP和ADP，其中ATP占52%（0.61 nmol）。与蛋白质结合的ADP的回收率从0.3 nmol/10⁸个细胞提高到了0.57 nmol/10⁸个细胞。这种方法首次成功地从血小板中回收了与蛋白质结合的ATP和ADP，其浓度符合肌动蛋白的预期浓度。
