Bork T, Mrsny R J
Department of Biology, University of Oregon, Eugene 97403.
Exp Cell Res. 1993 Sep;208(1):189-96. doi: 10.1006/excr.1993.1237.
Na,K-ATPase is an integral membrane protein complex involved in the regulation of intracellular Na+ and K+ ion levels. The number of accessible Na,K-ATPase complexes on the surface of human platelets before and after activation by adenosine 5'-diphosphate (ADP) was quantitated using ouabain, a highly specific inhibitor of this enzyme. Studies with [3H]ouabain revealed an increase in the number of accessible ouabain binding sites from approximately 55 per resting platelet to approximately 625 per ADP-activated platelet. Ultrastructural localization of this ion transport complex was also studied in resting human platelets or following activation by ADP. Localization was performed using a novel colloidal gold affinity probe which employed ouabain (ouabain-GAP) as a ligand specific for the Na,K-ATPase. Qualitatively, the number of surface-associated ouabain-GAP labels per platelet was observed to increase following activation by ADP. Fracture-labeling studies of frozen preparations of resting and freshly activated platelets suggested ouabain binding sites were associated with intracellular platelet structures during the early stages of activation. Together these results suggest an increase in the amount of ouabain binding sites at the surface of human platelets as an event of activation and that at least some of these additional sites may originate from intracellular sites.
钠钾-ATP酶是一种参与调节细胞内钠和钾离子水平的整合膜蛋白复合物。使用哇巴因（该酶的一种高度特异性抑制剂）对人血小板在被5'-二磷酸腺苷（ADP）激活前后表面可及的钠钾-ATP酶复合物数量进行了定量。用[3H]哇巴因进行的研究表明，可及的哇巴因结合位点数量从每个静息血小板约55个增加到每个ADP激活的血小板约625个。还对静息人血小板或ADP激活后的该离子转运复合物进行了超微结构定位研究。使用一种新型胶体金亲和探针进行定位，该探针采用哇巴因（哇巴因-金标蛋白A探针）作为钠钾-ATP酶的特异性配体。定性地观察到，每个血小板表面相关的哇巴因-金标蛋白A探针标记数量在ADP激活后增加。对静息和刚激活的血小板冷冻制剂进行的断裂标记研究表明，在激活早期，哇巴因结合位点与血小板细胞内结构相关。这些结果共同表明，人血小板表面哇巴因结合位点数量的增加是激活过程中的一个事件，并且这些额外位点中至少有一些可能源自细胞内位点。
