Fischer T H, White G C
Department of Medicine, University of North Carolina, Chapel Hill 27599.
Biochem Biophys Res Commun. 1989 Mar 15;159(2):644-50. doi: 10.1016/0006-291x(89)90043-0.
Inhibition of platelet function by cAMP is due at least in part to a reduction in the agonist stimulated increase in cytoplasmic calcium during cell activation. This inhibition is also associated with cAMP-dependent phosphorylation of thrombolamban, a 22 kDa phosphoprotein which is present in the same membrane fraction as the calcium-dependent ATPase. Phosphorylation of this protein has been correlated with increased uptake of calcium by microsomal membranes. The present study was undertaken to examine the interaction of thrombolamban with the Ca++-ATPase in order to assess the possibility that the increased calcium uptake was by a direct effect of thrombolamban on Ca++-ATPase activity or that thrombolamban was a component of the Ca++-ATPase. Several approaches were utilized to assess the interaction of thrombolamban with the microsomal Ca++-ATPase. Gel filtration of labeled microsomes solubilized under non-denaturing conditions showed a major peak of radioactivity (Kav 0.64) corresponding to thrombolamban which was well separated from the Ca++-ATPase activity (Kav 0.09). Chemical cross-linking studies using partially purified thrombolamban and intact microsomes showed incorporation of the phosphoprotein into a 147,000 dalton complex. Indirect immunostaining with an anti-Ca++-ATPase antibody failed to demonstrate the Ca++-ATPase in the 147,000 dalton complex. Recombination of the phosphorylated thrombolamban with the Ca++-ATPase had no effect on Ca++-ATPase activity. These results indicate that, under the conditions used in these experiments, there was no apparent interaction between thrombolamban and the microsomal Ca++-ATPase. We conclude that thrombolamban is covalently bound to the Ca++-ATPase.
环磷酸腺苷（cAMP）对血小板功能的抑制作用至少部分归因于细胞活化过程中激动剂刺激引起的细胞质钙增加的减少。这种抑制作用还与血栓调节蛋白（一种22 kDa的磷蛋白）的cAMP依赖性磷酸化有关，该蛋白与钙依赖性ATP酶存在于同一膜组分中。该蛋白的磷酸化与微粒体膜对钙的摄取增加相关。本研究旨在研究血栓调节蛋白与Ca++ -ATP酶的相互作用，以评估钙摄取增加是由于血栓调节蛋白对Ca++ -ATP酶活性的直接作用，还是血栓调节蛋白是Ca++ -ATP酶的一个组成部分。采用了几种方法来评估血栓调节蛋白与微粒体Ca++ -ATP酶的相互作用。在非变性条件下溶解的标记微粒体的凝胶过滤显示，对应于血栓调节蛋白的主要放射性峰（洗脱体积0.64）与Ca++ -ATP酶活性（洗脱体积0.09）分离良好。使用部分纯化的血栓调节蛋白和完整微粒体的化学交联研究表明，磷蛋白掺入了一个147,000道尔顿的复合物中。用抗Ca++ -ATP酶抗体进行间接免疫染色未能在147,000道尔顿的复合物中检测到Ca++ -ATP酶。磷酸化的血栓调节蛋白与Ca++ -ATP酶的重组对Ca++ -ATP酶活性没有影响。这些结果表明，在这些实验所用的条件下，血栓调节蛋白与微粒体Ca++ -ATP酶之间没有明显的相互作用。我们得出结论，血栓调节蛋白与Ca++ -ATP酶共价结合。
