Fischer T H, White G C
Department of Medicine, University of North Carolina, Chapel Hill 27514.
Biochem Biophys Res Commun. 1987 Dec 16;149(2):700-6. doi: 10.1016/0006-291x(87)90424-4.
In preparations of human platelet microsomes, cyclic AMP-dependent protein kinase induced the rapid phosphorylation of a single protein that was electrophoretically identical to the 22,000 dalton protein (P22) phosphorylated by cAMP in intact platelets. Phosphorylation of the microsomal protein was maximal at one minute and was followed by slow dephosphorylation. Although the protein was associated with a microsomal fraction, it could be separated from the membrane by 2 M NaCl indicating that it was a peripheral protein. Molecular weight was estimated by NaDodSO4-PAGE and by gel filtration chromatography. The molecular weight estimated by NaDodSO4-PAGE was 22,400 daltons and was somewhat larger than the 16,000 molecular weight estimated by gel filtration in the presence of NaDodSO4. In the absence of NaDodSO4, the protein chromatographed as a 36,000 dalton form. The presence of the 36,000 dalton form was not dependent on the phosphorylation state of the protein. The partially purified protein contained phosphoserine, but no phosphothreonine or phosphotyrosine. Two dimensional NaDodSO4-PAGE and isoelectric focusing of the phosphorylated protein revealed isomers with pl values of 5.9 and 6.3. These studies indicate that the 22 kDa microsomal protein and P22 in intact platelets are the same protein and that the 22 kDa protein is tightly bound to the microsomal membrane although the nature of this binding and the microsomal component(s) to which it is bound remain to be determined. We conclude that the 22 kDa protein in platelet microsomes is structurally distinct from, but functionally similar to, phospholamban, the cAMP-dependent protein kinase substrate in muscle, and may play a similar role in calcium transport. Based on this similarity, it is proposed that the 22 kDa protein in platelets be called thrombolamban.
在人血小板微粒体制备物中，环磷酸腺苷（cAMP）依赖性蛋白激酶可诱导一种单一蛋白质的快速磷酸化，该蛋白质在电泳上与完整血小板中被cAMP磷酸化的22,000道尔顿蛋白质（P22）相同。微粒体蛋白质的磷酸化在1分钟时达到最大值，随后缓慢去磷酸化。尽管该蛋白质与微粒体部分相关，但它可以通过2M氯化钠与膜分离，表明它是一种外周蛋白。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（NaDodSO4-PAGE）和凝胶过滤色谱法估计分子量。通过NaDodSO4-PAGE估计的分子量为22,400道尔顿，略大于在存在NaDodSO4的情况下通过凝胶过滤估计的16,000分子量。在不存在NaDodSO4的情况下，该蛋白质以36,000道尔顿的形式进行色谱分离。36,000道尔顿形式的存在不依赖于蛋白质的磷酸化状态。部分纯化的蛋白质含有磷酸丝氨酸，但不含磷酸苏氨酸或磷酸酪氨酸。磷酸化蛋白质的二维NaDodSO4-PAGE和等电聚焦显示等电点（pI）值为5.9和6.3的异构体。这些研究表明，完整血小板中的22kDa微粒体蛋白质和P22是同一种蛋白质，并且22kDa蛋白质紧密结合于微粒体膜，尽管这种结合的性质以及它所结合的微粒体成分仍有待确定。我们得出结论，血小板微粒体中的22kDa蛋白质在结构上与肌肉中cAMP依赖性蛋白激酶底物受磷蛋白不同，但在功能上相似，并且可能在钙转运中发挥类似作用。基于这种相似性，建议将血小板中的22kDa蛋白质称为血栓受磷蛋白。
