Horne W C, Miettinen H, Marchesi V T
Department of Pathology, Yale University School of Medicine, New Haven, CT 06510.
Biochim Biophys Acta. 1988 Oct 6;944(2):135-43. doi: 10.1016/0005-2736(88)90426-9.
Human erythrocyte membrane band 4.9 is phosphorylated by several erythrocyte protein kinases. Chromatography of erythrocyte membrane skeleton proteins on DEAE-Sephacel produces two proteins with relative mobilities, on gel electrophoresis, similar to that of band 4.9. The first, with a molecular mass of 49 kDa, is quite basic (pI greater than 8) while the second, 50.5 kDa, is slightly acidic (pI = 6.2). Comparative two-dimensional peptide mapping reveals that both proteins are present in band 4.9 on one-dimensional gels of total erythrocyte membrane proteins and membrane skeleton proteins. The 49 kDa protein, but not the 50.5 kDa protein, binds to actin filaments in a sedimentation assay. In intact erythrocytes metabolically labeled with [32P]orthophosphate, the 49 kDa protein is phosphorylated by protein kinase C, cAMP-dependent protein kinase, and protein kinases which are active in the absence of exogenous kinase activators. In contrast, the 50.5 kDa protein is phosphorylated by protein kinase C but not by the other protein kinases examined. Finally, two-dimensional peptide mapping was employed to compare the 49 kDa protein and a 57 kDa protein which copurifies with, and has many characteristics of, the 49 kDa protein. Significant similarities were found in both 125I-labeled chymotryptic peptide maps and 32P-labeled tryptic peptide maps, suggesting that the 49 kDa and 57 kDa proteins are closely related.
人类红细胞膜带4.9可被多种红细胞蛋白激酶磷酸化。用DEAE-琼脂糖凝胶对红细胞膜骨架蛋白进行色谱分析，在凝胶电泳上产生两种相对迁移率与带4.9相似的蛋白质。第一种蛋白质分子量为49 kDa，呈碱性（pI大于8），而第二种蛋白质分子量为50.5 kDa，呈弱酸性（pI = 6.2）。二维肽图比较显示，这两种蛋白质都存在于全红细胞膜蛋白和膜骨架蛋白的一维凝胶中的带4.9中。在沉降分析中，49 kDa的蛋白质能与肌动蛋白丝结合，而50.5 kDa的蛋白质则不能。在用[32P]正磷酸盐进行代谢标记的完整红细胞中，49 kDa的蛋白质可被蛋白激酶C、cAMP依赖性蛋白激酶以及在无外源激酶激活剂时仍有活性的蛋白激酶磷酸化。相比之下，50.5 kDa的蛋白质仅被蛋白激酶C磷酸化，而不被所检测的其他蛋白激酶磷酸化。最后，利用二维肽图比较49 kDa的蛋白质和一种与49 kDa蛋白质共纯化且具有许多49 kDa蛋白质特征的57 kDa蛋白质。在125I标记的胰凝乳蛋白酶肽图和32P标记的胰蛋白酶肽图中均发现了显著相似性，表明49 kDa和57 kDa的蛋白质密切相关。
