McCaffery P J, Fraser J K, Lin F K, Berridge M V
Malaghan Institute of Medical Research, Wellington School of Medicine, Wellington Hospital, New Zealand.
J Biol Chem. 1989 Jun 25;264(18):10507-12.
Chemical cross-linking of the red blood cell hormone, erythropoietin (Epo), to its receptor on erythroid cells has revealed the presence of two proteins closely associated with Epo, but the relationship between these two proteins is controversial. Using the cross-linking reagents disuccinimidyl suberate and dithiobissuccinimidyl propionate, we show that 125I-Epo can be specifically conjugated in a complex of 224kDa using mouse fetal liver cells, bone marrow cells, and Friend virus-induced splenic erythroblasts as demonstrated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. Under reducing conditions, the 224-kDa complex appeared as two Epo conjugates of 136 kDa and 119 kDa, and these bands were also observed to a variable extent in some nonreducing gels. Disulfide linking of the 136-kDa and 119-kDa bands was confirmed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis run under nonreducing followed by reducing conditions. With increasing time of 125I-Epo binding to Friend virus erythroblasts in the presence of sodium azide to inhibit receptor internalization, the 136-kDa and 119-kDa bands seen under reducing conditions increased markedly in intensity, whereas the 224-kDa band seen under nonreducing conditions declined. These results suggest that the 224-kDa Epo conjugate is inefficiently solubilized under nonreducing conditions following prolonged periods of Epo binding. A single class of saturable, high affinity receptors for Epo on each of the cell types tested is demonstrated. It is concluded that the two disulfide-linked Epo-binding proteins which can be independently cross-linked to Epo form a single ligand binding site.
红细胞生成素（Epo）这种红细胞激素与红系细胞上的受体进行化学交联后，发现有两种与Epo紧密相关的蛋白质，但这两种蛋白质之间的关系存在争议。使用交联剂辛二酸二琥珀酰亚胺酯和二硫双（琥珀酰亚胺基丙酸酯），我们发现，在非还原条件下通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳证明，125I-Epo可与小鼠胎肝细胞、骨髓细胞以及弗氏病毒诱导的脾成红细胞特异性结合形成224kDa的复合物。在还原条件下，224kDa的复合物呈现为136kDa和119kDa的两种Epo结合物，并且在一些非还原凝胶中也不同程度地观察到了这些条带。通过先在非还原条件下然后在还原条件下进行的二维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，证实了136kDa和119kDa条带之间存在二硫键连接。在存在叠氮化钠以抑制受体内化的情况下，随着125I-Epo与弗氏病毒成红细胞结合时间的增加，在还原条件下看到的136kDa和119kDa条带强度显著增加，而在非还原条件下看到的224kDa条带则下降。这些结果表明，在Epo长时间结合后，224kDa的Epo结合物在非还原条件下难以有效溶解。在所测试的每种细胞类型上都证明了存在一类单一的、可饱和的、高亲和力的Epo受体。得出的结论是，可以独立与Epo交联的两种二硫键连接的Epo结合蛋白形成一个单一的配体结合位点。
