Gahmberg C G, Karhi K K
J Immunol. 1984 Jul;133(1):334-7.
The Rho(D) antigen was recently identified as a 28,000 to 33,000 m.w. polypeptide expressed on the surface of human Rho(D)+ cells. We now show that 70 to 80% of the Rho(D) polypeptides remain firmly associated with the membrane skeleton (detergent-insoluble matrix) obtained after treatment of isolated membranes with Triton X-100. The same treatment solubilized most of the major sialoglycoprotein, glycophorin A. The membrane skeleton-bound Rho(D) polypeptides were not solubilized by procedures that dissociated spectrin, actin, and glyceraldehyde-3-phosphate dehydrogenase from the membrane. Affinity-purified 125I-labeled anti-Rho(D) antibodies bound to intact Rho(D)+ cells, Rho(D)+ membranes, and isolated membrane skeletons from Rho(D)+ cells, but not to Rho(D)- cells. The binding to Rho(D)+ cells was competitively inhibited efficiently by Rho(D)+ membranes and weakly by Rho(D)- membranes. When isolated unsealed Rho(D)+ and Rho(D)- membranes were labeled by lactoperoxidase-catalyzed iodination and solubilized in Triton X-100, Rho(D) polypeptides were immune precipitated only from Rho(D)+ membranes.
Rho(D)抗原最近被鉴定为一种分子量为28,000至33,000的多肽，在人类Rho(D)+细胞表面表达。我们现在发现，70%至80%的Rho(D)多肽与用Triton X-100处理分离膜后获得的膜骨架（去污剂不溶性基质）紧密结合。相同处理使大多数主要唾液酸糖蛋白即血型糖蛋白A溶解。与膜骨架结合的Rho(D)多肽不能通过使血影蛋白、肌动蛋白和甘油醛-3-磷酸脱氢酶从膜上解离的方法溶解。亲和纯化的125I标记抗Rho(D)抗体可与完整的Rho(D)+细胞、Rho(D)+细胞膜以及从Rho(D)+细胞分离的膜骨架结合，但不与Rho(D)-细胞结合。与Rho(D)+细胞的结合可被Rho(D)+细胞膜有效竞争性抑制，被Rho(D)-细胞膜弱竞争性抑制。当分离的未封闭Rho(D)+和Rho(D)-细胞膜通过乳过氧化物酶催化碘化进行标记并在Triton X-100中溶解时，Rho(D)多肽仅从Rho(D)+细胞膜中被免疫沉淀。
