Evans R M, Fink L M
Proc Natl Acad Sci U S A. 1977 Dec;74(12):5341-4. doi: 10.1073/pnas.74.12.5341.
Studies were carried out to identify transmembrane bridging proteins in the plasma membrane of mouse L-929 cells. Cells grown in suspension culture were 125I-labeled by lactoperoxidase and allowed to ingest latex particles to produce inside-out membrane phagosome preparations. Phagosomes were isolated and the inner membrane surface was labeled with N-(5'-aminopentyl)-5-dimethylamino-1-naphthalenesulfonamide (dansylcadavarine) by a transglutaminase-catalyzed reaction. The phagosome membrane proteins were solubilized and dansylcadavarine-labeled proteins were isolated by anti-dansyl immunoadsorbent affinity chromatography. Dansylcadavarine-labeled proteins were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography for the presence of 125I-labeled material. By this technique, two iodinated proteins with molecular weights of approximately 50,000 and 80,000 appear to be selectively retained by the anti-dansyl immunoadsorbent, suggesting that these proteins span the plasma membrane.
开展了多项研究以鉴定小鼠L-929细胞膜中的跨膜桥接蛋白。悬浮培养的细胞经乳过氧化物酶进行¹²⁵I标记，并使其摄取乳胶颗粒以制备内膜外翻的膜吞噬体。分离吞噬体，通过转谷氨酰胺酶催化反应，用N-(5'-氨基戊基)-5-二甲氨基-1-萘磺酰胺（丹磺尸胺）标记内膜表面。使吞噬体膜蛋白溶解，通过抗丹磺免疫吸附亲和层析分离丹磺尸胺标记的蛋白。通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳和放射自显影分析丹磺尸胺标记的蛋白，以检测¹²⁵I标记物质的存在。通过该技术，抗丹磺免疫吸附剂似乎选择性保留了两种分子量约为50,000和80,000的碘化蛋白，表明这些蛋白跨越质膜。
