Sears D A, Friedman J M, George J N
J Biol Chem. 1977 Jan 25;252(2):712-20.
The topography of the external surface of the human red cell membrane has been studied using an impermeant radioactive probe, [125I]diazodiiodosulfanilic acid, which binds covalently to protein groups of the membrane following reaction with intact cells. The pattern of labeling was assessed by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis followed by sequential analysis of single gels for carbohydrates (by staining with the periodic acid-Schiff (PAS) reagent), for proteins (by staining with Coomassie blue), and for radioactivity (by counting gels sliced in 2-mm segments). The radioactive probe bound to membrane polypeptides with apparent molecular weights of 94,200, 58,100, and 46,500 (Peaks A, B, and C, respectively). Peak A co-migrated with a small periodic acid-Schiff-positive band and protein Band 3 (nomenclature of Steck) (Steck, T.L. (1974)J. Cell Biol. 62: 1-19). Peak B migrated with protein Band(s) 4.5 slightly ahead of the major membrane glycoprotein (PAS-1). Peak C migrated like glycoprotein PAS-2 and protein Band 5, the actin-like, water-soluble membrane protein. In contrast to lactoperoxidase iodination and a number of other probes, [125I]diazodiiodosulfanilic acid reacted minimally with the major membrane glycoprotein, glycophorin. When it was reacted with isolated ghosts, all molecular weight classes of polypeptides were labeled. Treatment of labeled cells with neuraminidase or trypsin altered the glycoprotein staining pattern, but not the radioactive peaks. On the other hand, Pronase eliminated the Mr=94,200 radioactive peak, diminished the other two radioactive peaks, and profoundly changed the glycoprotein and protein staining patterns. Treatment of the membranes of labeled cells in a low ionic strength alkaline medium did not alter radioactive peaks and demonstrated that Peak C differed from the actin-like membrane protein. A nonionic detergent, Triton X-100, solubilized all radioactive components. The studies have defined the binding of [125I]diazodiiodosulfanilic acid to external proteins of the human red cell membrane. Its pattern of reaction differs quantitatively and qualitatively from other commonly used reagents, and it provides a useful additional vectorial probe for the study of membrane topography. Its reactions provide further evidence of the organizational complexity of the red cell membrane and emphasize the fact that interpretation of information derived from the use of membrane probes must take into account the differences resulting from the properties of the probing reagents themselves.
利用一种非渗透性放射性探针[125I]重氮二碘对氨基苯磺酸，对人红细胞膜外表面的拓扑结构进行了研究。该探针在与完整细胞反应后，会与膜上的蛋白质基团共价结合。标记模式通过十二烷基硫酸钠-聚丙烯酰胺圆盘凝胶电泳进行评估，随后依次对单一凝胶进行碳水化合物（用高碘酸-希夫（PAS）试剂染色）、蛋白质（用考马斯亮蓝染色）以及放射性（对切成2毫米片段的凝胶进行计数）分析。放射性探针与表观分子量分别为94,200、58,100和46,500的膜多肽结合（分别为峰A、峰B和峰C）。峰A与一条小的高碘酸-希夫阳性条带及蛋白质带3（Steck命名法）（Steck, T.L. (1974)J. Cell Biol. 62: 1 - 19）共迁移。峰B与蛋白质带4.5一起迁移，略先于主要的膜糖蛋白（PAS - 1）。峰C的迁移情况与糖蛋白PAS - 2及蛋白质带5相同，后者是肌动蛋白样的水溶性膜蛋白。与乳过氧化物酶碘化及其他一些探针不同，[125I]重氮二碘对氨基苯磺酸与主要的膜糖蛋白血型糖蛋白反应极少。当它与分离的血影反应时，所有分子量类别的多肽都被标记。用神经氨酸酶或胰蛋白酶处理标记细胞会改变糖蛋白染色模式，但不会改变放射性峰。另一方面，链霉蛋白酶消除了分子量为94,200的放射性峰，使另外两个放射性峰减弱，并深刻改变了糖蛋白和蛋白质染色模式。在低离子强度碱性介质中处理标记细胞的膜不会改变放射性峰，这表明峰C与肌动蛋白样膜蛋白不同。一种非离子去污剂Triton X - 100溶解了所有放射性成分。这些研究确定了[125I]重氮二碘对氨基苯磺酸与人红细胞膜外部蛋白质的结合。其反应模式在数量和质量上与其他常用试剂不同，它为研究膜拓扑结构提供了一种有用的额外的向量探针。其反应进一步证明了红细胞膜组织的复杂性，并强调了这样一个事实，即对来自膜探针使用信息的解释必须考虑到探测试剂自身性质所导致的差异。
