Thompson J A, Lau A L, Cunningham D D
Biochemistry. 1987 Feb 10;26(3):743-50. doi: 10.1021/bi00377a014.
A procedure was developed for selective radiolabeling of membrane proteins on cells to higher specific activities than possible with available techniques. Cell surface amino groups were derivatized with 125I-(hydroxyphenyl)propionyl groups via 125I-sulfosuccinimidyl (hydroxyphenyl)propionate (125I-sulfo-SHPP). This reagent preferentially labeled membrane proteins exposed at the cell surface of erythrocytes as assessed by the degree of radiolabel incorporation into erythrocyte ghost proteins and hemoglobin. Comparison with the lactoperoxidase-[125I]iodide labeling technique revealed that 125I-sulfo-SHPP labeled cell surface proteins to a much higher specific activity and hemoglobin to a much lower specific activity. Additionally, this reagent was used for selective radiolabeling of membrane proteins on the cytoplasmic face of the plasma membrane by blocking exofacial amino groups with uniodinated sulfo-SHPP, lysing the cells, and then incubating them with 125I-sulfo-SHPP. Exclusive labeling of either side of the plasma membrane was demonstrated by the labeling of some marker proteins with well-defined spatial orientations on erythrocytes. Transmembrane proteins such as the epidermal growth factor receptor on cultured cells could also be labeled differentially from either side of the plasma membrane.
开发了一种程序，用于对细胞上的膜蛋白进行选择性放射性标记，使其具有比现有技术更高的比活度。细胞表面氨基通过125I-磺基琥珀酰亚胺基（羟苯基）丙酸酯（125I-磺基-SHPP）用125I-（羟苯基）丙酰基进行衍生化。通过放射性标记掺入红细胞血影蛋白和血红蛋白的程度评估，该试剂优先标记暴露于红细胞表面的膜蛋白。与乳过氧化物酶-[125I]碘化物标记技术相比，发现125I-磺基-SHPP标记细胞表面蛋白的比活度高得多，标记血红蛋白的比活度低得多。此外，该试剂通过用未碘化的磺基-SHPP封闭外表面氨基、裂解细胞，然后用125I-磺基-SHPP孵育，用于对质膜细胞质面的膜蛋白进行选择性放射性标记。通过对红细胞上具有明确空间取向的一些标记蛋白进行标记，证明了质膜两侧的特异性标记。培养细胞上的跨膜蛋白，如表皮生长因子受体，也可以从质膜的两侧进行差异标记。
