Bretscher M S, Lutter R
Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK.
EMBO J. 1988 Dec 20;7(13):4087-92. doi: 10.1002/j.1460-2075.1988.tb03302.x.
A new reagent, DPSgt, is described which has been designed to label cell surface proteins at 0 degree C. The reagent is easily made; it is water soluble and contains a reactive impermeant ester at one end, a tyrosine which can be radioiodinated at the other, and a disulphide in-between. The label can be removed from cells by cleaving the disulphide linkage in it with glutathione at 0 degree C. When cells are warmed to 37 degrees C between labelling and reduction, labelled proteins which are endocytosed acquire resistance to reduction. This provides a simple way of measuring the endocytosis of surface proteins. The intracellular pools of transferrin and LDL receptors in K562 cells and fibroblasts have been estimated. The results indicate that intracellular receptors are in non-reducing compartments, and that uptake of average cell surface (by non-coated pit processes) in K562 cells is small.
描述了一种新试剂DPSgt，其设计用于在0摄氏度下标记细胞表面蛋白。该试剂易于制备；它可溶于水，一端含有反应性非渗透性酯，另一端含有可进行放射性碘化的酪氨酸，中间还有一个二硫键。通过在0摄氏度下用谷胱甘肽切割其中的二硫键，可以从细胞中去除该标记。当细胞在标记和还原之间升温至37摄氏度时，被内吞的标记蛋白获得抗还原性。这提供了一种测量表面蛋白内吞作用的简单方法。已经估计了K562细胞和成纤维细胞中转铁蛋白和低密度脂蛋白受体的细胞内池。结果表明，细胞内受体存在于非还原性区室中，并且K562细胞中平均细胞表面（通过非包被小窝过程）的摄取量很小。
