Baumann H, Hou E, Jahreis G P
J Cell Biol. 1983 Jan;96(1):139-50. doi: 10.1083/jcb.96.1.139.
Glycoproteins in the plasma membrane of rat hepatoma cells were labeled at their externally exposed tyrosine residues with 131I and at their galactose and sialic acid residues with 3H. The degradation of both isotopes in the total cell protein fraction, in glycoproteins purified by concanavalin A, and in glycoproteins separated on two-dimensional gels was determined. Similarly, the total cellular membrane glycoproteins were metabolically labeled with [35S]methionine and [3H]fucose. The fate of both incorporated labels was followed by lectin chromatography or by precipitation of the proteins with specific antibodies followed by electrophoretic gel separation. In both labeling experiments, the carbohydrate markers were lost from the ligand-recognized fraction with similar kinetics as from the total cell protein fraction. In some glycoprotein species which were separated by two-dimensional gel electrophoresis, the polypeptide portion exhibited up to a twofold slower rate of degradation relative to that of the carbohydrate moiety. This difference is most pronounced in carbohydrate-rich glycoproteins. To corroborate this finding, double-labeled membrane glycoproteins were incorporated into reconstituted phospholipid vesicles which were then transferred via fusion into the plasma membrane of mouse fibroblasts. Both the polypeptide and carbohydrate moieties of the transferred membrane glycoproteins were degraded with the same relative kinetics as in the original hepatoma cells. The rate of degradation is mostly a function of the structural properties of the membrane components as shown by the preservation of metabolically stable fucogangliosides of Reuber H-35 hepatoma cells transferred onto the fibroblasts. The technique of insertion of membrane components into the plasma membrane of another cell should assist in the elucidation of the exact route and mechanism of membrane protein destruction.
大鼠肝癌细胞质膜中的糖蛋白，其暴露于外部的酪氨酸残基用¹³¹I标记，半乳糖和唾液酸残基用³H标记。测定了这两种同位素在总细胞蛋白组分、伴刀豆球蛋白A纯化的糖蛋白以及二维凝胶上分离的糖蛋白中的降解情况。同样，总细胞膜糖蛋白用[³⁵S]甲硫氨酸和[³H]岩藻糖进行代谢标记。通过凝集素色谱法或用特异性抗体沉淀蛋白质后进行电泳凝胶分离，追踪这两种掺入标记物的去向。在这两个标记实验中，碳水化合物标记物从配体识别组分中丢失的动力学与从总细胞蛋白组分中丢失的动力学相似。在通过二维凝胶电泳分离的一些糖蛋白种类中，相对于碳水化合物部分，多肽部分的降解速率慢至两倍。这种差异在富含碳水化合物的糖蛋白中最为明显。为了证实这一发现，将双标记的膜糖蛋白掺入重构的磷脂囊泡中，然后通过融合将其转移到小鼠成纤维细胞的质膜中。转移的膜糖蛋白的多肽和碳水化合物部分的降解相对动力学与原始肝癌细胞中的相同。降解速率主要是膜成分结构特性的函数，如转移到成纤维细胞上的鲁伯H - 35肝癌细胞的代谢稳定岩藻糖神经节苷脂的保留所示。将膜成分插入另一个细胞质膜的技术应有助于阐明膜蛋白破坏的确切途径和机制。
