Hare J F, Holocher A
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97201.
J Biol Chem. 1994 Feb 25;269(8):5981-8.
Four cell surface-exposed, integral membrane proteins from H4-II-E-3 hepatoma monolayer cultures, derivatized by the membrane-impermeant reagent sulfosuccinimidyl 2-(biotin-amido)ethyl-1,3-dithioproprionate, were resistant to extraction with Triton X-100 at 0 degrees C. Thirty-three other similarly derivatized proteins were solubilized under these same conditions. Antisera were prepared that reacted only with Triton X-100-insoluble proteins. All four Triton X-100-insoluble proteins precipitated with the antibody were slowly degraded (t1/2 > 100 h). By contrast, all but four Triton X-100-soluble proteins were rapidly degraded (t1/2 = 24 h). The detergent-insoluble proteins did not possess glycosylphosphatidylinositol anchors nor were they solubilized by Triton X-100 after disruption of the cytoskeleton. In addition, they were insoluble in Triton X-100 in isolated membrane preparations but soluble when isolated on streptavidin-agarose and removed from other membrane proteins. We conclude that protein-protein interactions within the membrane itself result in insolubility in non-ionic detergents for a small cohort of plasma membrane proteins and that this may be directly related to the increased metabolic stability for this class of proteins.
来自H4-II-E-3肝癌单层培养物的四种细胞表面暴露的整合膜蛋白，经膜不透性试剂磺基琥珀酰亚胺基2-（生物素酰胺基）乙基-1,3-二硫代丙酸酯衍生化后，在0℃下对Triton X-100提取具有抗性。在相同条件下，其他33种类似衍生化的蛋白质被溶解。制备了仅与Triton X-100不溶性蛋白质反应的抗血清。与抗体沉淀的所有四种Triton X-100不溶性蛋白质均缓慢降解（半衰期>100小时）。相比之下，除四种外的所有Triton X-100可溶性蛋白质均迅速降解（半衰期=24小时）。去污剂不溶性蛋白质不具有糖基磷脂酰肌醇锚定，并且在细胞骨架破坏后也不被Triton X-100溶解。此外，它们在分离的膜制剂中不溶于Triton X-100，但在链霉亲和素-琼脂糖上分离并与其他膜蛋白分离时可溶。我们得出结论，膜本身内的蛋白质-蛋白质相互作用导致一小部分质膜蛋白在非离子去污剂中不溶，这可能与这类蛋白质代谢稳定性的增加直接相关。
