Christiansen K, Carlsen J
Department of Biochemistry C, University of Copenhagen, Denmark.
Biochem J. 1992 Jan 15;281 ( Pt 2)(Pt 2):425-30. doi: 10.1042/bj2810425.
Purified human insulin receptors were inserted into placental plasma-membrane vesicles by fusion of membranes with receptor-lysophosphatidylcholine micelles. Scatchard analysis of insulin binding showed that about 10-15% of the added receptors became inserted into the membrane. The receptor number could be increased about 3-fold, corresponding to approx. 5 pmol of receptor/mg of membrane protein. The receptors became firmly bound to the membrane, as they could not be removed by extensive wash. The insertion of exogenous receptors could be demonstrated by immunoblotting. The inserted insulin receptor had the same insulin-binding affinity as the isolated receptor and the endogenous receptor of the membrane. Insulin binding in the presence or absence of Triton X-100 revealed that more than 80% of the exogenous receptors had a right-side-out orientation. Function of the inserted receptors, as observed by insulin-stimulated autophosphorylation, could be demonstrated. About 80% of the added lysophospholipid, corresponding to approx. 160 nmol of lysophospholipid/mg of membrane protein, became integrated into the membrane and was partly metabolized to phospholipid and to non-esterified fatty acid. The method of insertion of isolated insulin receptors using the natural detergent, lysophospholipid, may be a method for insertion of receptors into intact cells, where the lysophospholipid, as in the plasma-membrane vesicles, will be acylated to phospholipid.
通过将膜与受体 - 溶血磷脂酰胆碱胶束融合，将纯化的人胰岛素受体插入胎盘质膜囊泡中。胰岛素结合的Scatchard分析表明，约10 - 15%添加的受体插入到膜中。受体数量可增加约3倍，相当于每毫克膜蛋白约5 pmol受体。受体与膜紧密结合，因为通过大量洗涤无法将其去除。通过免疫印迹可证明外源受体的插入。插入的胰岛素受体与分离的受体以及膜的内源性受体具有相同的胰岛素结合亲和力。在有或没有Triton X - 100存在的情况下进行胰岛素结合实验表明，超过80%的外源受体具有外翻取向。通过胰岛素刺激的自身磷酸化观察到，插入的受体功能可以得到证明。约80%添加的溶血磷脂，相当于每毫克膜蛋白约160 nmol溶血磷脂，整合到膜中，并部分代谢为磷脂和非酯化脂肪酸。使用天然去污剂溶血磷脂插入分离的胰岛素受体的方法，可能是一种将受体插入完整细胞的方法，在完整细胞中，溶血磷脂就像在质膜囊泡中一样，会被酰化为磷脂。
