MEMBRANE LIPID EFFECTS ON THE INSULIN RECEPTOR

这个项目的目的是为了更好地了解 胰岛素受体与膜脂之间的分子相互作用 它所接触的。 是否受体的定位在 不同的脂质微环境影响胰岛素结合， 评估。 所有研究都将使用从以下获得的胰岛素受体进行： 火鸡红细胞膜，一种广泛使用的实验模型。 β-辛基葡糖苷将用于增溶受体， 溶解的物质将相对于两种膜脂质被纯化 和蛋白质的一系列柱层析步骤 含有这种洗涤剂。 将在给药期间测量胰岛素结合率。 纯化过程，以确定结合特性是否 受体受到影响。 每一个连续纯化的制剂将 在蛋白质和脂质含量方面进行表征， 混合物. 将进行受体在脂质体中的重构 用最好的准备，可以获得保留约束力 活性，相对于蛋白质，纯度可能为10-50%， 每摩尔受体的磷脂和胆固醇含量低于1摩尔（Mr 350，000）。 的 磷脂头基组成、脂肪酰基链 成分、胆固醇含量、鞘磷脂含量和 鞘糖脂含量和组成对胰岛素结合的影响 将测定重组受体。 将试图 测量受体磷酸化对胰岛素结合的反应， 通过将32 P-ATP捕获在水性核中的重构脂质体制剂 在脂质体形成期间。 脂质体脂质组合物对脂质体的影响 将测试胰岛素刺激的磷酸化。 磷酸化 对胰岛素结合的反应也将在 含有不同添加膜的洗涤剂增溶制剂 脂质。 该项目将提供有关以下方面的基本信息： 胰岛素受体的可能膜脂质调节。 等 信息将增强我们对膜的影响的基本知识， 胰岛素及其受体之间相互作用的结构。 这将 在分子水平上提供有关激素调节的信息， 可能最终会增加我们对与癌症相关的异常的理解， 患有糖尿病

The purpose of this project is to gain a better understanding of the molecular interactions between the insulin receptor and the membrane lipids with which it is in contact. Whether the localization of the receptor in different lipid microenvironments influences insulin binding will be assessed. All studies will be done with insulin receptors obtained from the turkey erythrocyte membrane, a widely used experimental model. Beta-Octylglucoside will be used to solubilize the receptor, and the solubilized material will be purified with respect to both membrane lipids and proteins by a series of column chromatographic steps in solutions containing this detergent. Insulin binding will be measured during the course of purification in order to determine whether the binding properties of the receptor are affected. Each successively purified preparation will be characterized with respect to protein as well as lipid content and composition. Reconstitution of the receptor in liposomes will be carried out with the best preparation that can be obtained which retains binding activity, probably 10-50% pure with respect to protein and containing less than 1 mol phospholipid and cholesterol per mol receptor (Mr 350,000). The effect of phospholipid head group composition, fatty acyl chain composition, cholesterol content, sphingomyelin content and glycosphingolipid content and composition on insulin binding to the reconstituted receptor will be determined. Attempts will be made to measure receptor phosphorylation in response to insulin binding in the reconstituted liposome preparation by trapping 32P-ATP in the aqueous core during liposome formation. The effect of liposomal lipid composition on insulin-stimulated phosphorylation will be tested. Phosphorylation in response to insulin binding also will be assessed in the detergent-solubilized preparation containing different added membrane lipids. This project will provide fundamental information regarding possible membrane lipid modulation of the insulin receptor. Such information will enhance our basic knowledge of the effects of membrane structure on the interaction between insulin and its receptor. This will provide information about hormonal regulation at the molecular level and may ultimately add to our understanding of the abnormalities associated with diabetes.