INTERACTION OF VINCULIN WITH MUSCLE CELL MEMBRANES

拟议项目的目标是更好地了解 肌动蛋白-膜相互作用的分子水平，通过研究协会 黏着斑蛋白，一种肌动蛋白结合蛋白，与肌肉细胞膜的结合。 我们 我建议以下列方式处理这个问题：1） 来自平滑肌或心肌组织的含胰高血糖素的膜 将通过均质化，然后进行微分和密度 梯度离心步骤。 2)将从这些植物中提取甘草素 膜使用已知的许多不同的条件溶解 外周膜蛋白 比较黏着斑蛋白的提取方法 以确定肌动蛋白与膜接触的比例 依赖于纽蛋白 3)放射性标记黏着斑蛋白的结合 将研究和分析斯卡查德 图分析 将采用具体方法处理下列问题： 黏着斑蛋白-膜与黏着斑蛋白-肌动蛋白的膜缔合。 四、 在对膜的结合研究之后，我们将鉴定和分离 黏着斑蛋白受体 将使用各种提取方法来去除 黏着斑蛋白受体。 浸提后， 放射活性标记的粘着斑蛋白与提取的 膜。 受体的鉴定将通过使用 技术，如放射性标记黏着斑蛋白的凝胶覆盖， 黏着斑蛋白和兔抗黏着斑蛋白树脂上的亲和层析。 受体的分离将允许表征其分子结构。 大小、形状和氨基酸含量。 5)黏着斑蛋白受体抗体 将被提升并用于将受体定位在非肌肉细胞中， 免疫荧光技术。 通过这些研究，我们希望确定 细胞收缩器如何附着在细胞膜上 它必须与之相互作用以传递力。 因此，这些研究可能 导致更好地了解疾病状态和受损的功能， 肌肉. 在肌肉研究中获得的信息也将有助于回答 关于非肌肉组织结构和运动功能改变的问题 细胞

The goal of the proposed project is to gain a better understanding at the molecular level of actin-membrane interactions by studying the association of vinculin, an actin binding protein, with membranes of muscle cells. We propose to approach the problem in the following way: 1) Vinculin-containing membranes from either smooth or cardiac muscle tissue will be prepared by homogenization followed by differential and density gradient centrifugation steps. 2) Vinculin will be extracted from these membranes using a number of different conditions known to solubilize peripheral membrane proteins. The extraction of vinculin will be compared with that of actin to determine what proportion of actin-membrane contacts is vinculin-dependent. 3) The binding of radioactively labelled vinculin to vinculin-depleted membranes will be studied and analyzed by Scatchard plot analyses. Specific methods will be used to deal with the question of vinculin-membrane vs. vinculin-actin association of the membranes. 4) Following binding studies to membranes, we will identify and isolate the vinculin receptor. Various extraction methods will be used to remove the vinculin receptor from the membrane. Extraction may be followed by measurement of radoactively labelled vinculin binding to extracted membranes. The identification of the receptor will be accomplished by use of technniques such as gel overlay with radioactively labelled vinculin and affinity chromatography on vinculin and rabbit anti-vinculin resins. Isolation of the receptor will allow characterization of its molecular size, shape and amino acid content. 5) Antibodies to the vinculin receptor will be raised and used to localize the receptor in nonmuscle cells by immunofluorescence techniques. Through these studies we hope to determine how the cellular contractile apparatus is attached to the cell membrane with which it must interact to transmit force. These studies may therefore lead to a better understanding of diseased states and impaired function of muscle. Information gained in the study of muscle will also help to answer questions regarding altered structural and motile functions in nonmuscle cells.