CA TRANSPORT IN CARDIAC MEMBRANE VESICLES

我们将从猪心制备肌膜囊泡，分析其结构， 纯度和开发方法，用于测定和分离由内而外的囊泡 和右侧向外的方向。 我们将使用囊泡来研究两个 运输过程，被认为是重要的，在去除钙， 心肌细胞：钠钙交换和ATP依赖性钙泵。 我们将 研究了Na-Ca交换体系在两种温度下的动力学特征， 细胞质和细胞外膜表面，并评估ATP的作用， 这一运输过程的运作。 我们还将进行详细的 ATP依赖性钙转运系统的生化表征， 特别强调将其性质与类似情况的性质区分开来 心脏肌浆网中的转运系统。 的可能作用 cAMP和膜磷酸化，以及钙-钙调素系统， 将探讨调节这两种运输过程的活动。 最后，我们将尝试重建这些运输过程， 在用去污剂溶解膜之后，将磷脂膜溶解。 重组将是迈向纯化和分子生物学的第一步。 表征负责转运活性的膜组分。

We will prepare sarcolemmal membrane vesicles from pig heart, analyze their purity and develop methods for assaying and separating vesicles of inside-out and right-side-out orientations. We will use the vesicles to study two transport processes that are thought to be important in removing Ca from myocardial cells: Na-Ca exchange and the ATP-dependent Ca pump. We will investigate the kinetic characteristics of the Na-Ca exchange system at both the cytoplasmic and extracellular membrane surfaces and evaluate the role of ATP in the operation of this transport process. We will also undertake a detailed biochemical characterization of the ATP-dependent Ca transport system, with a special emphasis on differentiating its properties from those of a similar Ca transport system in the cardiac sarcoplasmic reticulum. The possible roles of cAMP and membrane phosphorylation, and of the Ca-calmodulin system, in regulating the activity of these two transport processes will be explored. Finally, we will attempt to reconstitute these transport processes in phospholipid membranes following membrane solubilization with detergents. Reconstitution will be the first step toward the purification and molecular characterization of the membrane components responsible for transport activity.