EPR STUDIES OF MEMBRANE PROTEINS

拟议的研究将为这些安排提供新的见解， 蛋白质在完整细胞膜中的运动和相互作用。 初始 研究将针对阴离子转运的特异性标记 人红细胞（RBC）中的蛋白质（带3）。 新合成 双功能自旋标记将与电子 顺磁共振（EPR）光谱定量和比较 完整红细胞中带3的旋转运动与血影膜中的运动 以及去除了选定的膜骨架蛋白的鬼膜。 这些研究将有助于定义蛋白质-蛋白质和蛋白质-脂质 红细胞中的相互作用决定了细胞的形状和变形能力。 在 这些研究的结论，应建立充分的方法 为了允许对来自A型糖尿病患者的异常RBC进行比较测量， 各种溶血性贫血 这些数据可能有助于确定 结构缺陷导致形状异常和细胞增加 对各种RBC紊乱的脆弱性。 这些相同的标记将用于标记阴离子结合蛋白， 已经在脂肪细胞膜中观察到。 许多交通工具的运输功能 脂肪细胞中的膜蛋白是通过激素结合来调节的， 表面受体 量化的旋转运动，这些 在基础和刺激条件下的蛋白质将有助于理解 激素对运输活动的作用机制。 红细胞的EPR数据 将使用高分辨率N15自旋收集脂肪细胞研究 标签 这将使更严格的数据分析，直接计算机 提取旋转扩散的实验谱的模拟 系数，因此，确定聚集的状态， 标记蛋白质。 自旋标记的ATP类似物将被用来检查功能 膜结合糖酵解酶之间的关系 甘油醛-3-磷酸脱氢酶、磷酸甘油酸激酶和 Na+，K+-ATP酶。 实验提出了确定的特点 和ATP的膜相关区室的微环境， 被观察到。 这些研究将提供深入了解生产和 RBC中ATP的利用方式。

The proposed research will provide new insights into the arrangements, motions and interactions of proteins in intact cell membranes. Initial studies will be directed toward specific labeling of the anion transport protein (band 3) in the human red blood cell (rbc). Newly synthesized bifunctional spin labels will be employed in conjunction with electron paramagnetic resonance (EPR) spectroscopy to quantitate and compare the rotational motions of band 3 in intact rbc with motions in ghost membranes and ghost membranes having selected membrane skeleton proteins removed. These studies will help define protein-protein and protein-lipid interactions in rbc which determine cell shape and deformability. At the conclusion of these studies, sufficient methodology should be established to permit comparative measurements on abnormal rbc from patients with a variety of hemolytic anemias. These data may aid in establishing what structural defects are responsible for abnormal shape and increased cell fragility for a variety of rbc disorders. These same labels will be employed to label anion binding proteins which have been observed in adipocyte membranes. The transport functions of many membrane proteins in adipocytes are modulated by binding of hormones to surface receptors. Quantitation of the rotational motions of these proteins under basal and stimulated conditions will aid in understanding mechanisms of hormone action on transport activity. EPR data from the rbc and adipocyte studies will be collected using high resolution N15 spin labels. This will enable more rigorous data analysis by direct computer simulation of experimental spectra for extracting rotational diffusion coefficients and, hence, determining the states of aggregation of the labeled proteins. Spin labeled ATP analogues will be employed to examine the functional relationship between membrane bound glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and the Na+, K+-ATPase. Experiments are proposed to determine the characteristics and microenvironment of membrane associated compartments of ATP which have been observed. These studies will provide insight into the production and mode of utilization of ATP in the RBC.