A STUDY OF RETINAL PHOTORECEPTOR MEMBRANE PROTEINS

视网膜感光膜蛋白的研究

• 批准号: 3256760
• 负责人: ROBERT S MOLDAY
• 金额: $ 6.66万
• 依托单位: UNIVERSITY OF BRITISH COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-04-01 至 1990-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3256760
• 关键词: actins; affinity chromatography; calmodulin; cell membrane; chemical association; chromatography; conformation; cow; density gradient ultracentrifugation; divalent cations; electron microscopy; electrophoresis; gel filtration chromatography; glycoproteins; immunochemistry; immunoelectron microscopy; ion exchange chromatography; ionic strengths; laboratory mouse; membrane channels; membrane permeability; membrane proteins; monoclonal antibody; myosins; nucleic acid sequence; protein sequence; protein structure; proteolysis; radioimmunoassay; retina; retina degeneration; retinal adaptation; rhodopsin; rod cell; spectrin; visual photoreceptor

The long term objective of this research program is to study the molecular structure and properties of rod outer segment (ROS) disk and plasma membrane proteins in order to elucidate their role in visual excitation, in the organization and renewal of the ROS organelle and in retinal degenerative diseases. Four related areas of research will be investigated in this grant period: 1) The ROS plasma membrane and plasma membrane specific proteins will be purified and characterized. This will be carried out by labeling ROS plasma membrane specific glycoproteins with recently developed Ricin-gold-dextran particles and separating the 'dense' labeled plasma membrane from disks by gradient centrifugation. The protein and lipid composition of the plasma membrane will be determined and compared with disk membranes using biochemical, immunochemical and electron microscopic techniques developed over the past grant period. Emphasis will be directed toward identifying and characterizing the c-GMP binding protein, Na+ channels and ROS plasma membrane-specific glycoproteins. The generation of monoclonal antibodies (Mab) against these proteins for use as specific probes for studying their distribution, structure and function will be carried out. 2) ROS spectrin recently identified in this laboratory will be purified by gel filtration, ion exchange and immunoaffinity chromatography and characterized with regard to its subunit structure by SDS-gel electrophoresis, its size and shape by Platinum rotary shadowing for electron microscopy and its interaction with other membrane and cytoskeletal proteins by binding analysis and reconstitution methods. The possible role of ROS spectrin and other cytoskeletal proteins in disk-disk and disk-plasma membrane interactions will also be studied. (3)Peripherin, a newly discovered ROS rim protein, will be further characterized using biochemical and DNA recombinant techniques. The gene will be cloned and its nucleotide and amino acid sequence determined in order to obtain information about its membrane organization and relation to other known proteins. 4) Antirhodopsin Mabs will be further characterized and used as immunological probes to analyze the structural and functional domains of rhodopsin.

该研究计划的长期目标是研究 杆外段（ROS）的分子结构和性质 盘和质膜蛋白，以阐明它们的 在视觉兴奋、组织和更新中的作用 ROS 细胞器和视网膜退行性疾病。 四相关 在此资助期内将调查研究领域： 1) ROS质膜和质膜特异性蛋白 将被纯化和表征。 这将由 标记 ROS 质膜特异性糖蛋白 最近开发的蓖麻毒素-金-葡聚糖颗粒和分离 通过梯度从圆盘中获得“致密”标记的质膜 离心。 血浆的蛋白质和脂质成分 膜将被确定并与盘膜进行比较 使用生化、免疫化学和电子显微镜 在过去的资助期内开发的技术。 重点将是 旨在识别和表征 c-GMP 结合 蛋白质、Na+ 通道和 ROS 质膜特异性 糖蛋白。 单克隆抗体 (Mab) 的产生 针对这些蛋白质用作研究其的特异性探针 将进行分布、结构和功能。 2）活性氧 该实验室最近鉴定出的血影蛋白将通过以下方法纯化 凝胶过滤、离子交换和免疫亲和层析 并通过 SDS-gel 表征其亚基结构 电泳，其大小和形状通过铂旋转阴影 用于电子显微镜及其与其他膜的相互作用 和细胞骨架蛋白通过结合分析和重构 方法。 ROS血影蛋白和其他物质的可能作用 盘-盘和盘-质膜中的细胞骨架蛋白 相互作用也将被研究。 (3)外周蛋白，一种新的 发现了 ROS 边缘蛋白，将使用进一步表征 生物化学和DNA重组技术。 该基因将是 克隆并确定其核苷酸和氨基酸序列 为了获得有关其膜组织的信息和 与其他已知蛋白质的关系。 4) 抗视紫红质单克隆抗体将 进一步表征并用作免疫探针 分析视紫质的结构和功能域。