CELL & MOLECULAR BIOLOGY OF (NA+ + K+) ATPASE

细胞

• 批准号: 3082470
• 负责人: CHARLES F SIMMONS
• 金额: $ 6.35万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-30 至 1992-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3082470
• 关键词: adenosinetriphosphatase; antibody formation; chimeric proteins; complementary DNA; dogs; electron microscopy; gel electrophoresis; gene expression; genetic manipulation; genetic transcription; genetic translation; immunochemistry; immunocytochemistry; immunoelectron microscopy; immunofluorescence technique; isozymes; laboratory rabbit; laboratory rat; membrane model; membrane proteins; molecular cloning; mutagens; neoplastic cell culture for noncancer research; nucleic acid probes; oligopeptides; peptide chemical synthesis; point mutation; protein biosynthesis; protein engineering; protein sequence; protein structure function; protein transport; site directed mutagenesis; sodium potassium exchanging ATPase; tissue /cell culture; transfection; transposon /insertion element; western blottings

The (Na+ and K+) ATPase is an oligomeric plasma membrane protein complex which catalyzes the ATP-dependent exchange of Na+ and K+ ions across the plasma membrane. Recent experimental evidence suggests that this enzyme exists as several distinct isoforms in mammalian tissues. In order to characterize the structure-function relationships of (Na+ and K+) ATPase isoforms, we will isolate and sequence full length complementary DNA (cDNA) clones of the alpha (catalytic) and beta subunits of rat (Na+ and K+) ATPase. Based upon the deduced amino acid sequences from these clones, site specific, affinity purified polyclonal antibodies will be raised against appropriate synthetic peptides and/or fusion proteins. These antibodies will be utilized to characterize protein expression, targeting, and processing in cultured rat cells and tissues by immunoprecipitation of metabolically labelled proteins, immunofluorescence, colloidal gold immunocytochemistry, and immunoblot analysis. In vitro transcription vectors will be constructed from full length alpha and beta subunit cDNA clones. RNA from in vitro transcription of these vectors will be translated in wheat germ and/or reticulocyte lysate microsomal membrane systems. We will characterize membrane insertion, protein topology, and post- translational modification of each subunit, as well as subunit association and functions of the (Na+ and K+) ATPase. We will evaluate (Na+ and K+) ATPase expression, targeting, and function in heterologous eukaryotic cells transfected with eukaryotic expression vectors containing full length rat alpha and/or beta subunit cDNAs. Site directed mutagenesis and gene fusion strategies will be utilized to extend in vitro translation and cell expression approaches. We will seek to directly examine how changes in subunit primary structure lead to changes in enzyme targeting, processing, subunit association, and function. These complementary approaches will combine molecular biology, protein biochemistry, and cell biology to further elucidate the function of the (Na+ and K+) ATPase family of proteins. These studies will lead to a better understanding of the biology of (Na+ and K+) ATPase in normal and disease states, both in epithelial and excitable tissues (such as nerve or muscle). This enzyme provides an excellent model for studying the targeting, processing, and function of polarized plasma membrane proteins.

(Na+和K+)ATPase是一种低聚质膜 催化依赖于三磷酸腺苷交换的蛋白质复合体 Na+和K+离子穿过质膜。近期 实验证据表明，这种酶以几种形式存在 哺乳动物组织中不同的异构体。为了刻画出 (Na+和K+)ATPase的结构与功能关系 异构体，我们将分离并测序全长互补 猪瘟病毒α(催化)亚基和β亚基的DNA(CDNA3)克隆 大鼠(Na+、K+)ATPase。根据推导出的氨基酸 这些克隆的序列，位点特定，亲和力纯化 多克隆抗体将被用来对抗适当的合成 多肽和/或融合蛋白。这些抗体将被利用 对蛋白质的表达、靶向和加工进行表征 免疫沉淀法培养大鼠细胞和组织 代谢性标记蛋白，免疫荧光，胶体 金免疫细胞化学和免疫印迹分析。离体 转录载体将由全长的阿尔法基因构建 和β亚基基因克隆。来自体外转录的RNA 这些载体中的一部分将被翻译成小麦胚芽和/或 网织红细胞裂解微粒体膜系统。我们会 表征膜插入、蛋白质拓扑结构和后处理 每个亚基以及亚基的翻译修饰 (Na+和K+)ATPase的结合和功能。我们会 评估(Na+和K+)ATPase的表达、靶向和功能 在异种真核细胞中导入真核细胞 含有全长大鼠α和/或β基因的表达载体 亚单位cDNA。定点突变与基因融合 将利用策略来扩展体外翻译和细胞 表达方式接近。我们将寻求直接研究如何 亚基一级结构的变化导致酶的变化 目标、处理、亚单位关联和功能。这些 互补的方法将结合分子生物学， 蛋白质生物化学和细胞生物学，以进一步阐明 (Na+和K+)ATPase蛋白家族的功能。这些 研究将有助于更好地理解(Na+)的生物学 和K+)ATPase在正常和疾病状态下，都在上皮中 和可兴奋的组织(如神经或肌肉)。这种酶 为研究靶向提供了一个很好的模型， 极化质膜蛋白的加工和功能。