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

细胞

• 批准号: 3082468
• 负责人: CHARLES F SIMMONS
• 金额: $ 6.33万
• 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/3082468
• 关键词: adenosinetriphosphatase; antibody formation; chemical structure function; complementary DNA; electron microscopy; gel electrophoresis; gene expression; genetic manipulation; genetic transcription; genetic translation; immunochemistry; immunoelectron microscopy; immunofluorescence technique; isozymes; membrane model; molecular cloning; mutagens; neoplastic cell culture for noncancer research; oligopeptides; peptide chemical synthesis; point mutation; tissue /cell culture; transposon /insertion element

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+）ATP酶是一种寡聚质膜 蛋白质复合物，催化ATP依赖的交换， Na+和K+离子穿过质膜。 最近 实验证据表明，这种酶以多种形式存在， 哺乳动物组织中的不同亚型。 为了表征 （Na ~+和K ~+）ATP酶结构与功能关系 异构体，我们将分离和测序全长互补 DNA（cDNA）克隆的α（催化）和β亚基， 大鼠（Na+和K+）ATP酶。 根据推测的氨基酸 来自这些克隆的序列，位点特异性，亲和纯化 多克隆抗体将针对适当的合成的 肽和/或融合蛋白。 这些抗体将用于 以表征蛋白质表达、靶向和加工， 通过免疫沉淀培养的大鼠细胞和组织 代谢标记蛋白，免疫荧光，胶体 胶体金免疫细胞化学和免疫印迹分析。 体外 转录载体将从全长α-淀粉酶基因构建 和β亚基cDNA克隆。 体外转录RNA 这些载体中的一种将在小麦胚芽中翻译，和/或 网织红细胞裂解物微粒体膜系统。 我们将 表征膜插入，蛋白质拓扑结构，和后- 每个亚基的翻译修饰，以及亚基 （Na+和K+）ATP酶的功能。 我们将 评价（Na+和K+）ATP酶表达、靶向和功能 在用真核细胞转染的异源真核细胞中， 含有全长大鼠α和/或β的表达载体 亚基cDNA。 定点突变与基因融合 策略将被用来扩展体外翻译和细胞 表达方法。 我们将直接研究 亚基一级结构的变化导致酶的变化 靶向、加工、亚基缔合和功能。 这些 互补方法将结合联合收割机分子生物学， 蛋白质生物化学和细胞生物学，以进一步阐明 （Na+和K+）ATP酶家族蛋白质的功能。 这些 研究将有助于更好地了解（Na+）的生物学 和K+）ATP酶在正常和疾病状态下，无论是在上皮细胞 和可兴奋组织（如神经或肌肉）。 这种酶 提供了一个很好的研究靶向的模型， 加工和极化质膜蛋白的功能。