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

细胞

• 批准号: 3082472
• 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/3082472
• 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+) ATP 酶是一种寡聚质膜 催化 ATP 依赖性交换的蛋白质复合物 Na+ 和 K+ 离子穿过质膜。 最近的 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 酶的结构-功能关系 同种型，我们将分离全长互补序列并对其进行测序 α（催化）和 β 亚基的 DNA（cDNA）克隆 大鼠（Na+ 和 K+）ATP 酶。 根据推导的氨基酸 来自这些克隆的序列，位点特异性，亲和纯化 将针对适当的合成产生多克隆抗体 肽和/或融合蛋白。 这些抗体将被利用 表征蛋白质表达、靶向和加工 免疫沉淀法培养大鼠细胞和组织 代谢标记蛋白、免疫荧光、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 alpha 构建 和β亚基cDNA克隆。 来自体外转录的RNA 这些载体中的一些将被翻译到小麦胚芽中和/或 网织红细胞裂解物微粒体膜系统。 我们将 表征膜插入、蛋白质拓扑和后 每个亚基以及亚基的翻译修饰 （Na+ 和 K+）ATP 酶的关联和功能。 我们将 评估（Na+ 和 K+）ATP 酶表达、靶向和功能 在转染真核细胞的异源真核细胞中 含有全长大鼠α和/或β的表达载体 亚基 cDNA。 定点诱变和基因融合 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 靶向、加工、亚基关联和功能。 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+ 和 K+) 正常和疾病状态下的 ATP 酶，均在上皮细胞中 和兴奋组织（如神经或肌肉）。 这种酶 为研究目标提供了一个很好的模型， 极化质膜蛋白的加工和功能。