SHORT TERM REGULATION OF THE NA+ PUMP

NA 泵的短期调节

• 批准号: 6177550
• 负责人: CARLOS H PEDEMONTE
• 金额: $ 14.32万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-18 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6177550
• 关键词: crosslink; enzyme activity; enzyme mechanism; enzyme structure; intermolecular interaction; protein kinase C; protein purification; protein sequence; protein structure function; renal tubular transport; sodium potassium exchanging ATPase; tissue /cell culture; yeast two hybrid system

DESCRIPTION (Adapted from the Applicant's Abstract): This is the first amendment of a new submission designed to explore the mechanisms through which protein kinase C regulates the sodium pump in a proximal tubule model. There are three Specific Aims. The first is to determine the mechanism whereby phosphorylation of the alpha subunit serine-11 and/or serine-18 residue by protein kinase C alters the activity of the sodium pump. These studies will involve assessing the phosphorylation of transfected wild-type and amino-terminus mutants of the rodent alpha subunit expressed in OK cells and will involve measurements of rubidium, ATPase activities and changes in intracellular sodium concentrations. The second aim has two parts. The first is to determine which amino acids at the amino-terminus interact with other cytosolic domains of the Alpha subunit. The second is to determine whether the aminoterminus specifically interacts with other cytosolic domains of the alpha subunit through the phosphates linked to serine 11 and/or serine 18. Those studies will require methods outlined under Specific Aim #1. Specific Aim #3 is to determine the cytosolic domains of the alpha subunit that interact with the amino terminus. These studies will use a variety of techniques to determine the interaction or closeness of polypeptide segments of the sodium pump. These will involve affinity purification of the peptides, use of the yeast two-hybrid system and chemical cross linking studies.

描述(改编自申请者摘要)：这是第一个 修正一项旨在通过以下途径探讨机制的新划界案 在近端小管模型中，哪种蛋白激酶C调节钠泵。 有三个具体目标。一是确定机制 从而使α亚基丝氨酸-11和/或丝氨酸-18磷酸化 蛋白激酶C的残基改变了钠泵的活性。这些 研究将包括评估转基因野生型的磷酸化。 和啮齿动物α亚基氨基端突变体在OK细胞中的表达 并将涉及测量Rb，ATPase活性和在 细胞内钠离子浓度。第二个目标包括两个部分。这个 首先是确定氨基末端的哪些氨基酸与之相互作用 Alpha亚基的其他胞液结构域。二是确定 氨基末端是否与其他胞浆特异地相互作用 通过与丝氨酸11相连的磷酸盐的α亚基的结构域 和/或丝氨酸18。这些研究将需要下列方法 特定目标#1.特定目标#3确定细胞胞浆结构域 与氨基末端相互作用的阿尔法亚单位。这些研究将 使用各种技术来确定 钠泵的多肽片段。这些将涉及亲和力 多肽的纯化，酵母双杂交系统的使用和 化学交联剂研究。