PHYSICAL PROPERTIES OF AN ION PUMP

离子泵的物理特性

• 批准号: 3280775
• 负责人: GENE A SCARBOROUGH
• 金额: $ 9.18万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-08-01 至 1990-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3280775
• 关键词: Neurospora; adenosinetriphosphatase; conformation; detergents; enzyme complex; enzyme structure; membrane permeability; molecular weight; ultracentrifugation

An understanding of transmembrane transport phenomena at the molecular level is an important objective of contemporary biology. A major category of biological transport molecules is the transport ATPases, the most thoroughly studied of which can be subdivided into the FIFO ATPAse/ATP synthases of mitochondria, bacteria and chloroplasts, and the aspartyl-phosphoryl-enzyme intermediate family, which includes the Na+/K+-translocating ATPase of animal cell plasma membranes, the CA2+-translocating ATPase of sarcoplasmic reticulum, the H+/K+-translocating ATPase of gastric mucosa, and the H+-ATPase in the plasma membrane of Neurospora and yeast. In this laboratory we are working toward an understanding of the molecular mechanism of the Neurospora plasma membrane H+-ATPase anticipating that what is learned about the mechanism of this enzyme will contribute to our understanding of the molecular mechanisms of the other ATPases in the aspartyl-phosphoryl-enzyme intermediate family, and possibly transport ATPases in general. Ongoing research in this laboratory is attempting to elucidate several aspects of the chemistry of the H+-ATPase, including the identification of active site residues, determination of the primary amino acid sequence, and topological mapping of the orientation of the polypeptide chain in the membrane at several stages of the catalytic cycle. The studies proposed in this application will investigate the physical properties of this transport molecule. Specifically, we hope to 1) identify several detergents or other amphiphiles that will facilitate preparation of the ATPase, which is oligomeric as isolated, as stable, active, lipid-free, water soluble monomers or low molecular weight multimers thereof (protomers) and determine their molecular weights and thus monomer copy numbers, 2) characterize a variety of other physical properties of the ATPase monomers (protomers) obtained, including their shape and secondary structural composition in the presence and absence of ligands known to induce enzyme conformational changes, and their possible multiple domain structure, and 3) develop procedures for crystallizing the ATPase monomers (protomers) that are obtained. Our long term goal, outside the scope of this proposal, is an elucidation of the molecular structure of this ion pump.

对分子上跨膜转运现象的理解 水平是当代生物学的重要目标。 主要类别 生物运输分子的是运输ATPases，最多 可以将其细分为FIFO ATPase/ATP进行彻底研究 线粒体，细菌和叶绿体的合成酶，以及 天冬氨酸 - 磷酸酶中间家族，其中包括 Na+/k+ - 转移动物细胞质膜的ATPase， Ca2+ - 肌质网的ATPase， H+/K+ - 胃粘膜的ATPase和H+-ATPase在 神经和酵母的质膜。 在这个实验室中，我们正在工作 了解神经孢子血浆的分子机制 膜H+-ATPase预见到有关机制的知识 这种酶将有助于我们对分子的理解 黄体基磷酸酶中其他ATP酶的机理 中间家庭，总体上可能运输ATPases。 正在进行 该实验室的研究试图阐明 H+-ATPase的化学，包括活性位点的识别 残留物，初级氨基酸序列的测定和拓扑 在膜上多肽链方向的映射 催化周期的几个阶段。 提出的研究 应用程序将调查此运输的物理特性 分子。 具体来说，我们希望1）确定几种洗涤剂或其他 将有助于准备ATPase的两亲物 孤立，稳定，活跃，无脂溶性，水溶性的寡聚体 单体或低分子量多聚体（原始体）和 确定其分子量，从而确定单体拷贝数，2） 表征ATPase单体的各种其他物理特性 （原始物质）获得的，包括其形状和次要结构 在存在和不存在已知诱导酶的配体的情况下组成 构象变化及其可能的多个领域结构，以及 3）制定结晶ATPase单体（原始物）的程序 获得的。 我们的长期目标，超出了该提议的范围， 是该离子泵的分子结构的阐明。