MECHANISM OF ACTIVE ION TRANSPORT--STRUCTURAL STUDIES

活性离子传输机制--结构研究

• 批准号: 6056358
• 负责人: J. KENT BLASIE
• 金额: $ 9.87万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6056358
• 关键词: X ray crystallography; active sites; active transport; calcium transporting ATPase; enzyme mechanism; enzyme structure; flash photolysis; fluorescence microscopy; ion transport; laboratory rabbit; sarcoplasmic reticulum; time resolved data

DESCRIPTION: The goal of this project is to determine the actual mechanism of active (energy dependent) ion transport across biological membranes to at least the submolecular level. The sarcoplasmic reticulum Ca+2ATPase is employed as the prototypical ion pump. Recent time-resolved synchrotron x-ray diffraction studies determined the cylindrically-averaged profile structure for the calcium pump to moderate resolution (ca. 15 percent) within fully-functional isolated sarcoplasmic reticulum membranes for three important, transiently-trapped enzyme intermediates occurring within the cyclic series of partial reactions responsible for active calcium transport, the so-called E1, (Ca+2)xE1 and (Ca+2)xE1-P intermediates. These studies were extended to determine the locations of lanthanide ions (La+3, Tb+3), replacing calcium ions on the enzyme high-affinity metal binding sites, within the profile structure of the calcium pump AND the effect of enzyme phosphorylation on the positions and metal ion occupancies of these sites. Collectively, these studies suggest a novel mechanism for active ion transport. As a result of this work, together with the critical developments of high count-rate, rapid time-framing x-ray detector and vectorially-oriented single monolayers of the detergent-solubilized Ca+2ATPase, Dr. Blasie is now poised to investigate, not only the profile structures of all enzyme intermediates within the dynamic active calcium transport process (as opposed to only a few selected transiently trapped intermediates), but also the positions and metal ion occupancies of the calcium binding sites themselves within the profile structures of each of these enzyme intermediates. This key time-resolved structural information, combined with the emerging high-resolution 3-dimensional structure for the enzyme, will undoubtedly provide deep insight into the mechanism of active calcium transport by the Ca+2ATPase, most likely high relevant to the transport mechanism for other members of the P-type family of ion pumps as well, including the plasma membrane Na+1/K+1ATPases and Ca+2ATPases. Such knowledge concerning the actual mechanism for the formation of transmembrane ionic gradients is an important step toward understanding their regulation as central to cardiac bioenergetics at the cellular level.

描述：该项目的目的是确定实际机制 跨生物膜的活性（能量依赖）离子转运 至少分子水平。 肌质网Ca+2atpase是 用作原型离子泵。 最近分辨的同步加速器 X射线衍射研究确定了圆柱冲积的轮廓 钙泵至中度分辨率的结构（约15％） 在功能齐全的孤立核质网中，三个 重要的，瞬间捕获的酶中间体发生在 负责主动钙转运的一系列循环系列部分反应， 所谓的E1，（Ca+2）Xe1和（Ca+2）Xe1-P中间体。 这些研究 被扩展以确定灯笼离子的位置（LA+3，TB+3）， 在酶高亲和力金属结合位点上取代钙离子， 在钙泵的轮廓结构内和酶的作用 这些位点的位置和金属离子占用物上的磷酸化。 总的来说，这些研究提出了一种活跃离子的新机制 运输。 由于这项工作，以及关键 高计数，快速定义的X射线检测器的发展 洗涤剂 - 溶解化的矢量为导向的单一单层 CA+2ATPase，Blasie博士现在准备调查，不仅是个人资料 动态活性钙中所有酶中间体的结构 运输过程（而不是只有少数瞬时捕获 中间体），以及位置和金属离子占用 钙结合位点本身在每个的轮廓结构中 这些酶中间体。 这个关键的时间分辨结构信息， 与新兴的高分辨率3维结构相结合 酶，无疑将深入了解主动机制 CA+2ATPase的钙转运，很可能与 P型离子泵家族的其他成员的运输机制作为 好吧，包括质膜Na+1/K+1ATPases和Ca+2ATPase。 这样的 有关跨膜形成的实际机制的知识 离子梯度是了解其法规的重要一步 在细胞水平上是心脏生物能学的中心。