MECHANISM OF ACTIVE ION TRANSPORT--STRUCTURAL STUDIES

描述：该项目的目标是确定实际的机制 活性(依赖于能量的)离子通过生物膜传输到AT 至少在亚分子水平上。肌浆网Ca+2ATPase是 被用作原型离子泵。最新时间分辨同步加速器 X射线衍射研究确定了柱面平均轮廓 钙泵的结构达到中等分辨率(约15%) 在功能齐全的分离肌浆网膜内 重要的、瞬间捕获的酶中间体出现在 负责主动钙转运的一系列循环部分反应， 即所谓的E1、(Ca+2)XE1和(Ca+2)XE1-P中间体。这些研究 用于确定稀土离子(La+3，Tb+3)的位置 取代酶高亲和力金属结合部位上的钙离子， 在钙泵的轮廓结构和酶的作用下 这些位置上的磷酸化和金属离子的占有率。 总而言之，这些研究提出了一种新的活性离子机制 运输。作为这项工作的结果，连同关键的 高计数率、快速定帧X射线探测器的研制和应用 洗涤剂增溶的矢量取向单层膜 CA+2ATPase，布莱西博士现在准备调查，不仅仅是档案 动态活性钙内各酶中间体的结构 传输过程(而不是仅选择几个被暂时困住的对象 中间体)的位置和金属离子的占有率 钙结合位点本身在每个的轮廓结构中 这些酶的中间体。该关键的时间分辨结构信息， 结合新出现的高分辨率三维结构 酶，无疑将提供对活性机制的深刻洞察 通过Ca+2ATPase进行的钙转运，很可能与 P型离子泵AS系列其他成员的输运机制 包括质膜Na+1/K+1 ATPase和Ca+2 ATPase。是这样的 关于跨膜形成的实际机制的认识 离子梯度是了解其调节的重要一步。 在细胞水平上作为心脏生物能量学的中心。

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.