CRYSTALLIZATION OF A CALCIUM PUMP

快收缩肌肌浆网Ca-ATP酶活性与肌浆网Ca-ATP酶活性呈负相关。 离子泵是参与体内平衡的一类离子泵的代表 Na+、K+、H+和Ca+2的阳离子。 的相似性 这些离子泵（Na/K-ATP酶，H/K- ATP酶和Ca-ATP酶）已被解释为表明， 它们都来自于一个共同的祖先基因（1）。 CA+2 在这些阳离子中是独一无二的， 对激素和递质的反应。 钙泵损失 活动与杜氏肌营养不良等疾病有关， 营养不良（2），布罗迪病（3）和实验性肌强直（4）。 这里提出的工作有明确的目标， 家兔肌浆网Ca-ATPase的长期 通过以下方式获得详细结构信息的目的 衍射方法在这类晶体中的应用。 ca-atp酶 将在F.N.教授的实验室进行纯化。布里格斯使用 该实验室开发的程序（5）， 均质酶 该材料在存在下溶解， 洗涤剂，并将以这种形式作为起始 用于结晶试验的材料。 Ca-ATPase存在于两个 稳定的构象形式，并在不同的状态下， 聚合来 对于结晶，那些已知有利于 将使用单体。 跟踪优化这些条件 将通过结合和交换洗涤剂制成， 在孔蛋白结晶的情况下进行。 聚合状态将 通过分析型HPLC或离心法进行监测 洗涤剂的存在。 温和的琥珀酰化和脒基化 Ca-ATPase也将被尝试，因为前者已被证明是 减少聚集。 两种构象状态：高（E1） 和低（E2）钙亲和力，将通过结合 已知的稳定两种状态之一的配体。 越稳定， 首先筛选钙结合的E1型以进行结晶。 二价过渡金属和几种不同的核苷酸将 测试它们对结晶的影响。 E2状态存在 作为囊泡中的二聚体，并通过钒酸盐稳定。 钒酸盐- 结合的酶将在结晶试验中筛选， 洗涤剂、沉淀剂浓度和pH。结晶 试验将通过坐滴或微透析方法进行。 由于Ca-ATP酶在细胞质中具有大致相等的程度， 暴露的和内在的膜区域，小的两亲性 分子如庚烷三醇将被测试其 促进结晶的作用。 通过以下方式获得的任何晶体 这些方法将通过X射线测量晶格有序度 衍射静止或进动照片。

The Ca-ATPase of fast twitch muscle sarcoplasmic reticulum is representative of a class of ion pumps involved in the homeostasis of the cations Na+, K+, H+ and Ca+2. The similarities in structures and kinetics of these ion pumps (Na/K-ATPase, H/K- ATPase and Ca-ATPase) have been interpreted to indicate that they have been derived from a common ancestral gene (1). Ca+2 is unique among these cations in that if functions as a messenger in response to hormones and transmitters. Loss of Ca-pump activity is associated with such diseases as Duchenne muscular dystrophy (2), Brody's disease (3) and experimental myotonia (4). The work proposed here has the specific aim of crystallizing rabbit sarcoplasmic reticulum Ca-ATPase with the long-term objective of obtaining detailed structural information through application of diffraction methods to such crystals. Ca-ATPase will be purified in the laboratory of Prof. F.N. Briggs using procedures developed in that laboratory (5), which yield homogeneous enzyme. This material is solubilized in the presence of detergents, and will be used in this form as the starting material for crystallization trials. Ca-ATPase exists in two stable conformational forms, and in different states of aggregation. For crystallization, those conditions known to favor the monomer will be used. Trails to optimize these conditions will be made by combining and exchanging detergents, as was done in the case of porin crystallization. Aggregation state will be monitored either by analytical HPLC or centrifugation in the presence of detergent. Mild succinylation and amidination of the Ca-ATPase will also be tried, since the former has been shown to reduce aggregation. The two conformational states: high (E1) and low (E2) calcium affinity, will be selected through binding of ligands known to stabilize one of the two states. The more stable, Ca-bound E1 form will be screened for crystallization first. Divalent transition metals and several different nucleotides will be tested for their effects on crystallization. The E2 state exists as a dimer in vesicles and is stabilized by vanadate. Vanadate- bound enzyme will be screened in crystallization trials, varying detergent, precipitant concentrations and pH. Crystallization trials will be done by the sitting drop or microdialysis methods. Since Ca-ATPase has roughly equal extent of cytoplasmically exposed and intrinsic membrane regions, small amphiphilic molecules such as the heptane-triols will be tested for their effects in promoting crystallization. Any crystals obtained by these methods will be surveyed for lattice order by x-ray diffraction still or precession photographs.