SARCOPLASMIC RETICULUM CALCIUM ATPASE

肌浆网钙ATP酶

• 批准号: 6349159
• 负责人: GIUSEPPE INESI
• 金额: $ 22.3万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6349159
• 关键词: active sites; calcium flux; calcium transporting ATPase; cardiac myocytes; enzyme inhibitors; enzyme mechanism; enzyme structure; heart function; immunofluorescence technique; intracellular transport; ion transport; molecular cloning; protein structure function; sarcoplasmic reticulum; site directed mutagenesis; thapsigargin; tissue /cell culture; transfection

The sarcoplasmic reticulum ATPase concentrates cytosolic Ca2+ into intracellular compartments, thereby permitting relaxation and subsequent tension development of cardiac and skeletal muscle. We will use ATPase proteins and cDNA clones to study catalytic, transport and inhibitory mechanisms, as well as ATPase expression following gene transfer, as follows: 1-Characterization of the mechanism of Ca2+ binding, ATP utilization, intermediate enzyme phosphorylation and Ca2+ transport, as they relate to ATPase topology and structure. Following previous information obtained by indirect measurement, we will extend our mutational analysis by expressing mutated ATPase in large quantities for direct measurements of stoichiometry, equilibrium and kinetic constants, and the effects of specific amino acid mutations of the partial reactions of the catalytic and transport cycle. ATPase mutants will be also used for spectroscopic studies to relate functional steps to structural features, an explain intermolecular linkages of separate function domains. 2-Topology of the specific ATPase inhibition by thapsigargin. We will synthesize high affinity thapsigargin analogs, including a radioactive azido derivative for (covalent) photo labeling of the native ATPase, proteolytic digestion, proteolytic digestion, proteolytic digestion, sequencing and determination of labeled amino acid(s) as the specific binding site. Furthermore guided by previous findings on the effects of large chimerical exchanges, we will test the effects of specific amino acid mutations on the sensitivity of the Ca2+ ATPase to thapsigargin. The specific aim is to define the topology of the thapsigargin inhibitory site and characterize the inhibitory mechanism. 3-Gene transfer and ATPase expression in foreign cells and cardiac muscle. A variety of cultured cells including cardiac myocytes will be transfected with SERCA1 or SERCA2A cDNA using various methods to obtain maximal efficiency and expression of Ca2+ ATPase. A variety of DNA constructs will be used, including dual gene constructs for expression under selective pressure, high yield promoters for overexpression, and tissue specific promoters for cardiac cells. Expression and targeting will be monitored by immunofluorescence in permeabilized cells and in isolated subcellular fractions. The specific aims are to: (a) recover large amounts of expressed ATPase for mutational studies of transport and catalytic function by the isolated enzyme, and (b) used gene transfer to established functional intracellular organelles and to influence cytosolic Ca2+ homeostasis.

肌浆网ATP酶将胞浆Ca 2+浓缩成 细胞内区室，从而允许松弛和随后的 心肌和骨骼肌张力发展。 我们将使用ATP酶 蛋白质和cDNA克隆研究催化，运输和抑制 机制，以及基因转移后的ATP酶表达， 如下：1-Ca 2+结合机制的表征，ATP 利用，中间酶磷酸化和Ca 2+转运，作为 它们涉及ATP酶的拓扑学和结构。 按照以往 通过间接测量获得的信息，我们将扩展我们的突变 通过大量表达突变的ATP酶进行直接分析， 化学计量、平衡和动力学常数的测量，以及 特定氨基酸突变的部分反应的影响， 催化和运输循环。 ATP酶突变体也将用于 光谱研究，将功能步骤与结构特征联系起来， 解释独立功能域的分子间连接。 2-拓扑 毒胡萝卜素对ATP酶的特异性抑制作用。 我们将合成高 亲和毒胡萝卜素类似物，包括放射性叠氮衍生物， 天然ATP酶的（共价）光标记，蛋白水解消化， 蛋白水解消化、蛋白水解消化、测序和测定 标记的氨基酸作为特异性结合位点。 此外， 根据先前关于大规模虚拟交换影响的研究结果，我们将 测试特定氨基酸突变对检测灵敏度的影响。 Ca 2 + ATP酶对毒胡萝卜素的作用。 具体目标是定义 thapsigargin抑制位点，并表征抑制机制。 3-外源细胞和心肌中的基因转移和ATP酶表达。 将转染包括心肌细胞在内的各种培养细胞 用SERCA 1或SERCA 2A cDNA，使用各种方法， Ca ~（2+）ATP酶的表达。 各种DNA构建体将 包括用于在选择性条件下表达的双基因构建体。 压力，高产量启动子过表达，和组织特异性 心脏细胞的启动子。 表达和靶向将由 透化细胞和分离的亚细胞中的免疫荧光 分数 具体目标是：（a）收回大量的 表达的ATP酶，用于转运和催化功能的突变研究 通过分离的酶，和（B）使用基因转移建立 功能性细胞内细胞器，并影响胞质Ca 2 + 体内平衡