MOLECULAR BIOLOGY OF ORGANELLAR CA-ATPASE ISOFORMS

这个应用程序的长期目标是理解 细胞内钙泵分子的结构和功能 细胞内细胞器，并了解它们的调节如何影响 平滑肌细胞和非肌肉细胞的细胞生理学。 具体目标如下： 1.分离和鉴定编码细胞内钙离子的所有异构体 泵，以确定它们在不同组织和组织中的表达水平 细胞系，并定义它们在细胞内的位置。这件事会做到的 利用cDNA克隆技术确定异构体，并使用 对稳态消息级别(即核糖核酸酶保护)进行量化分析 实验)用于鉴定的每个不同的cDNA克隆。我们将解决 不同亚型在细胞内的位置问题， 尤其是关于钙存储的不同隔室，通过 将同种异型特异性抗体与 免疫组织化学和免疫电子显微镜方法及细胞膜 分馏程序。 2.表达编码不同Ca-ATPase异构体的c DNA分子 COS-1细胞，从而测定其各种生化参数。 每种产品的功能和结构。这些参数将包括：范围(如果 任何)糖基化，在COS-1细胞内的定位，定量 分子的翻转数，无论是对三磷酸腺苷水解还是对三磷酸腺苷 钙转运，转运功能对钙的依赖 浓度、三磷酸腺苷(和其他核苷酸)浓度以及pH值。 结构与功能的关系将针对属性进行讨论 它们在使用嵌合突变和定点突变的异构体中有所不同 分子。 3.检测Ca-ATPase的泵浦特性 异构体在细胞内受到调节。钙摄取的特性 将使用抑制亚型来检测进入皂苷渗透性的细胞 评估每个物种对总体的贡献的特定抗体 摄取，在各种条件下的钙浓度，pH等。 配体如PMA，cGMP，cAMP对钙摄取的影响将是 被监视着。不同的钙泵亚型与不同的 将解决细胞内钙池的问题。这部小说的目标 肿瘤促进剂thapsigargin也将被调查。 这些研究将阐明控制细胞质的机制。 钙浓度--控制多种细胞的关键因素 事件--以及荷尔蒙效应器对其的调节。

The long-term objectives of this application are to understand the structure and function of calcium pump molecules which reside in intracellular organelles, and to understand how their regulation influences cell physiology in both smooth muscle and non-muscle cells. The specific aims are as follows: 1. To isolate and characterize all isoforms encoding intracellular calcium pumps, to determine the level of their expression in different tissues and cell lines, and to define their location within the cell. This will be done using techniques of cDNA cloning to define the isoforms, and using quantitative analyses of steady state message levels (i.e. RNase protection assay) for each of the different cDNA clones identified. We will address the question of intracellular location of the different isoforms, particularly with regard to distinct compartments of calcium storage, by employing isoform specific antibodies in conjunction with immunohistochemical and immunoelectron microscopic methods and membrane fractionation procedures. 2. To express cDNA molecules encoding the different Ca-ATPase isoforms in COS-1 cells, and thus to determine various biochemical parameters of function and structure for each. These parameters will include: extent (if any) of glycosylation, localization within the COS-1 cells, quantitation of the turn-over number of the molecules, both for ATP hydrolysis and for calcium transport, the dependence of the transport function upon calcium concentration, upon ATP (and other nucleotide) concentration, and upon pH. The relation of structure to function will be addressed for properties which differ among the isoforms using chimeric and site-directed mutated molecules. 3. To examine the extent to which the pumping properties of Ca-ATPase isoforms are regulated within the cell. The properties of calcium uptake into saponin permeabilized cells will be examined, using inhibitory isoform specific antibodies to assess the contribution of each species to overall uptake, under a variety of conditions of calcium concentration, pH, etc. The effect of ligands such as PMA, cGMP, cAMP on calcium uptake will be monitored. The association of different calcium pump isoforms with distinct intracellular calcium pools will be addressed. The target for the novel tumour promoter, thapsigargin, will also be investigated. These studies will shed light on the mechanisms governing cytoplasmic calcium concentration - the key element controlling a variety of cellular events - and its regulation by hormonal effectors.