MOLECULAR BIOLOGY OF ORGANELLAR CA-ATPASE ISOFORMS

本应用程序的长期目标是了解 钙泵分子结构和功能 细胞内的细胞器，并了解它们的调节如何影响 平滑肌和非肌肉细胞的细胞生理学。 具体目标如下： 1.分离和表征编码细胞内钙的所有亚型 泵，以确定其在不同组织中的表达水平， 细胞系，并定义它们在细胞内的位置。为此将 使用cDNA克隆技术来定义同种型，并使用 稳态信息水平（即RNA酶保护）的定量分析 测定）。我们将解决 不同亚型的细胞内定位问题， 特别是关于钙储存的不同隔室， 使用同种型特异性抗体结合 免疫组化和免疫电镜方法和膜 分馏程序。 2.为了表达编码不同Ca-ATP酶亚型的cDNA分子， COS-1细胞，从而确定各种生化参数， 每个人的功能和结构。这些参数将包括：范围（如果 任何）糖基化，COS-1细胞内的定位， 分子的周转数，无论是ATP水解， 钙转运，转运功能对钙的依赖性 在一些实施方案中，所述方法基于ATP（和其他核苷酸）浓度、基于ATP（和其他核苷酸）浓度和基于pH。 结构与功能的关系将在性能方面得到解决 其在使用嵌合和定点突变的同种型之间不同， 分子。 3.为了检测Ca-ATPase的泵送特性在多大程度上 同种型在细胞内受到调节。钙吸收特性 将使用抑制性同种型， 特异性抗体，以评估每个物种对总体 吸收，在各种条件下的钙浓度，pH值等。 配体如PMA、cGMP、cAMP对钙摄取的影响将在本文中详细讨论。 监测。不同的钙泵亚型与不同的 细胞内钙池将被解决。小说的目标 还将研究肿瘤促进剂毒胡萝卜素。 这些研究将有助于阐明细胞质内 钙浓度-控制各种细胞的关键因素 事件-和它的调节激素效应。

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.