CALCIUM BINDING PROTEIN FUNCTION AND VITAMIN D

钙结合蛋白功能和维​​生素 D

• 批准号: 3245352
• 负责人: MARIAN R WALTERS
• 金额: $ 12.48万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-03 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3245352
• 关键词: 1,25 dihydroxycholecalciferol; active sites; affinity chromatography; animal tissue; antibody; biological signal transduction; calcium; calcium binding protein; calmodulin; cycloheximide; endocrine gland /system; hormone regulation /control mechanism; kidney; laboratory rabbit; laboratory rat; protein biosynthesis; protein purification; protein structure function; radiotracer; receptor; receptor sensitivity; renal glomerulus; renal tubule; tissue /cell culture; tritium; western blottings

There are important links between the biochemical regulators, calcium (Ca++) and calcium binding proteins (CaBP's), and the vitamin D endocrine system. However, the precise functions of many of the CaBP's are unknown, as are details of the mechanism(s) by which 1,25(OH)2D participates in their regulation. The overall goal of this proposal is to understand two aspects of these complex and interrelated problems: (a) the functions and characteristics of 1,25(OH)2D-induced calmodulin (CaM)-acceptor proteins (185 KD and 115 KD) which we recently identified by the 125-I-CaM gel overlay procedure in the rat kidney cytosol, and (b) whether the functions of the vitamin D-related calbindin-D28K (CaBP-D28K) also involve specific acceptor proteins. Whether 1,25(OH)2D induction of the CaM-acceptor sites is a 1,25(OH)2D3 receptor-mediated event will be investigated by comparing the time course of induction of the CaM-acceptors to those of known biochemical sequelae of the 1,25(OH)2D receptors. In addition, we will determine the steroid specificity of their induction, whether the degree of their induction correlates with receptor levels, and whether protein and RNA synthesis are required for their induction. Both in vivo (vitamin D deficient rats vs. 1,25(OH)2D-stimulated rats) and in vitro (kidney cell lines known to contain 1,25(OH)2D receptors) models will be tested. Functional characteristics of the 1,25(OH)2D-induced CaM-acceptors will be investigated by establishing their location along the nephron and in other subcellular fractions, by testing for re-distribution within the cell after 1,25(OH)2D stimulation, and by determining whether they bind CaBP's other than CaM. The 1,25(OH)2D-induced CaM acceptors will be purified for antibody production and for determining a partial amino acid sequence to compare to other known sequences. With the antibodies, 1,25(OH)2D- induction of the CaM-acceptor proteins will be compared to 1,25(OH)2D enhancement of their CaM-binding capability. Finally, whether CaBP-D28K also interacts with specific acceptor proteins will be tested by modifying the gel overlay procedure and testing for binding under a variety of different biochemical and physiological conditions. In particular, these studies will utilize CaBP-D28K purified without exposure to Ca-chelating agents, which seem to complex to the protein. Further, possible weak (and thus non-functional) interactions of the CaBP-D28K with CaM-acceptors will also be tested. If putative CaBP-D28K acceptors are identified, they will be characterized by techniques similar to those proposed for the CaM- acceptors. These studies will provide a solid basis for understanding the functions of CaBP-D28K and CaM-acceptor sites and their roles in mediating the effects of 1,25(OH)2D in its principal target tissues.

生化调节剂，钙之间存在重要联系 （Ca ++）和钙结合蛋白（CABP）以及维生素D内分泌 系统。 但是，许多CABP的确切功能是未知的， 正如1,25（OH）2d参与的机制的细节 他们的监管。 该提议的总体目标是了解两个 这些复杂和相互关联的问题的各个方面：（a）功能和功能 1,25（OH）2D诱导的钙调蛋白（CAM） - 受体蛋白的特征 （185 kd和115 kd），我们最近通过125-i-Cam凝胶确定了这一点 大鼠肾脏细胞质中的叠加过程，以及（b）功能是否功能 维生素D相关的Calbindin-D28K（CABP-D28K）也涉及特定 受体蛋白。 是否1,25（OH）2D诱导CAM-Acceptor站点 是1,25（OH）2d3受体介导的事件将通过比较 CAM受访者归纳的时间过程 1,25（OH）2D受体的生化后遗症。 此外，我们将 确定其诱导的类固醇特异性，是否程度 它们的诱导与受体水平以及蛋白质和是否相关 RNA合成是其诱导所必需的。 两者都体内（维生素D 不足的大鼠与1,25（OH）2D刺激的大鼠）和体外（肾细胞 将测试已知包含1,25（OH）2D受体）模型的线。 1,25（OH）2D引起的CAM受体的功能特性将是 通过沿肾单位的位置建立其位置来调查 亚细胞分数，通过测试细胞内的重新分布之后 1,25（OH）2D刺激，并确定它们是否绑定CABP的其他 比凸轮。 1,25（OH）2D引起的凸轮受体将被净化 抗体产生和确定部分氨基酸序列 与其他已知序列进行比较。 使用抗体，1,25（OH）2d- CAM受体蛋白的诱导将与1,25（OH）2D进行比较 增强其凸轮结合能力。 最后，是否CABP-D28K 还将通过修改来测试与特定受体蛋白的相互作用 凝胶叠加程序和测试在各种下结合 不同的生化和生理条件。 特别是这些 研究将利用CABP-D28K纯化而不会暴露于Ca-Chelating 代理，似乎对蛋白质复杂。 此外，可能的弱（和 因此，CABP-D28K与CAM受体的无功能相互作用将 也可以测试。 如果确定了假定的CABP-D28K受体，他们将 具有类似于凸轮的技术的特征 受体。 这些研究将为理解 CABP-D28K和CAM-Acceptor站点的功能及其在中介中的作用 1,25（OH）2d在其主要目标组织中的影响。