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.

在生化调节剂，钙， (Ca++）和钙结合蛋白（CaBP），以及维生素D内分泌 系统 然而，许多CaBP的精确功能是未知的， 如1，25（OH）2D参与的机制的细节 他们的规则。 本提案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： 1，25（OH）2D诱导的钙调素受体蛋白的特性 (185 KD和115 KD），我们最近用125-I-CaM凝胶鉴定了它们 覆盖程序在大鼠肾细胞质中的作用，以及（B）是否 维生素D相关的钙结合蛋白-D28 K（CaBP-D28 K）也涉及特异性 受体蛋白 1，25（OH）2D是否诱导了钙调素受体位点 是1，25（OH）2D 3受体介导的事件，将通过比较 钙调素受体诱导到已知受体的时间过程 1，25（OH）2D受体的生化后遗症。 此外，我们会 确定其诱导类固醇特异性，是否程度 它们的诱导与受体水平相关， RNA合成是其诱导所必需的。 体内（维生素D 缺陷大鼠与1，25（OH）2D刺激的大鼠）和体外（肾细胞 已知含有1，25（OH）2D受体的细胞系）模型进行测试。 1，25（OH）2D诱导的钙调素受体的功能特性将是 通过确定它们在肾单位和其他组织中的位置来研究 亚细胞部分，通过测试细胞内的再分布， 1，25（OH）2D刺激，并通过确定它们是否结合CaBP的其他功能， 比CaM 将纯化1，25（OH）2D诱导的CaM受体， 抗体产生和测定部分氨基酸序列， 与其他已知序列相比。 有了抗体，1，25（OH）2D- CaM受体蛋白的诱导将与1，25（OH）2D进行比较 增强其钙调素结合能力。 最后，CaBP-D28 K 也与特定受体蛋白相互作用，将通过修饰 凝胶覆盖程序和在各种条件下的结合测试 不同的生化和生理条件。 特别是这些 研究将使用未暴露于Ca螯合物的纯化CaBP-D28 K 试剂，这些试剂似乎对蛋白质来说很复杂。 此外，可能的弱（和 因此，CaBP-D28 K与CaM受体的非功能性相互作用将 也被测试。 如果鉴定出推定的CaBP-D28 K受体，它们将 其特征在于与CaM提出的技术相似的技术- 受体。 这些研究将为理解 CaBP-D28 K和CaM受体位点的功能及其在介导 1，25（OH）2D在其主要靶组织中的作用。