CALCIUM BINDING PROTEIN FUNCTION AND VITAMIN D

在生化调节剂和钙之间有重要联系 (Ca++)和钙结合蛋白(CABP)，以及维生素D内分泌 系统。然而，许多CABP的确切功能尚不清楚， 如1，25(OH)2D参与的机制(S)的细节 他们的规定。这项建议的总体目标是理解两个 这些复杂和相互关联的问题的各个方面：(A)职能和 1，25(OH)2D诱导的钙调素受体蛋白的特性 (185kD和115kD)，我们最近通过125I-CaM凝胶鉴定了它 在大鼠肾脏胞浆中的覆盖程序，以及(B)是否有功能 与维生素D相关的钙结合蛋白-D28K(CABP-D28K)也涉及特定的 受体蛋白。1，25(OH)2D对CaM受体的诱导作用 是1，25(OH)2D3受体介导的事件将通过比较 钙调素受体诱导为已知钙调素受体的时间进程 1，25(OH)2D受体的生化后遗症。此外，我们还将 确定类固醇对其诱导的特异性，是否程度 它们的诱导与受体水平相关，以及蛋白质和 它们的诱导需要RNA的合成。两者都在体内(维生素D 缺陷大鼠与1，25(OH)2D刺激大鼠)和体外(肾细胞 已知含有1，25(OH)2D受体的线条将被测试。 1，25(OH)2D诱导的钙调素受体的功能特性 通过确定它们在肾单位和其他部位的位置进行调查 亚细胞组分，通过测试后细胞内的再分布 1，25(OH)2D刺激，并通过确定它们是否与CABP的其他 而不是卡姆。1，25(OH)2D诱导的CaM受体将被纯化 产生抗体，并测定部分氨基酸序列以 与其他已知序列进行比较。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 增强了它们与CaM结合的能力。最后，无论CABP-D28K 也将通过修饰来测试与特定受体蛋白的相互作用 凝胶覆盖程序和在各种情况下结合的测试 不同的生化和生理条件。尤其是，这些 研究将利用纯化的CABP-D28K，而不暴露于钙螯合 似乎与蛋白质有复杂关系的试剂。此外，可能的疲软(和 因此，CABP-D28K与CaM受体的相互作用 也要接受测试。如果确定了假定的CABP-D28K受体，他们将 其特点是采用类似于为凸轮提出的技术- 接受者。这些研究将为理解 CABP-D28K和CaM受体位点的功能及其在调节中的作用 1，25(OH)2D在其主要靶组织中的作用。

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.