MECHANISMS OF D-STIMULATED INTESTINAL CALCIUM TRANSPORT

D-刺激肠道钙转运的机制

• 批准号: 3228600
• 负责人: DANIEL David BIKLE
• 金额: $ 14.12万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-04-01 至 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3228600
• 关键词: 1,25 dihydroxycholecalciferol; SDS polyacrylamide gel electrophoresis; adenosinetriphosphatase; alkaline phosphatase; basolateral membrane; brush border membrane; calcium channel; calcium metabolism; calmodulin; chickens; cyclic AMP; cyclic nucleoside monophosphate; cycloheximide; dietary calcium; duodenum; epithelium; gastrointestinal nutrient absorption; gel electrophoresis; hormone regulation /control mechanism; intestines; laboratory rabbit; membrane permeability; mitochondria; nutrition related tag; phosphorylation; protein biosynthesis; protein kinase

Calcium transport across the intestine requires the entry of calcium into the intestinal epithelial cell across the brush border membrane (BBM), passage through the cell, and removal at the basolateral membrane, 1,25(OH)2D regulates calcium transport at each of these steps but the molecular mechanisms are poorly understood. At the BBM calcium transport is increased by 1,25(OH)2D presumably by changes in the function of an as yet unidentified carrier or channel. Associated with the increases in calcium transport across the BBM is an increase in calmodulin (CaM) binding to a 102,000 mol wt protein in the BBM; CaM antagonists block 1,25(OH)2D stimulated calcium transport across the BBM and block the binding of CaM to this protein. Neither the increased calcium transport nor the increased CaM binding to this protein following 1,25(OH)2D are blocked by bycloheximide despite marked inhibition of protein synthesis. Our hypothesis is that this 102,000 mol wt protein is the calcium channel or a modulator of the calcium channel in the BBM and is central to 1,25(OH)2D regulation of calcium transport across the BBM. We plan to purify this protein by a combination of membrane extraction, salt precipitation, HPLC, and SDS polyacrylamide gel electrophoresis. The purified protein will be evaluated for its capacity to act as a calcium channel or a regulator of such a channel by reconstitution experiments with liposomes. Comparison of the protein isolated from vitamin-D deficient and vitamin-D replete chicks will be made with respect to CaM binding and calcium inophore activity. Changes in phosphorylation and glycosylation of this protein after 1,25(OH)2D administration will be sought. The protein will be used as antigen to develop a polyclonal antiserum for immunolocalization studies, immunoassays, Western blots, and affinity chromatography. From the purified protein partial amino acid sequences will be determined to guide the synthesis of oligonucleotide probes which will be used to select the appropriate clone(s) from a cDNA library prepared from chick intestinal mRNA. After identifying and sequencing the clones spanning the reading frame for the protein, the primary amino acid structure will be deduced, and computer searches made to identify homologous sequences in other proteins. Regions of particular interest such as the CaM binding domain and transmembrane domains will be sought and compared to other CaM binding proteins and ion channels. From these studies a clearer molecular understanding of calcium transport across the BBM should emerge.

跨肠道的钙运输需要将钙进入 横跨刷子边界膜（BBM）的肠上皮细胞， 通过细胞，并在基底外侧膜上去除， 1,25（OH）2D在每个步骤中调节钙的运输，但 分子机制知之甚少。 在BBM钙运输 大概会随着AS的功能的变化而增加1,25（OH）2D 但身份不明的载体或渠道。 与增加有关 跨BBM的钙转运是钙调蛋白（CAM）结合的增加 BBM中的102,000 mol WT蛋白；凸轮拮抗剂阻塞1,25（OH）2D 刺激钙在BBM上的钙转运，并阻止CAM与 该蛋白质。 钙转运增加也不增加 1,25（OH）2D后，凸轮与该蛋白质结合被阻塞 尽管明显抑制了蛋白质合成，但旁己二酰亚胺尽管有明显的抑制作用。 我们的 假设是，这种102,000 mol WT蛋白是钙通道或A BBM中钙通道的调节剂，为1,25（OH）2D中心 调节整个BBM的钙转运。 我们计划净化这一点 蛋白质结合膜提取，盐沉淀，HPLC， 和SDS聚丙烯酰胺凝胶电泳。 纯化的蛋白质将是 评估其充当钙通道或调节器的能力 通过对脂质体进行重构实验的通道。 比较 从维生素-D缺乏和维生素-D的鸡中分离出的蛋白质 将在CAM结合和钙无体面活性方面制作。 该蛋白的磷酸化和糖基化的变化 将寻求1,25（OH）2D管理。 该蛋白质将被用作 抗原开发多克隆抗血清进行免疫定位研究， 免疫测定，蛋白质印迹和亲和色谱法。 来自 纯化的蛋白质部分氨基酸序列将确定引导 寡核苷酸探针的合成，该探针将用于选择 由雏鸡肠道制备的cDNA文库的适当克隆 mRNA。 识别和测序跨越读数的克隆 蛋白质的框架，将推导主要的氨基酸结构， 和计算机搜索以识别其他 蛋白质。 特别感兴趣的区域，例如凸轮结合域 将寻求跨膜结构域并将其与其他CAM结合进行比较 蛋白质和离子通道。 从这些研究中，分子更清晰 了解跨BBM的钙转运应出现。