Pansini A R, Christakos S
J Biol Chem. 1984 Aug 10;259(15):9735-41.
A vitamin D-dependent Mr = 28,000 calcium-binding protein (CaBP) has been isolated from rat kidney. Rat renal CaBP was purified from heat-treated post-mitochondrial supernatants by gel filtration on Sephadex G-100 followed by preparative gel electrophoresis. The specific properties and characteristics of the protein were examined. Rat renal CaBP was found to have a pI of 4.8 and showed increased electrophoretic mobility during polyacrylamide gel electrophoresis in the presence of 1 mM EDTA. Amino acid analysis of renal CaBP revealed a high content of glutamic and aspartic acids and a low level of methionine, histidine, cysteine, and tyrosine, similar but not identical to chick intestinal CaBP. Circular dichroism studies indicated that the alpha-helical content of renal CaBP was of the order of 31% and was changed to a minor degree by the addition of calcium. A study of the thermal stability indicated that renal CaBP is heat-stable up to 75 degrees C. Binding studies utilizing the technique of equilibrium dialysis established a dissociation constant of 2.1 X 10(-6) M and binding sites with a capacity of approximately 4 mol Ca2+/mol of CaBP. Immunologically, using Ouchterlony immunodiffusion, a precipitin line which joined with total coalescence with that due to purified renal CaBP was observed with extracts of rat brain and kidney but not with extracts of rat parathyroid, skeletal muscle, myocardium, bone, pancreas, intestine, and liver. In summary, these studies present the first purification and characterization of vitamin D-dependent rat renal calcium-binding protein. The antibody as well as the protein will be useful for the development of a radioimmunoassay and for the determination of the molecular mechanisms of induction of renal calcium-binding protein.
已从大鼠肾脏中分离出一种维生素D依赖性的分子量为28,000的钙结合蛋白（CaBP）。大鼠肾脏CaBP是通过在Sephadex G - 100上进行凝胶过滤，随后进行制备性凝胶电泳，从热处理后的线粒体后上清液中纯化得到的。对该蛋白质的特定性质和特征进行了检测。发现大鼠肾脏CaBP的pI为4.8，并且在1 mM EDTA存在下进行聚丙烯酰胺凝胶电泳时，其电泳迁移率增加。对肾脏CaBP的氨基酸分析显示，谷氨酸和天冬氨酸含量高，而甲硫氨酸、组氨酸、半胱氨酸和酪氨酸水平低，与鸡肠道CaBP相似但不完全相同。圆二色性研究表明，肾脏CaBP的α - 螺旋含量约为31%，添加钙后略有变化。热稳定性研究表明，肾脏CaBP在高达75摄氏度时是热稳定的。利用平衡透析技术进行的结合研究确定解离常数为2.1×10⁻⁶ M，结合位点的容量约为每摩尔CaBP结合4摩尔Ca²⁺。在免疫方面，使用双向免疫扩散法，观察到大鼠脑和肾脏提取物与纯化的肾脏CaBP产生的沉淀线完全融合，而大鼠甲状旁腺、骨骼肌、心肌、骨骼、胰腺、肠道和肝脏的提取物则没有。总之，这些研究首次对维生素D依赖性大鼠肾脏钙结合蛋白进行了纯化和表征。该抗体以及该蛋白质将有助于开发放射免疫测定法，并用于确定肾脏钙结合蛋白诱导的分子机制。
