摘要
降钙素基因相关肽（ＣＧＲＰ）为３７肽，它在体内主要分布于神经系统和心血管系统，是较强的扩张血管物质。人们已认识到脑部的ＣＧＲＰ最多，由于新鲜猪脑来源丰富，故我们选用猪脑为材料来纯化ＣＧＲＰ受体。以期为该受体的深入研究提供足够的材料和奠定基础，纯化的受体将用于制备抗ＣＧＲＰ受体的单克隆抗体，为了从猪脑中纯化ＣＲＲＰ受体，首先用含有胆盐的磷酸盐缓冲液将该受体从猪脑细胞膜上解离下来，解离下来的受体量较多，而杂蛋白较少，且受体活性可保持较长时间不降低。以离子交换柱层析（ＱＡＥ－Ｓｅｐｈａｄｅｘ－Ａ－２５）进行初步分离纯化，再用化学合成的ＣＧＲＰ与活化的琼脂糖（Ａｆｆｉ－Ｇｅｌ－１０，Ｂｉｏ－Ｒａｄ公司产品）制备的亲和层析柱进行亲和层析，取得的纯化受体保留了与１２５Ｉ－ＣａＧＲＰ结合的能力，而与降钙素（Ｃａｌｃｉｔｏｎｉｎ）神经肽Ｙ（ＮｅｕｒｏｐｅｐｔｉｄｅＹ）和Ｋａｔａｃａｌｃｉｎ等无交叉反应，但与有４６％氨基酸序列同源性的Ａｍｙｌｉｎ有一定交叉反应。纯化的受体经ＳＤＳ－聚丙烯酰胺凝胶电泳和高压液相色谱（ＨＰＬＣ）鉴定呈现单一蛋白条带或蛋白峰，其位置对应分子量为６６ｋＤ。
Intact calcitonin gene-related peptide (CGRP) receptors were solubilised from porcine neural membranes using sodium cholate: potassium buffer (K2HPO4 : Na chloate=125 : 3 mmol/L). Under this buffer concentration the highest number of CGRP receptors and the lowest number of proteins were solubilised from the cerebellar membranes, and the maximum stability of CGRP receptors was maintained during storage. CGRP receptors were unstable at higher temperatures and at higher ionic concentrations of buffer and detergents. The addition of protease inhibitors (e.g.aprotinin 100 kIU/ml) , or protectants such as 20% glycerol and 10 mmol/ldithioerythretol, afforded parital protection and increased the stability of CGRP receptors. Specitic affinity gel columns were prepared with CGRP (1-37) for purification of CGRP receptor from porcine cerebellar membranes. Synthetic CGRP(1-37) was covalently coupled to activated agarose (Affi-Gel 10) at PH 7 and 4 ℃. The efficiency of the affinity column decreased to 60 % and 20% after 10 and 20 runs respectively. The isolated receptors retained their capacity of binding i25 I-CGRP, were specific to CGRP and showed no cross reactivity to a number of other peptides,including calcitonin, katacalcin and neuropeptide Y. However, some cross-reactivity was observed wity peptide-amylin having 46 % amino acid sequence homology to hCGRP. CGRP receptor were identified and characterized as a monomeric membrane protein with Mr 66kD by both SDSPAGE and gel permeation chromatography. We have developed and optimised the procedures for isolation, purification and characterization of functional CGRP receptors.
