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Basic Study on Structure and Function of Mammalian Kidney Lectin.

哺乳动物肾凝集素结构与功能的基础研究。

基本信息

批准号：
62580113
负责人：
MATSUMOTO Isamu
金额：
$ 0.96万
依托单位：
Ochanomizu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

We have reported the partial purification and characterization of mammalian kidney lectins specific to sialic acid. Recently we have found the similar lectins also in chicken kidney. The common distribution of the kidney lectins in all the animals examined suggests that they play an importantrole in kidney specific functions. In this study, we developed new methods to detect and purify the kidney lectins and found their novel properties. In the new lectin detection method, the lectin sample was spotted on nitrocellulose membrane, washed with the solution of bovine serum albumin, reconstituted with phosphatidylethanolamine (PE), and allowed to react with horseradish peroxidase-labeled glycoproteins, and then the resulting binding was detected by coloration with diaminobenzidine. By this method the kidney extract devoid of lipids was found to bind strongly sialoglycoprotein only upon reconstitution with PE. Therefore, the inactivation of the lectins during purification procedures such as ion exchange chromatography and affinity chromatography could be explained by the removal of phospholipids essential for the lectin activity. One of the chicken lectins was recovered in fractions eluted with tris-buffered saline on hydrophobic chromatography using C18-Sepharose column and shown to contain several protein bands on SDS-PAGE analysis. When these bands were transfered to nitrocellulose membrane and examined by the new method, 31kDa protein was found to be a sialic acid specific lectin. furthermore, the lectin activity of the 31kDa protein was reactivated not only with PE, but also with PC, PI and PS. Therefore, the polar head group charge of phospholipids seemsnot to be directly concerned in the binding with sialic acid. It was also shown that the 31kDa protein has O<@D5-@>D5-linked oligosaccharides, and isoelectric point of <@DBca(/)-@>DDB.7, and its amino terminal blocked.

我们已经报道了哺乳动物肾脏凝集素特异性唾液酸的部分纯化和表征。最近在鸡肾中也发现了类似的凝集素。肾凝集素在所有被检查动物中的共同分布表明它们在肾脏特异性功能中发挥重要作用。本研究建立了新的检测和纯化肾凝集素的方法，并发现了其新的性质。在新的凝集素检测方法中，凝集素样品点在硝酸纤维素膜上，用牛血清白蛋白溶液洗涤，用磷脂酰乙醇胺（PE）重构，并允许与辣根过氧化物酶标记的糖蛋白反应，然后通过二氨基联苯胺显色检测所产生的结合。通过该方法，发现不含脂质的肾提取物仅在用PE重构时才与唾液酸糖蛋白强烈结合。因此，在纯化过程中，如离子交换层析和亲和层析的凝集素的失活可以解释为磷脂的凝集素活性所必需的去除。其中一种鸡凝集素在使用C18-Sepharose柱的疏水层析上用Tris缓冲盐水洗脱的级分中回收，并在SDS-PAGE分析上显示含有几个蛋白条带。将这些条带转移到硝酸纤维素膜上，用新的方法检测，发现31 kDa的蛋白是唾液酸特异性凝集素。此外，31 kDa蛋白的凝集素活性不仅被PE激活，而且被PC、PI和PS激活。因此，磷脂的极性头基电荷与唾液酸的结合没有直接关系。该蛋白分子量为31 kDa，具有O<@D5-@>D5连接的寡糖，等电点为<@DBca（/）-@>DDB.7，氨基端封闭。