Basic Study on Structure and Function of Mammalian Kidney Lectin.

我们报道了哺乳动物肾脏唾液酸凝集素的部分纯化和性质。最近，我们在鸡肾中也发现了类似的凝集素。肾脏凝集素在所有被研究动物中的共同分布表明，它们在肾脏的特定功能中发挥着重要的调控作用。在本研究中，我们开发了检测和纯化肾脏凝集素的新方法，并发现了它们的新性质。在新的凝集素检测方法中，凝集素样品在硝酸纤维素膜上斑点，用牛血清白蛋白溶液洗涤，与磷脂酰乙醇胺(PE)重组，允许与辣根过氧化物酶标记的糖蛋白反应，然后用二氨基联胺显色检测结合情况。用这种方法发现，去脂肾提取物只有在与PE重组时才能与唾液糖蛋白强烈结合。因此，在离子交换层析和亲和层析等纯化过程中凝集素的失活可以通过去除凝集素活性所必需的磷脂来解释。鸡肉凝集素经C18-Sepharose柱疏水层析后，经三缓冲液洗脱，分离得到一种凝集素，经SDS-PAGE分析显示含有多条蛋白带。将这些条带转移到硝酸纤维素膜上，用新的方法进行检测，发现31 kDa的蛋白质是唾液酸特异的凝集素。此外，31 kDa蛋白的凝集素活性不仅被PE激活，而且还被PC、PI和PS激活。因此，磷脂的极性头基电荷似乎与唾液酸的结合没有直接关系。31 kDa蛋白含有O&lt；@D5-@&gt；D5连接的寡糖，等电点为&lt；@DBca(/)-@&gt；DDB.7，其氨基末端封闭。

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.