从犬肾传代细胞（MDCK）中分离并鉴定顶端分泌的80千道尔顿糖蛋白

Purification and characterization of the apically secreted 80 KDa glycoprotein from Madin-Darby canine kidney (MDCK) cells.

作者信息

Liu J W, Lu R L, Han J R, Liu M C

机构信息

Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019.

出版信息

Biochem Int. 1991 Sep;25(1):109-21.

PMID:1772437

Abstract

An 80 KDa glycoprotein (gp 80), known to be released predominantly from the apical surface by filter-grown Madin-Darby canine kidney cells, was purified to electrophoretic homogeneity. Purified gp 80 was found to have a disulfide-bonded dimeric structure, and appeared to exist in two molecular forms, a major (high-molecular weight) form consisting of a 46 KDa subunit and a 39 KDa subunit and a minor (low-molecular weight) form consisting of a 46 KDa subunit and a 33 KDa subunit. Upon de-glycosylation by N-glycanase treatment, the 46 KDa subunit was converted to a 25.6 KDa form, whereas both the 39 KDa and the 33 KDa subunit gave rise to a 21.1 KDa form. V8 protease mapping of deglycosylated polypeptides revealed the 39 KDa and the 33 KDa subunit to have nearly identical band patterns, which also exhibited a high degree of homology to that derived from the 46 KDa subunit. Radioimmunoassays revealed that the binding of the purified gp 80 to fibrinogen (or heparin) was dependent on both pH and divalent cations. Furthermore, binding of gp 80 to immobilized fibrinogen (or heparin) was inhibited in the presence of free fibrinogen (or heparin) added in the assay mixture.

摘要

一种80千道尔顿的糖蛋白（gp 80），已知主要由滤过生长的麦迪逊-达比犬肾细胞从顶端表面释放，已被纯化至电泳纯。纯化后的gp 80被发现具有二硫键连接的二聚体结构，并且似乎以两种分子形式存在，一种主要的（高分子量）形式由一个46千道尔顿的亚基和一个39千道尔顿的亚基组成，另一种次要的（低分子量）形式由一个46千道尔顿的亚基和一个33千道尔顿的亚基组成。经N-糖苷酶处理去糖基化后，46千道尔顿的亚基转变为25.6千道尔顿的形式，而39千道尔顿和33千道尔顿的亚基均产生21.1千道尔顿的形式。对去糖基化多肽进行V8蛋白酶图谱分析显示，39千道尔顿和33千道尔顿的亚基具有几乎相同的条带模式，与源自46千道尔顿亚基的条带模式也表现出高度同源性。放射免疫分析表明，纯化后的gp 80与纤维蛋白原（或肝素）的结合取决于pH值和二价阳离子。此外，在测定混合物中加入游离纤维蛋白原（或肝素）时，gp 80与固定化纤维蛋白原（或肝素）的结合受到抑制。