Troccoli N M, Hausman R E
Biological Science Center, Boston University, MA 02215.
Cell Differ. 1988 Feb;22(3):225-31. doi: 10.1016/0045-6039(88)90014-0.
Retina cognin (R-cognin) is an intrinsic membrane protein of vertebrate retinal cells which supports tissue-specific cell adhesion and mediates cell type-specific associations during development. As a first step in understanding how R-cognin mediates specific adhesion of retinal cell membranes, we asked if cognin bound to another cognin molecule or to a different macromolecule, a possible cognin-binding protein. To do this, we constructed an affinity column with retinal cell membrane proteins (enriched for cognin) bound to the matrix. Proteins in a detergent extract of retinal cell membranes were exposed to this matrix and those which bound specifically eluted and identified by immunoelectrophoresis. Most prominent among these was a protein with an apparent mass of 64 kDa. The binding of this material to the column was blocked by cognin antibody. To eliminate possible artifacts of molecular interactions in vitro, we sought independent confirmation that 64 kDa protein actually bound R-cognin. Using a modified retina membrane vesicle system, we asked what proteins could be photoaffinity cross-linked to cognin during vesicle aggregation. Cross-linking produced a 114 kDa complex on gels which could be resolved into a 50 kDa (cognin) and a 64 kDa band under reducing conditions. Identification of a 64 kDa protein by independent techniques suggests that cognin promotes association of embryonic chick neural retina cells by binding to this macromolecule or these molecules. Identification of a second component in the mechanism should allow elucidation of cognin's role in mediating cell-cell interactions in developing neural retina.
视网膜认知蛋白（R-认知蛋白）是脊椎动物视网膜细胞的一种内在膜蛋白，它支持组织特异性细胞黏附，并在发育过程中介导细胞类型特异性关联。作为理解R-认知蛋白如何介导视网膜细胞膜特异性黏附的第一步，我们询问认知蛋白是否与另一个认知蛋白分子或不同的大分子（一种可能的认知蛋白结合蛋白）结合。为此，我们构建了一个亲和柱，其中视网膜细胞膜蛋白（富含认知蛋白）与基质结合。视网膜细胞膜去污剂提取物中的蛋白质与该基质接触，特异性结合的蛋白质被洗脱，并通过免疫电泳进行鉴定。其中最突出的是一种表观质量为64 kDa的蛋白质。该物质与柱的结合被认知蛋白抗体阻断。为了消除体外分子相互作用可能产生的假象，我们寻求独立证据来证实64 kDa蛋白质确实与R-认知蛋白结合。使用改良的视网膜膜囊泡系统，我们询问在囊泡聚集过程中哪些蛋白质可以与认知蛋白进行光亲和交联。交联在凝胶上产生了一个114 kDa的复合物，在还原条件下可分解为一个50 kDa（认知蛋白）和一个64 kDa的条带。通过独立技术鉴定出一种64 kDa的蛋白质，这表明认知蛋白通过与这种大分子或这些分子结合来促进胚胎鸡神经视网膜细胞的关联。确定该机制中的第二个成分应有助于阐明认知蛋白在发育中的神经视网膜中介导细胞间相互作用的作用。
