Miller J D, Hadley R D
Department of Anatomy and Cell Biology, Medical University of South Carolina, Charleston 29425.
J Neurobiol. 1991 Jul;22(5):431-42. doi: 10.1002/neu.480220502.
Polyclonal antibodies directed against laminin (LM), and against the A and B chains of reduced LM were used to identify antigenically related proteins in the extracellular matrix (ECM) of the snail Helisoma trivolvis. Immunofluorescence of snail central ganglionic rings using either the anti-LM or anti-B chain antibodies labeled the ECM within ganglionic sheaths as well as basal laminae surrounding the ganglia. Both the anti-LM and anti-B chain antibodies recognized a prominent, approximately 300-kD protein on immunoblots of a snail central ganglion preparation enriched in ECM components. The anti-A chain antibody failed to label any structures in sections of snail ganglia or to recognize any proteins on immunoblots of ganglionic ECM. A polyclonal antibody was raised against the approximately 300-kD snail protein. Immunofluorescence of snail ganglia with the anti- approximately 300-kD antibody gave a distribution of labeled structures comparable to that obtained with the anti-LM antibody. Immunofluorescent labeling of sections of snail muscle and salivary gland with the anti- approximately 300-kD antibody revealed a distribution of reactive protein characteristic of an ECM component. Probing immunoblots of ganglionic ECM with the anti- approximately 300-kD antibody revealed the recognition of the same approximately 300-kD protein as identified by the anti-LM antibodies. Media conditioned by Helisoma central ganglionic rings (CM) contains an unidentified neurite outgrowth promoting factor (NOPF). Immunoblots of CM probed with the anti-B chain and anti- approximately 300-kD antibodies reveal the recognition of a soluble approximately 300-kD protein similar to the approximately 300-kD protein identified in snail ECM. The ganglionic ECM preparation containing the approximately 300-kD protein supported outgrowth from cultured snail buccal neurons B5, and addition of anti- approximately 300-kD Fab fragments to CM abolished its outgrowth promoting activity. These results suggest that the approximately 300-kD ECM protein may be the NOPF in CM and/or functions in promoting neurite outgrowth.
针对层粘连蛋白（LM）以及还原型LM的A链和B链的多克隆抗体，被用于鉴定蜗牛三角帆蚌细胞外基质（ECM）中抗原相关蛋白。使用抗LM或抗B链抗体对蜗牛中枢神经节环进行免疫荧光检测，标记了神经节鞘内以及神经节周围基膜中的ECM。抗LM和抗B链抗体在富含ECM成分的蜗牛中枢神经节制剂的免疫印迹上均识别出一种突出的、约300-kD的蛋白。抗A链抗体未能标记蜗牛神经节切片中的任何结构，也未能在神经节ECM的免疫印迹上识别任何蛋白。制备了针对约300-kD蜗牛蛋白的多克隆抗体。用抗约300-kD抗体对蜗牛神经节进行免疫荧光检测，得到的标记结构分布与用抗LM抗体得到的相似。用抗约300-kD抗体对蜗牛肌肉和唾液腺切片进行免疫荧光标记，显示出一种具有ECM成分特征的反应性蛋白分布。用抗约300-kD抗体探测神经节ECM的免疫印迹，显示识别出与抗LM抗体鉴定出的相同的约300-kD蛋白。由蜗牛中枢神经节环（CM）条件培养基含有一种未鉴定的神经突生长促进因子（NOPF）。用抗B链和抗约300-kD抗体探测CM的免疫印迹，显示识别出一种可溶性约300-kD蛋白，类似于在蜗牛ECM中鉴定出的约300-kD蛋白。含有约300-kD蛋白的神经节ECM制剂支持培养的蜗牛颊神经元B5的生长，并且向CM中添加抗约300-kD Fab片段消除了其生长促进活性。这些结果表明，约300-kD的ECM蛋白可能是CM中的NOPF和/或在促进神经突生长中起作用。
