Gurd J W, Fu S C
J Neurochem. 1982 Sep;39(3):719-25. doi: 10.1111/j.1471-4159.1982.tb07951.x.
Glycoproteins were isolated from a rat brain synaptic junction fraction by affinity chromatography on Concanavalin A-agarose. The isolated glycoproteins were digested with pronase and radiolabeled with 125I-Bolton Hunter reagent, and 125I-Concanavalin A-binding glycopeptides were isolated by chromatography on Concanavalin A-agarose. Treatment of the 125I-Concanavalin A-binding glycopeptides with either alpha-mannosidase or endo-beta-N-acetylglucosaminidase-C11 abolished their interaction with Concanavalin A. The pronase digest was reacted with endo-beta-N-acetylglucosaminidase-C11 and released oligosaccharides were reduced with NaB3H4. Following affinity chromatography on Concanavalin A-agarose, Concanavalin A-binding [3H]oligosaccharides were chromatographed on Biogel P4. Two major oligosaccharides corresponding to standard carbohydrates containing eight and five mannose residues were identified. Treatment of these oligosaccharides with alpha-mannosidase converted them to smaller saccharides having a mobility on Biogel P4 columns equal to the standard disaccharide mannose-beta-1-4-N'-acetylglucosamine. These results demonstrate that the Concanavalin A receptor activity associated with CNS synaptic junctions resides in asparagine-linked oligosaccharides of the high-mannose type.
通过伴刀豆球蛋白A - 琼脂糖亲和层析从大鼠脑突触连接部分分离糖蛋白。将分离得到的糖蛋白用链霉蛋白酶消化，并用125I - 博尔顿·亨特试剂进行放射性标记，然后通过伴刀豆球蛋白A - 琼脂糖层析分离125I - 伴刀豆球蛋白A结合糖肽。用α - 甘露糖苷酶或内切β - N - 乙酰葡糖胺酶 - C11处理125I - 伴刀豆球蛋白A结合糖肽，可消除它们与伴刀豆球蛋白A的相互作用。将链霉蛋白酶消化产物与内切β - N - 乙酰葡糖胺酶 - C11反应，释放的寡糖用NaB3H4还原。在伴刀豆球蛋白A - 琼脂糖上进行亲和层析后，将伴刀豆球蛋白A结合的[3H]寡糖在Biogel P4上进行层析。鉴定出两种主要的寡糖，分别对应于含有八个和五个甘露糖残基的标准碳水化合物。用α - 甘露糖苷酶处理这些寡糖，可将它们转化为在Biogel P4柱上迁移率与标准二糖甘露糖 - β - 1 - 4 - N'- 乙酰葡糖胺相等的较小糖类。这些结果表明，与中枢神经系统突触连接相关的伴刀豆球蛋白A受体活性存在于高甘露糖型的天冬酰胺连接寡糖中。
