Howes S, Bissoon N, Ito M, Beesley P W, Gurd J W
Department of Biochemistry, University of Toronto, Ontario, Canada.
Neurochem Res. 1990 Mar;15(3):257-63. doi: 10.1007/BF00968669.
Slices were prepared from rat forebrains and the incorporation of [3H]mannose and [35S]methionine into proteins and glycoproteins determined. The incorporation of methionine continued to increase for up to 8 hours whereas mannose incorporation was maximal between 2 and 4 hours and declined thereafter. Glycopeptides prepared by pronase digestion of [3H]mannose-labeled glycoproteins were digested with endoglucosaminidase H (endo H) and analysed by gel filtration. The major endo H-sensitive oligosaccharide eluted in a position similar to standard Man8GlcNAc. In the presence of castanospermine, which inhibits glucosidase I, the first enzymatic step in the processing of N-linked oligosaccharides, a new endo H-sensitive glycan similar in size to standard Glc3Man9GlcNAc2 accumulated. Synaptic membranes (SMs) were isolated from slices which had been incubated with either [3H]mannose or [35S]methionine in the presence and absence of castanospermine. In the presence of inhibitor the relative incorporation of [3H]mannose into high-mannose glycans of synaptic glycoproteins was increased. The incorporation of newly synthesized, [35S] methionine-labeled, Con A-binding glycoproteins into SMs was not affected by the addition of inhibitor. Many of the glycoproteins synthesized in the presence of castanospermine exhibited a decreased electrophoretic mobility indicative of the presence of altered oligosaccharide chains. The results indicate that changes in oligosaccharide composition produced by castanospermine had little effect on the subsequent transport and incorporation of glycoproteins into synaptic membranes.
从大鼠前脑制备切片，并测定[3H]甘露糖和[35S]甲硫氨酸掺入蛋白质和糖蛋白的情况。甲硫氨酸的掺入在长达8小时内持续增加，而甘露糖的掺入在2至4小时达到最大值，此后下降。用链霉蛋白酶消化[3H]甘露糖标记的糖蛋白制备的糖肽，用内切葡糖胺聚糖酶H（endo H）消化并通过凝胶过滤分析。主要的endo H敏感寡糖在与标准Man8GlcNAc相似的位置洗脱。在抑制N-连接寡糖加工的第一步酶促反应即葡糖苷酶I的栗精胺存在下，一种大小与标准Glc3Man9GlcNAc2相似的新的endo H敏感聚糖积累。从在有和没有栗精胺存在的情况下用[3H]甘露糖或[35S]甲硫氨酸孵育过的切片中分离突触膜（SMs）。在抑制剂存在下，[3H]甘露糖掺入突触糖蛋白的高甘露糖聚糖中的相对量增加。新合成的、[35S]甲硫氨酸标记的、伴刀豆球蛋白A结合糖蛋白掺入SMs不受抑制剂添加的影响。在栗精胺存在下合成的许多糖蛋白表现出电泳迁移率降低，表明存在改变的寡糖链。结果表明，栗精胺产生的寡糖组成变化对糖蛋白随后向突触膜的转运和掺入影响很小。
