Van Schaftigen E
Laboratoire de Chimie Physiologique, Université Catholique de Louvain, Brussels, Belgium.
Biochem J. 1995 May 15;308 ( Pt 1)(Pt 1):23-9. doi: 10.1042/bj3080023.
Glucosamine, a potent inhibitor of glucokinase (hexokinase IV or D), was used to estimate the contribution of this enzyme to glucose phosphorylation in freshly isolated rat hepatocytes and its sensitivity to fructose 6-phosphate in situ. Experiments with radiolabelled glucosamine indicated that this amino sugar, at concentrations of 5 or 40 mM, readily penetrated hepatocytes to reach in 1 min a total (i.e., glucosamine+metabolites) intracellular concentration equal to 0.8-1.2-fold its extracellular concentration. In marked contrast, N-acetylglucosamine barely penetrated the cells. The detritiation of [2-3H]glucose, used to estimate glucose phosphorylation in intact cells, was inhibited by glucosamine much more potently than by N-acetylglucosamine, half-maximal effects being reached at about 2.5 and 30 mM respectively. Extrapolation of the data indicated that about 12% of the detritiation was resistant to glucosamine. Dihydroxyacetone (10 mM), lactate (10 mM) + pyruvate (1 mM), and glucagon (1 microM) increased up to 8-fold the concentration of hexose 6-phosphates (glucose 6-phosphate+fructose 6-phosphate) and, against expectations, modestly decreased the detritiation rate measured in the absence of glucosamine. In the presence of 40 mM glucosamine, these agents increased the detritiation rate, which then positively correlated with the concentration of hexose 6-phosphates. This hexose 6-phosphates-dependent detritiation was sensitive to inhibition by vanadate, and was also catalysed by gel-filtered cell-free extracts, as well as by liver microsomes in the presence of phosphoglucoisomerase; it can be explained by an exchange reaction catalysed by glucose-6-phosphatase. When this exchange reaction is taken into account, it appears that the rate of glucose detritiation attributable to glucokinase decreases when the concentration of hexose 6-phosphates increases. This is in agreement with the known effect of fructose 6-phosphate to potentiate the inhibition of glucokinase by its regulatory protein.
氨基葡萄糖是葡萄糖激酶（己糖激酶IV或D）的强效抑制剂，用于评估该酶对新鲜分离的大鼠肝细胞中葡萄糖磷酸化的贡献及其在原位对6-磷酸果糖的敏感性。用放射性标记的氨基葡萄糖进行的实验表明，这种氨基糖在5或40 mM的浓度下很容易穿透肝细胞，在1分钟内达到的细胞内总浓度（即氨基葡萄糖+代谢产物）等于其细胞外浓度的0.8 - 1.2倍。与之形成鲜明对比的是，N-乙酰氨基葡萄糖几乎不能穿透细胞。用于评估完整细胞中葡萄糖磷酸化的[2-³H]葡萄糖的脱氚作用，被氨基葡萄糖抑制的程度比N-乙酰氨基葡萄糖强得多，半最大效应分别在约2.5和30 mM时达到。数据外推表明，约12%的脱氚作用对氨基葡萄糖有抗性。二羟基丙酮（10 mM）、乳酸（10 mM）+丙酮酸（1 mM）和胰高血糖素（1 μM）使6-磷酸己糖（6-磷酸葡萄糖+6-磷酸果糖）的浓度增加了8倍，并且出乎意料的是，在没有氨基葡萄糖的情况下测量的脱氚速率略有下降。在存在40 mM氨基葡萄糖的情况下，这些试剂增加了脱氚速率，然后该速率与6-磷酸己糖的浓度呈正相关。这种依赖于6-磷酸己糖的脱氚作用对钒酸盐的抑制敏感，并且也由凝胶过滤的无细胞提取物以及在磷酸葡萄糖异构酶存在下的肝微粒体催化；这可以通过葡萄糖-6-磷酸酶催化的交换反应来解释。当考虑到这种交换反应时，似乎当6-磷酸己糖的浓度增加时，归因于葡萄糖激酶的葡萄糖脱氚速率会降低。这与6-磷酸果糖增强其调节蛋白对葡萄糖激酶抑制作用的已知效应一致。
