Cadefau J, Bollen M, Stalmans W
Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, Herestraat 49, B-3000 Leuven, Belgium.
Biochem J. 1997 Mar 15;322 ( Pt 3)(Pt 3):745-50. doi: 10.1042/bj3220745.
Non-metabolized glucose derivatives may cause inactivation of phosphorylase but, unlike glucose, they are unable to elicit activation of glycogen synthase in isolated hepatocytes. We report here that, after the previous inactivation of phosphorylase by one of these glucose derivatives (2-deoxy-2-fluoro-alpha-glucosyl fluoride), glycogen synthase was progressively activated by addition of increasing concentrations of glucose. Under these conditions, the degree of activation of glycogen synthase was linearly correlated with the intracellular glucose-6-phosphate (Glc-6-P) concentration. Addition of glucosamine, an inhibitor of glucokinase, decreased both parameters in parallel. Further experiments using an inhibitor of either protein kinases (5-iodotubercidin) or protein phosphatases (microcystin) in isolated hepatocytes indicated that Glc-6-P does not affect glycogen-synthase kinase activity but enhances the glycogen-synthase phosphatase reaction. Experiments in vitro showed that the synthase phosphatase activity of glycogen-bound type-1 protein phosphatase was increased by physiological concentrations of Glc-6-P (0.1-0.5 mM), but not by 2.5 mM fructose-6-P, fructose-1-P or glucose-1-P. At physiological ionic strength, the glycogen-associated synthase phosphatase activity was nearly entirely Glc-6-P-dependent, but Glc-6-P did not relieve the strong inhibitory effect of phosphorylase a. The large stimulatory effects of 2.5 mM Glc-6-P, with glycogen synthase b and phosphorylase a as substrates, appeared to be mostly substrate-directed, while the modest effects observed with casein and histone IIA pointed to an additional stimulation of glycogen-bound protein phosphatase-1 by Glc-6-P. We conclude that glucose elicits hepatic synthase phosphatase activity both by removal of the inhibitor, phosphorylase a, and by generation of the stimulator, Glc-6-P.
非代谢性葡萄糖衍生物可能会导致磷酸化酶失活，但与葡萄糖不同的是，它们无法在分离的肝细胞中引发糖原合酶的激活。我们在此报告，在这些葡萄糖衍生物之一（2-脱氧-2-氟-α-葡萄糖基氟化物）使磷酸化酶先前失活后，通过添加浓度不断增加的葡萄糖，糖原合酶逐渐被激活。在这些条件下，糖原合酶的激活程度与细胞内葡萄糖-6-磷酸（Glc-6-P）浓度呈线性相关。添加葡萄糖激酶抑制剂氨基葡萄糖会使这两个参数同时降低。在分离的肝细胞中使用蛋白激酶抑制剂（5-碘结核菌素）或蛋白磷酸酶抑制剂（微囊藻毒素）进行的进一步实验表明，Glc-6-P不影响糖原合酶激酶活性，但会增强糖原合酶磷酸酶反应。体外实验表明，生理浓度的Glc-6-P（0.1 - 0.5 mM）可增加与糖原结合的1型蛋白磷酸酶的合酶磷酸酶活性，但2.5 mM的6-磷酸果糖、1-磷酸果糖或1-磷酸葡萄糖则不能。在生理离子强度下，与糖原相关的合酶磷酸酶活性几乎完全依赖于Glc-6-P，但Glc-6-P并不能解除磷酸化酶a的强烈抑制作用。以糖原合酶b和磷酸化酶a为底物时，2.5 mM Glc-6-P的巨大刺激作用似乎主要是底物导向的，而以酪蛋白和组蛋白IIA为底物时观察到的适度作用则表明Glc-6-P对与糖原结合的蛋白磷酸酶-1有额外的刺激作用。我们得出结论，葡萄糖通过去除抑制剂磷酸化酶a和生成刺激物Glc-6-P来引发肝脏合酶磷酸酶活性。
