Van Schaftingen E
Laboratoire de Chimie Physiologique, Université Catholique de Louvain, Belgium.
Eur J Biochem. 1995 Nov 15;234(1):301-7. doi: 10.1111/j.1432-1033.1995.301_c.x.
The purpose of this study was to identify the mechanism by which proglycosyn and resorcinol decrease the phosphorylase a content and the fructose 2,6-bisphosphate concentration in isolated hepatocytes. The intracellular concentrations of the glucuronide derivatives of proglycosyn and resorcinol have been measured by HPLC in hepatocytes incubated for 5 min or 30 min with different concentrations of these agents. At both times, there was a reciprocal relationship between the phosphorylase a content and the intracellular concentration of the glucuronidated metabolites, half-maximal inactivation being observed at about 2 mumol/g protein and 0.25 mumol/g protein for resorcinylglucuronide and proglycosyn-glucuronide, respectively. Glycogen synthase was not significantly activated by these agents after 5 min but was well activated after 30 min. Preincubation of hepatocytes with 1 mM resorcinol or with 100 microM proglycosyn resulted in a decrease in the rate at which phosphorylase was activated following the addition of glucagon, vasopressin, the protein phosphatase inhibitor calyculin A or the calcium ionophore A 23187, but did not reduce the rate of synthase inactivation. Proglycosynglucuronide and resorcinylglucuronide inhibited phosphorylase kinase in liver Sephadex filtrates, with Ki values of about 0.75 mM and 4 mM, respectively. Preincubation of the filtrates with ATP and cAMP decreased the sensitivity of phosphorylase kinase to resorcinylglucuronide by about fourfold. It is concluded that the effect of resorcinol and proglycosyn on the phosphorylase a content is due, at least partly, to an inhibition of phosphorylase kinase by their glucuronidated metabolites. Resorcinol and proglycosyn caused a parallel decrease in the concentration of fructose 2,6-bisphosphate and of hexose 6-phosphates, without significantly changing the activity of 6-phosphofructo-2-kinase. The decrease in the fructose 2,6-bisphosphate concentration appears therefore to be secondary to the decrease in the hexose 6-phosphate concentration.
本研究的目的是确定前糖原素和间苯二酚降低分离肝细胞中磷酸化酶a含量及果糖2,6 -二磷酸浓度的机制。通过高效液相色谱法测定了在不同浓度的这些试剂作用下孵育5分钟或30分钟的肝细胞中前糖原素和间苯二酚的葡萄糖醛酸苷衍生物的细胞内浓度。在这两个时间点，磷酸化酶a含量与葡萄糖醛酸化代谢物的细胞内浓度之间存在反比关系，间苯二酚葡萄糖醛酸苷和前糖原素 - 葡萄糖醛酸苷分别在约2 μmol/g蛋白质和0.25 μmol/g蛋白质时观察到半数最大失活。糖原合酶在5分钟后未被这些试剂显著激活，但在30分钟后被充分激活。用1 mM间苯二酚或100 μM前糖原素预孵育肝细胞，导致在添加胰高血糖素、血管加压素、蛋白磷酸酶抑制剂花萼海绵诱癌素A或钙离子载体A23187后磷酸化酶被激活的速率降低，但未降低合酶失活的速率。前糖原素 - 葡萄糖醛酸苷和间苯二酚葡萄糖醛酸苷抑制肝葡聚糖凝胶滤液中的磷酸化酶激酶，其抑制常数（Ki）值分别约为0.75 mM和4 mM。用ATP和cAMP预孵育滤液可使磷酸化酶激酶对间苯二酚葡萄糖醛酸苷的敏感性降低约四倍。得出的结论是，间苯二酚和前糖原素对磷酸化酶a含量的影响至少部分归因于其葡萄糖醛酸化代谢物对磷酸化酶激酶的抑制作用。间苯二酚和前糖原素使果糖2,6 -二磷酸和己糖6 -磷酸的浓度平行降低，而6 -磷酸果糖 - 2 -激酶的活性没有显著变化。因此，果糖2,6 -二磷酸浓度的降低似乎继发于己糖6 -磷酸浓度的降低。
