el-Maghrabi M R, Claus T H, Pilkis J, Pilkis S J
Proc Natl Acad Sci U S A. 1982 Jan;79(2):315-9. doi: 10.1073/pnas.79.2.315.
Addition of glucagon to isolated rat hepatocytes resulted in inhibition of 6-phosphofructo-2-kinase (ATP:D-fructose-6-phosphate-2-phosphotransferase) activity in extracts of the cells and in a decrease in the intracellular level of fructose 2,6-bisphosphate. The effect on 6-phosphofructo-2-kinase was characterized by a decrease in the affinity of the enzyme for fructose 6-phosphate. To investigate the mechanism of action of glucagon, 6-phosphofructo-2-kinase from rat liver was partially purified by polyethylene glycol precipitation, DEAE-cellulose chromatography, (NH4)2SO4 fractionation, Sephacryl S-200 gel filtration, DEAE-Sephadex chromatography, and Sephadex G-100 gel filtration. Incubation of the purified enzyme with the catalytic subunit of the cyclic AMP-dependent protein kinase from rat liver and [gamma-32P]ATP resulted in 32P incorporation into a protein with a subunit Mr of 49,000 as determined by NaDodSO4 disc gel electrophoresis. Associated with this phosphorylation was an inhibition of 6-phosphofructo-2-kinase activity that was also characterized by a decrease in the affinity of the enzyme for fructose-6-phosphate. Both the phosphorylation and the inhibition of the purified 6-phosphofructo-2-kinase were blocked by addition of the heat-stable protein kinase inhibitor. It is concluded that the glucagon-induced decrease in fructose 2,6-bisphosphate levels observed in isolated hepatocytes is due, at least in part, to cyclic AMP-dependent phosphorylation and inhibition of 6-phosphofructo-2-kinase.
向分离的大鼠肝细胞中添加胰高血糖素会导致细胞提取物中6-磷酸果糖-2-激酶(ATP:D-果糖-6-磷酸-2-磷酸转移酶)活性受到抑制,同时细胞内果糖2,6-二磷酸水平降低。对6-磷酸果糖-2-激酶的影响表现为该酶对6-磷酸果糖的亲和力下降。为了研究胰高血糖素的作用机制,通过聚乙二醇沉淀、DEAE-纤维素色谱法、硫酸铵分级分离、Sephacryl S-200凝胶过滤、DEAE-葡聚糖凝胶色谱法和Sephadex G-100凝胶过滤对大鼠肝脏的6-磷酸果糖-2-激酶进行了部分纯化。将纯化后的酶与大鼠肝脏的环磷酸腺苷依赖性蛋白激酶催化亚基以及[γ-32P]ATP一起孵育,通过十二烷基硫酸钠圆盘凝胶电泳测定,结果显示有32P掺入到一个亚基分子量为49,000的蛋白质中。与这种磷酸化相关的是6-磷酸果糖-2-激酶活性受到抑制,其特征同样是该酶对6-磷酸果糖亲和力下降。添加热稳定蛋白激酶抑制剂可阻断纯化的6-磷酸果糖-2-激酶磷酸化及活性抑制。结论是,在分离的肝细胞中观察到的胰高血糖素诱导的果糖2,6-二磷酸水平降低至少部分是由于环磷酸腺苷依赖性磷酸化以及6-磷酸果糖-2-激酶受到抑制所致。