REGULATION OF 6-PHOSPHOFRUCTO-2-KINASE ACTIVITY BY CYCLIC AMP-DEPENDENT PHOSPHORYLATION
REGULATION OF 6-PHOSPHOFRUCTO-2-KINASE ACTIVITY BY CYCLIC AMP-DEPENDENT PHOSPHORYLATION
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DOI：
10.1073/pnas.79.2.315
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发表时间：
1982-01-01
期刊：
影响因子：
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通讯作者：
PILKIS, SJ
中科院分区：
文献类型：
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作者：
ELMAGHRABI, MR;CLAUS, TH;PILKIS, SJ
在分离的大鼠肝细胞中加入高血糖素可抑制细胞提取物中6-磷酸果糖-2-激酶(ATP:D-fructose-6-phosphate-2-phosphotransferase)的活性，并降低细胞内2，6-二磷酸果糖的水平。对6-磷酸果糖-2-激酶的影响表现为该酶对6-磷酸果糖的亲和力降低。为探讨胰升糖素的作用机制，采用聚乙二醇沉淀、DEAE-纤维素层析、(NH4)2SO4分级沉淀、Sephacryl S-200凝胶过滤、DEAE-Sephadex层析和Sephadex G-100凝胶过滤等步骤，从大鼠肝脏中部分纯化了6-磷酸果糖-2-激酶。纯化的酶与大鼠肝脏cAMP依赖的蛋白激酶的催化亚基和[~(32)P]-ATP孵育，经NaDodSO_4圆盘凝胶电泳法测定，~(32)P掺入一个亚基为49,000的蛋白质中。与这种磷酸化相关的是6-磷酸果糖-2-激酶活性的抑制，这也是该酶对6-磷酸果糖亲和力降低的特征。6-磷酸果糖-2-激酶的磷酸化和抑制作用均可通过加入热稳定性蛋白激酶抑制剂来阻断。在分离的肝细胞中观察到的高血糖素诱导的2，6-二磷酸果糖水平的降低，至少部分是由于cAMP依赖的磷酸化和抑制6-磷酸果糖-2-激酶。
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 cAMP-dependent protein kinase from rat liver and [.gamma.32P]ATP resulted in 32P incorporation into a protein with a subunit MW 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. The glucagon-induced decrease in fructose 2,6-bisphosphate levels observed in isolated hepatocytes is due, at least in part, to cAMP-dependent phosphorylation and inhibition of 6-phosphofructo-2-kinase.