François J, Van Schaftingen E, Hers H G
Eur J Biochem. 1984 Nov 15;145(1):187-93. doi: 10.1111/j.1432-1033.1984.tb08539.x.
When glucose was added to a suspension of Saccharomyces cerevisiae in stationary phase, it caused a transient increase in the concentration of cyclic AMP and a more persistent increase in the concentration of hexose 6-phosphate and of fructose 2,6-bisphosphate. These effects of glucose on cyclic AMP and fructose 2,6-bisphosphate but not that on hexose 6-phosphate were greatly decreased in the presence of 0.15 mM acridine orange or when a temperature-sensitive mutant deficient in adenylate cyclase was used at the restrictive temperature. Incubation of the cells in the presence of dinitrophenol and in the absence of glucose increased the concentration of both cyclic AMP and fructose 2,6-bisphosphate, but with a minimal change in that of hexose 6-phosphate. Glucose induced also in less than 3 min a severalfold increase in the activity of 6-phosphofructo-2-kinase and this effect was counteracted by the presence of acridine orange. When a cell-free extract of yeast in the stationary phase was incubated with ATP-Mg and cyclic AMP, there was a 10-fold activation of 6-phosphofructo-2-kinase. Finally, the latter enzyme was purified 150-fold and its activity could then be increased about 10-fold upon incubation with ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase. This activation resulted from a 4.3-fold increase in V and a 2-fold decrease in Km. Both forms of the enzyme were inhibited by sn-glycerol 3-phosphate. From these results it is concluded that the effect of glucose in increasing the concentration of fructose 2,6-bisphosphate in S. cerevisiae is mediated by the successive activation of adenylate cyclase and of cyclic-AMP-dependent protein kinase and by the phosphorylation of 6-phosphofructo-2-kinase by the latter enzyme. In deep contrast with what is known of the liver enzyme, yeast 6-phosphofructo-2-kinase is activated by phosphorylation instead of being inactivated.
当向处于稳定期的酿酒酵母悬液中添加葡萄糖时，会导致环磷酸腺苷（cAMP）浓度短暂升高，以及6-磷酸己糖和2,6-二磷酸果糖浓度持续升高。在存在0.15 mM吖啶橙的情况下，或者当使用在限制温度下缺乏腺苷酸环化酶的温度敏感突变体时，葡萄糖对cAMP和2,6-二磷酸果糖的这些影响（但对6-磷酸己糖的影响并非如此）会大大降低。在二硝基苯酚存在且无葡萄糖的情况下培养细胞，会使cAMP和2,6-二磷酸果糖的浓度均升高，但6-磷酸己糖的浓度变化最小。葡萄糖还能在不到3分钟的时间内使6-磷酸果糖-2-激酶的活性增加数倍，而吖啶橙的存在会抵消这种作用。当将处于稳定期的酵母无细胞提取物与ATP-Mg和cAMP一起孵育时，6-磷酸果糖-2-激酶会被激活10倍。最后，将该酶纯化了150倍，然后在与ATP-Mg和环磷酸腺苷依赖性蛋白激酶的催化亚基一起孵育时，其活性可增加约10倍。这种激活是由于V增加了4.3倍，Km降低了2倍。两种形式的酶都受到3-磷酸甘油的抑制。从这些结果可以得出结论，葡萄糖增加酿酒酵母中2,6-二磷酸果糖浓度的作用是通过腺苷酸环化酶和环磷酸腺苷依赖性蛋白激酶的相继激活以及后者对6-磷酸果糖-2-激酶的磷酸化来介导的。与已知的肝脏酶形成鲜明对比的是，酵母6-磷酸果糖-2-激酶通过磷酸化被激活而非失活。
