Purwin C, Nicolay K, Scheffers W A, Holzer H
J Biol Chem. 1986 Jul 5;261(19):8744-9.
The phosphorylation of fructose-1,6-bisphosphatase is preceded by a transient increase in the intracellular level of cyclic AMP which activates a cyclic AMP-dependent protein kinase (Pohlig, G., and Holzer, H. (1985) J. Biol. Chem. 260, 13818-13823). Possible mechanisms by which sugars or ionophores might activate adenylate cyclase and thereby lead to an increase in cyclic AMP concentrations were studied. Studies with permeabilized yeast cells demonstrated that neither sugar intermediates nor carbonyl cyanide m-chlorophenylhydrazone are able to increase adenylate cyclase activity. In the light of striking differences of the effects of fermentable sugars and of carbonyl cyanide m-chlorophenylhydrazone on parameters characterizing the membrane potential, it seems not reasonable that the activity of adenylate is under control of the membrane potential. Rapid quenching of 9-aminoacridine fluorescence after addition of fermentable sugars to starved yeast cells indicated an intracellular acidification. The 31P NMR technique showed a fast drop of the intracellular pH from 6.9 to 6.55 or 6.4 immediately after addition of glucose or carbonyl cyanide m-chlorophenylhydrazone. The time course of the decrease of the cytosolic pH coincides with the transient increase of cyclic AMP concentration and the 50% inactivation of fructose-1,6-bisphosphatase under the conditions of the NMR experiments. Kinetic studies of adenylate cyclase activity showed an approximately 2-fold increase of activity when the pH was decreased from 7.0 to 6.5, which is the result of a decrease in the apparent Km for ATP with no change in Vmax. These studies suggest that activation of adenylate cyclase by decrease in the cytosolic pH starts a chain of events leading to accumulation of cyclic AMP and phosphorylation of fructose-1,6-bisphosphatase.
果糖-1,6-二磷酸酶的磷酸化之前，细胞内环状AMP水平会短暂升高，这会激活一种依赖环状AMP的蛋白激酶（波利格，G.，和霍尔泽，H.（1985年）《生物化学杂志》260卷，13818 - 13823页）。研究了糖类或离子载体可能激活腺苷酸环化酶从而导致环状AMP浓度升高的可能机制。对透性化酵母细胞的研究表明，糖类中间体和羰基氰化物间氯苯腙都不能增加腺苷酸环化酶的活性。鉴于可发酵糖类和羰基氰化物间氯苯腙对表征膜电位的参数的影响存在显著差异，腺苷酸环化酶的活性受膜电位控制似乎不合理。向饥饿的酵母细胞中添加可发酵糖类后，9-氨基吖啶荧光的快速淬灭表明细胞内酸化。31P NMR技术显示，添加葡萄糖或羰基氰化物间氯苯腙后，细胞内pH值立即从6.9快速下降到6.55或6.4。在NMR实验条件下，胞质pH值下降的时间进程与环状AMP浓度的短暂升高以及果糖-1,6-二磷酸酶50%的失活相吻合。腺苷酸环化酶活性的动力学研究表明，当pH值从7.0降至6.5时，活性大约增加2倍，这是由于ATP的表观Km降低而Vmax不变的结果。这些研究表明，胞质pH值降低激活腺苷酸环化酶引发了一系列事件，导致环状AMP积累和果糖-1,6-二磷酸酶磷酸化。
