Abstract
In Escherichia coli, adenylate cyclase activity in toluene-treated cells can be inhibited by glucose while the activity in a broken cell preparation cannot. Adenylate cyclase activity in the permeabilized but not in broken cells is stimulated somewhat specifically and additively by potassium and phosphate. Kinetic studies show sigmoid substrate-velocity curves for the toluene-treated cells but hyperbolic curves for the broken cells. The stimulatory effects of potassium and phosphate on adenylate cyclase activity in tolulene-treated cells are associated with increases in the Vmax and Km for ATP. While the enzyme activity in toluene-treated cells shows a preference for magnesium over manganese, the reverse is observed in broken cells. Stimulation of adenylate cyclase activity in toluene-treated cells requires the presence of the proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS proteins can be phosphorylated in a P-enolpyruvate-dependent reaction. The stimulatory effects of ions will not occur if the PTS proteins are not phosphorylated. Since potassium phosphate stimulates both adenylate cyclase and PTS activities in toluene-treated cells, it is proposed that the effect of potassium phosphate on adenylate cyclase activity is mediated through an effect on the PTS. A model for dual regulation by glucose of adenylate cyclase activity is proposed. This model involves regulation of both the condition of the PTS proteins as well as the cellular concentration of phosphate.
Highlights
From the Laboratow of Biochemical Genetics,National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205
Since potassium phosphate stimulates both adenylate cyclase and phosphotransferase system (PTS) activities in toluenetreated cells, it is proposed that the effect of potassium phosphate on adenylate cyclase activity is mediated through an effect on the PTS
We show that E. coli cells, permeabilizedwith toluene, contain an adenylate cyclase activity that is stimulated by both potassium and phosphate
Summary
I A A NOAOOKIONI in the presence of potassium. This was verifiedby demonstrating a substantial stimulation(approximately 5-fold) by potassium arsenate. There was no stimulation by sulfate even in the presence of potassium ion It appears that the anion-dependent stimulation of adenylate cyclase activity in permeable cells shows some degree of specificity. The calculated Hill coefficients ( N values) arein agreement with this (see Table I); permeable cells show N values from 1.4 to 1.7, whileextracts show N values from 1.1 to 1.3 Another major difference in the behavior of permeable cells compared to extracts is that the activity is saturated at substantially lower ATP concentrations in extracts than in FIG. 4. Effect of potassium and phosphate on adenylate cyclase kinetics in permeable cells and extracts of E. coli. In the absence of added potassium or phosphate, the V,, for adenylate cyclase in the extract was approximately threetimes higher than in permeable cells. Adenylate cyclase activity is expressed as nanomoles of cAMP formed per hour per milligram protein
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