The effect of potassium depletion on the initial kinetics of glycolysis in ascites tumor cells

Abstract

Ehrlich ascites carcinoma cells depleted of K + and provided with 5.5 mM K + in isosmotic 50 mM tris(hydroxymethyl)methylglycine buffer at pH 7.4 and 38 °C take up K + from the medium at a rate of 6 μmoles/ml intracellular fluid per min. Depleted cells exposed to K + for 2 min prior to glucose addition exhibit a higher initial rate of glycolysis, a lower glycose-6- P accumulation, and a higher fructose-1,6- P 2 accumulation than depleted cells incubated in a K +-free medium. Both the K + transport and the effect of K + on glycolysis are blocked by 2 mM oubain. Calculation of the in vitro velocities of glycolytic enzymes from the rates of accumulation of lactate and glycolytic intermediates shows that the presence of K + accelerates the velocities of fructose-6-phosphate kinase and lactate dehydrogenase about 2-fold and the velocity of hexokinase about 1.5-fold during the first 15 s. In either the presence or absence of K +, the hexokinase velocity is highest immediately after glucose addition and declines sharply with time; this decline is greater than would be predicted by product inhibition by the accumulated glucose-6- P. The maximal stimulation of fructose-6-phosphate kinase attibutable to the increasing intarcellular K + concentration is only 1.25-fold. These observations indicate that the initial acceleration in glycolysis is not simply mediated through a direct K + activation of fructose-6-phosphate kinase. The calculated theoretical rate of ATP generation by glycolysis shows that glycolysis is an ATP-utilizing system for the first 5–10 s both in the presence and in the absence of K +. Hence, the initial stimulation of glycolysis by K + is not a consequence of an increased rate of ATP hydrolysis associated with K + transport, although this mechanism may be responsible for the stimulation of steady-state glycolysis. The initial rate of phosphate ester (hexose and triose phosphates) accumulation corresponds to be rate of ATP generation by the “tail-end” of glycolysis, or twice the rate of lactate accumulation, in either the absence or presence of K +, but both the rate and the maximal level of ester accumulated are higher in the presence of K +. This implies that the oxidatively generated pool of ATP which is diverted from endogenous reactions to hexokinase and fructose-6-phosphate kinase on the introduction of glucose is larger in the presence of K +. Valinomycin (0.27 μM) under certain conditions can produce effects on the glycolysis of non-depleted cells which superficially resemble the effects of K + on depleted cells. However, unlike K +, valinomycin stimulates the initial rate of glycolytic ATP generation, and abolishes the initial correspondence between the ATP generation by the “tail-end” of glycolysis and phosphate ester accumulation. These observations are interpreted to mean that valinomycin introduces an ATPase activity effective on glycolytically generated ATP. Comparison of the theoretical ATP generation in the presence and absence of K + indicates that approximately one ATP is hydrolyzed for each K + transported.