Competition for ADP between Pyruvate Kinase and Mitochondrial Oxidative Phosphorylation as a Control Mechanism in Glycolysis

Abstract

To assess a possible role of pyruvate kinase as a site for respiratory and glycolytic interaction, competition for ADP between pyruvate kinase and respiratory phosphorylation was measured in a model system consisting of rat liver mitochondria respiring in the presence of pyruvate kinase, phosphoenolpyruvate, ATP, and an ADP-regenerating system consisting of glucose and purified yeast hexokinase. This system allowed determination of total ATP production, equivalent to ADP utilization, by measuring glucose 6-phosphate formation; ADP utilization by pyruvate kinase by pyruvate formation; and respiratory ADP utilization by Pi uptake. 0 2 uptake was measured by means of an oxygen electrode. In the presence of a respiratory substrate such as succinate or glutamatemalate, the addition of pyruvate kinase and phosphoenolpyruvate reduced 0 2 uptake as well as oxidative phosphorylation about 80 %. Respiration was increasingly inhibited with increasing pyruvate kinase, and this inhibition was decreased with increased hexokinase or ATP. Mitochondria1 respiratory inhibition by pyruvate kinase and phosphoenolpyruvate was accompanied by an increase in the ATP/ADP ratio from 0.3 to 32. These inhibitory effects of pyruvate kinase on respiration were abolished by addition of 2,4-dinitrophenol. These results are in acccord with a role of pyruvate kinase as a determinant of glycolytic activity by competing with oxidative phosphorylation for the available ADP; and provide additional support for our previous suggestion that high glycolysis of certain tumors may be attributable to their extraordinarily high pyruvate kinase activity.Ever since the epoch-making studies of Warburg, carried out over fifty years ago, the high aerobic glycolysis of tumors has remained a challenging mystery as well as a focal point for much effort, not only to control cancer, but to understand regulatory control mechanisms in carbohydrate metabolism.Recent work from our own and other laboratories on the Morris hepatomas, a series of chemicallyinduced transplantable liver neoplasms of the rat, [l-31, has shown that tumors arising from a single cell of origin may range widely in growth rate and degree of differentiation, paralleling wide variations in their enzyme and isozyme activities [4--81 and their rates of aerobic glycolysis [9 -11 1. In studies of the enzymatic bases for the differences in the glycolytic activity of these tumors, attention has been focussed on their pyruvate kinase activity. Two observations of particular significance are that those tumors having high Enzymes. Pyruvate kinase (EC 2.7.1.40); hexokinase (EC 2.7.1.1). glycolytic activity have extraordinarily high pyruvate kinase activities [4,12-141 and furthermore the molecular forms and kinetic properties of these pyruvate kinase differ from those of the adult liver [4,15,16].Johnson [17] and Lynen [18] many years ago called attention to the possibility that the decrease of glycolysis brought about by oxygen, a phenomenon termed by Warburg [19] the Pasteur effect, may have its enzymatic ...

Stimulation of Tumor‐Cell Respiration by Inhibitors of Pyruvate Kinase

“…Analysis of the cell contents (Table 1) revealed that with 3 pM rotenone, and glucose present, the pyruvate concentration was greatly increased from 2.7 to 4.8 nmol/106 cells, owing presumably to the increased glycolysis ; and there was also an approximate doubling of the ATP/ADP ratio from 2.1 to 3.7. These findings are in accord with our previous results [3] which showed that the ATP/ADP ratio increased with increasing pyruvate kinase activity in vitro. The increased glycolysis under the influence of rotenone, coupled with a marked increase in the ATPiADP ratio under conditions in which respiratory ATP formation is drastically lower- …”

“…With 20 pM rotenone present to inhibit the endogenous respiration, the pyruvate kinase activity was high and was not affected by the amount of mitochondria. The inhibition of mitochondrial respiration by increasing pyruvate kinase is accompanied by an increase in the ATP/ADP ratio due to competition for ADP favorable to pyruvate kinase, as shown previously [3].…”

“…The experiments in vitro were performed as described previously [3] except that pyruvate formation was continuously monitored by adding 5 U per ml lactate dehydrogenase and 0.6 mM NADH, and measuring the change in absorbance in a 3-ml cuvette of a Perkin-Elmer Hitachi 356 double-beam spectrophotometer, as descrived by us [4]. The cuvette was equipped with a stirring device and a micro Clarktype oxygen electrode supplied by Instech Laboratories 316 Stimulation of Tumor-Cell Respiration (Philadelphia, Pa.).…”

“…As described previously [3], ADP is continuously regenerated in this system by the addition of glucose, ATP, and purified yeast hexokinase, and the rate of pyruvate kinase activity is limited by the rate of the hexokinase reaction. The various components, added, in order, to a final volume of 3 ml in the cuvette, to give the designated final concentrations, were: 5 mM glutamate, 5 tnM malate, 5 mM glucose, 5 mM Pi, 12 mM phosphoenolpyruvate, the designated activities of pyruvate kinase.…”

“…In a previous report in this journal [3], we described a model system composed of intact, well-coupled rat liver mitochondria, together with variable quantities of purified rabbit muscle pyruvate kinase, and an ADP-regenerating system consisting of glucose and yeast hexokinase. In this system, the addition of increasing pyruvate kinase levels progressively lowered respiration, thus simulating the Crabtree effect; and also progressively increased the ATP/ADP ratio.…”