Coordination among Rate-limiting Steps of Glycolysis and Respiration in Intact Ascites Tumor Cells

Abstract

Abstract The accumulation of lactate and phosphorylated glycolytic intermediates, as well as the variation in adenosine triphosphate, diphosphate, and 5'-monophosphate, were measured over the first 4 min after addition of 0.77 mm glucose to Ehrlich ascites carcinoma cells in 54 mm phosphate buffer (pH 7.4) at 370 and 300. The rates of glucose flux through the phosphofructokinase step (Vpfk) and the lactate dehydrogenase step (Vldh) were calculated and compared with each other and the rate of oxygen consumption (Vr), measured by means of a bare, vibrating reed platinum electrode. During the period from 10 to 90 set or more after glucose addition, both Vr and Vpfk are linearly related to Vldh; at 370, Vpfk = 2.5 (Vldh - 1.1) and Vr = 0.38 (Vldh), where velocities are measured in micromoles per ml of cells per min; at 300, Vpfk = 4.1 (Vldh - 1.3) and Vr = 0.32 (Vldh). These results suggest a common controlling mechanism for all three rates. During the period from 10 to 60 sec approximately linear relationships between Vpfk, Vldh, and Vr and the ADP concentration are observed, but the slopes and intercepts of the lines vary considerably from one experiment to another unlike the relationships among the velocities themselves, which are almost identical in all experiments at the same temperature. In a given experiment, both Vldh and Vr extrapolate to the same ADP concentration (0 to 1.0 µmole per ml of cells) at zero velocity, which implies that if ADP is governing these rates only a fraction of the ADP may be involved in the control, but the same fraction or pool controls both respiration and glycolysis. The velocities are also approximately linear functions of esterified phosphate concentration. Since the increase in esters such as fructose diphosphate probably occurs at the expense of intracellular inorganic phosphate, this observation could be consistent with control of the rates by inorganic phosphate. The kinetic data do not identify either ADP or inorganic phosphate as the major controlling agent, but they do indicate a close coordination among the initial segment of glycolysis, the terminal segment of glycolysis, and respiration which is not fully explainable in terms of variations in either the ADP or phosphate levels in the whole cell.