Possible regulatory interactions between compartmentalized glycolytic systems during initiation of glycolylsis in ascites tumor cells

Changes were measured in the rates of respiration and in the levels of glycolytic intermediates during the first 5 min after addition of 1.6 mM glucose to a suspension (5%, v/v) of respiring Ehrlich ascites carcinoma cells incubated in an isotonic 50 mM tris(hydroxymethyl)methylglycine buffer (pH 7.4) at 38 °C. The rates of accumulation of lactate and glycolytic intermediates were used to calculate the in vitro velocities of glycolytic enzymes.The initial velocities of hexokinase (EC 2.7.1.1), fructose-6-phosphate kinase (EC 2.7.1.11) and lactate dehydrogenase (EC 1.1.1.27) in μmoles glucose equivalents/ ml cells per min were 14, 11 and 4, respectively. The velocities of the two kinases fell sharply to less than 5 between 5 and 10 s, while the velocity of the dehydrogenase declined gradually over the first minute. The initial burst of activity in the kinases, which lasted for about 8 s, was associated with a rapid accumulation of phosphate ester and a negative net ATP generation by glycolysis. The accumulation of phosphate ester is almost exactly matched by the generation of ATP by the “tail end” of glycolysis (triose-P to lactate) in this period. After this time (10–25 s) the rate of oxidative phosphorylation calculated as six times the rate of O₂ consumption, is nearly identical to the combined rate of ATP utilization by hexokinase and fructose-6-phosphate kinase. As observed previously, oxamate (42 mM) blocked lactate dehydrogenase but did not depress the rate of phosphate ester accumulation.These various observations and correlations can be interpreted in terms of a dual glycolytic system. The accumulation of phosphate ester during the first 8 s is attributed to the operation of a partial glycolytic system, System B, which includes only the first three or four enzymes of glycolysis, and which draws upon an ATP pool (Pool I) previously employed in assorted cytoplasmic phosphorylations. The ADP generated by System B is rephosphorylated by and regulates the rate of a complete glycolytic system A, which converts glucose to lactate with little intermediate accumulation. The tail end of System A generates a new pool of ATP (Pool II) and controls the rate of glucose input through its head end, which is supplied by ATP being produced by oxidative phosphorylation. This scheme of interlocking controls is transient and alters after 8 s, when System B slows to a stop.