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Glycolysis and Respiration

I. A Cell-free Sarcoma 37 Ascites Tumor Preparation¹

( Biochemistry Department, Upstate Medical College, State University of New York, Syracuse, New York)

A cell-free system of Sarcoma 37 ascites cells which respired and glycolyzed readily has been studied. The addition of DPN² to the system was required for maximal respiration. The fortified preparation compared favorably in magnitude of oxygen consumption and glucose utilization with the intact Sarcoma 37 cells. Citrate, glucose, glucose-1-P, glucose-6-P, fructose, and 2-deoxyglucose all stimulated respiration in the homogenate system beyond endogenous respiration. Acetate, pyruvate, and 2-deoxyglucose-6-P did not stimulate respiration.

Changes in the concentration of P_i in a glycylglycine-buffered medium affected glycolysis and respiration in intact cells and in the cell-free system. Respiration was decreased in whole cells at P_i concentrations greater than 100 µmoles per flask. The cell-free preparation showed diminished respiration at concentrations below 10 µmoles of P_i per vessel and above 50 µmoles per vessel. Optimal lactate production was obtained with 1 µmole of P_i per vessel in intact cells and in the cell-free system. Lactic acid production was decreased in a P_i-free medium in whole cells and in the broken cell system. Lactic acid production was also decreased in both systems at P_i levels above 100 µmoles per flask. The disappearance of glucose was directly related to the lactic acid produced; i.e., the more glucose used, the more lactate produced. Glucose oxidation was influenced by P_i concentration. Glucose-1-¹⁴C oxidation was decreased at high P_i concentration in the intact cells and in the homogenate; oxidation was maximal at concentrations greater than 100 µmoles per vessel. There was decreased oxidation of glucose-6-¹⁴C in both systems at high P_i concentration, i.e., greater than 50 µmoles per flask.

Oxygen consumption was unaffected by the addition of ATP, ADP, or AMP to a glycolyzing preparation of broken cells. However, ATP, ADP, and AMP did affect glycolysis in this system. Glucose disappeared faster and lactate was formed more rapidly in the presence of the adenine nucleotides. The intact cells and homogenate systems were shown to oxidize glucose by non-Embden-Meyerhof pathways. Respiration due to glucose utilization was shown to increase from more than 20% in the 1st hr of incubation to more than 70% during the 3rd hr in both systems. The addition of TPN to the cell-free preparation did not influence respiration but had an effect on glycolysis during the 30- to 60-min incubation period. The oxidation of glucose-1-¹⁴C to ¹⁴CO₂ was greatly enhanced in the presence of TPN, indicating that the level of TPN was limiting for glucose oxidation in the unfortified system.

¹ This investigation was supported in part by USPHS grant CA-06194, from the National Cancer Institute.

² The following abbreviations are used: DPN and DPNH, diphosphopyridine nucleotide and its reduced form; glucose-1-P, glucose-1-phosphate; P_i, orthophosphate; ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; TPN, triphosphopyridine nucleotide; P-enolpyruvate, phosphorylenolpyruvate.

Received 12/14/64.