Control of the adenylate charge in Novikoff ascites cells.

The activity of several enzymes that contribute to the cellular adenylate energy charge, the changes in the total pool of adenine nucleotides, and the relationship of cytoplasmic versus mitochondrial maintenance of the adenylate charge were studied in Novikoff ascites cells. The activities of the following substrate-level phosphorylating enzymes were analyzed: cytoplasmic adenylate kinase, pyruvate kinase, and phosphoglycerate kinase; mitochondrial adenylate kinase and nucleoside diphosphokinase. Tabulation of these enzymatic measurements were made by setting up a theoretical ratio of cytoplasmic to mitochondrial nonoxidative phosphorylation potential. The ratio was 0.44 in liver tissue and 72.2 in the Novikoff ascites cells. The activities of several enzymes that are involved in the transportation of reducing equivalents across the mitochondrial membranes were made: cytoplasmic malate dehydrogenase, glyceraldehyde phosphate dehydrogenases, lactate dehydrogenase, glucose 6-phosphate dehydrogenase, flavin adenine dinucleotide- and nicotinamide adenine dinucleotide-glycerophosphate dehydrogenases. NAD+-malate dehydrogenase was 500 units in liver tissue and 110 units in Novikoff ascites cells. Cytoplasmic NAD+-glycerophosphate dehydrogenase was 120 units in liver tissue and 11 units in Novikoff ascites cells. NAD+-malate dehydrogenase is a key enzyme in the malate-aspartate shuttle system; NAD+-glycerophosphate dehydrogenase is a key enzyme in the α-glycerophosphate shuttle system. Comparison of the ratio of the activities of NAD+-glycerophosphate dehydrogenase versus NAD+-lactate dehydrogenase allows for the examination of the competing hydrogen-accepting systems of phospholipid synthesis versus anaerobic metabolism. The ratio was 0.5 in liver tissue and 0.05 in Novikoff ascites cells. Measurement of adenosine mono-, di-, and triphosphates were made in Novikoff ascites cells incubated under various metabolic conditions. The reaction parameter ( Q ) as related to adenylate kinase reaction and the adenylate energy charge expression were calculated from these measurements. Anaerobically, and without a carbon source, the adenylate charge fell from 0.91 to 0.12 within 30 min of incubation; the Q decreased from 4 to near 1 ( Q for adenylate kinase at equilibrium is 1.2). Q and adenylate charge were maintained during anaerobic metabolism with glucose but not with pyruvate or succinate. Addition of 2-deoxyglucose to the anaerobic system caused the adenylate charge to fall even more rapidly, but this effect was overcome with a glucose:2-deoxyglucose ratio of 3:1. Aerobic metabolism in the presence of glyoxylate showed a decrease in the adenylate charge from 0.91 to 0.64. Glutamate but not pyruvate, citrate, or isocitrate could overcome the effect of glyoxylate. With a combination of carbon sources and pathway inhibitors, it would appear that either the cytoplasmic enzymatic systems or the mitochondrial enzymatic systems, by themselves, are capable of maintaining a high (>0.9) adenylate charge in Novikoff ascites cells, if they have the necessary and proper carbon source. Therefore, if the decrease in the potential of the malate-aspartate and α-glycerophosphate shuttle systems and the increase in the potential of the ability of the cytoplasmic enzymatic system to produce adenosine triphosphate are considered, it is likely that tumor mitochondria are functionally sound but are unable to function at maximum capacity because they are unable to obtain enough adequate precursor metabolites.