Leukocyte Energy Metabolism III. Anaerobic and Aerobic ATP Production and Related Enzymes

Abstract

We have measured anaerobic and aerobic energy metabolism in leukocytes from human and animal blood, using several methods. We have taken special care to correct our results for an unavoidable contamination with thrombocytes, which has been neglected in all previous work. White cells were isolated by a method combining sedimentation through Dextran with low speed centrifugations. A pellet of thrombocytes was prepared in parallel from the same blood. Mitochondria were isolated by differential centrifugation in 0.25 M sucrose. Firstly, we have specially studied two cytosolic glycolytic enzymes, phosphoglycerate kinase and pyruvate kinase, both of which catalyze a reaction producing ATP. These two enzymes are very active in leukocytes. Other nucleotides, such as CDP, GDP and UDP can also be phosphorylated. Secondly, we have measured the activity of several mitochondrial enzymes, the most active being α-glycerophosphate oxidase. Using an enzymatic test, we have measured the amount of ATP produced in oxidative phosphorylation; in these experiments it was necessary to consider the activity of adenylate kinase as well as correcting for thrombocyte contamination. Several metabolites in direct or indirect relation with the Krebs cycle were used as oxidizable substrates: glutamate, succinate and α-glycerophosphate gave the best results. Except for amytal, inhibitors of oxidative phosphorylation also inhibit ATP production in leukocyte mitochondria. In conclusion, it can be said that oxidative phosphorylation exists in leukocytes, but that the phosphorylation steps in glycolysis produce 1,000 to 10,000 times more ATP than aerobic energy metabolism.