THE ENZYME RESPONSIBLE FOR THE RESPIRATORY BURST IN ELICITED GUINEA-PIG PERITONEAL-MACROPHAGES

The enzymatic basis of the respiratory burst induced by phorbol myristate acetate in elicited peritoneal macrophages of the guinea pig was studied. A membrane-bound oxidase that perferentially uses NADPH as substrate is evidently the main enzyme responsible for activation of the oxidative metabolism. The supernatant of postnuclear fractions of resting macrophages oxidized NADH and NADPH with formation of O2-. The activity with both substrates was very low and did not change in the supernatant obtained from activated cells. The cell-free particles of resting macrophages also oxidized both NADH and NADPH with formation of O2-. The activity of the cell-free particles from activated macrophages did not change when NADH is the substrate. The activity of the cell-free particles from activated cells was markedly increased when NADPH is the substrate. In cell-free particles from activated macrophages, the Km for NADPH was .apprx. 1 order of magnitude lower than that for NADH and the Vmax with NADPH is double that with NADH. The NADPH oxidase of cell-free particles was insensitive to azide, cyanide, antimycin A and rotenone and was sensitive to the SH reagent PCMB. All these drugs have the same effect on the respiratory response of intact macrophages. A direct correlation was found between the degree of activation of the respiratory metabolism of intact macrophages and the extent of activation of the NADPH oxidase. A new approach, designed to measure the activity of the oxidase soon after the activation of the enzyme has taken place, shows that the NADPH oxidase can account for the respiratory burst of intact macrophages.