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The enzyme responsible for the respiratory burst in elicited guinea pig peritoneal macrophages
Author(s) -
Berton Giorgio,
Bellavite Paolo,
Dri Pietro,
de Togni Piero,
Rossi Filippo
Publication year - 1982
Publication title -
the journal of pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.964
H-Index - 184
eISSN - 1096-9896
pISSN - 0022-3417
DOI - 10.1002/path.1711360403
Subject(s) - respiratory burst , nadph oxidase , antimycin a , biochemistry , enzyme , chemistry , oxidative phosphorylation , phorbol , substrate (aquarium) , oxidase test , biology , mitochondrion , ecology , protein kinase c
The enzymatic basis of the respiratory burst induced by phorbol myristate acetate in elicited peritoneal macrophages of the guinea‐pig has been studied. The following evidence suggests that a membrane‐bound oxidase that preferentially uses NADPH as substrate is the main enzyme responsible for activation of the oxidative metabolism: (1) The supernatant of postnuclear fractions of resting macrophages oxidises NADH and NADPH with formation of O 2 − . The activity with both substrates is very low and does not change in the supernatant obtained from activated cells. (2) The cell‐free particles of resting macrophages also oxidise both NADH and NADPH with formation of O 2 − . The activity of the cell‐free particles from activated macrophages does not change when NADH is the substrate. By contrast, the activity of the cell‐free particles from activated cells is markedly increased when NADPH is the substrate. (3) In cell‐free particles from activated macrophages the K m for NADPH is about one order of magnitude lower than that for NADH and the V max with NADPH is double that with NADH. (4) The NADPH oxidase of cell‐free particles is insensitive to azide, cyanide, antimycin A and rotenone and is sensitive to the sulphydryl reagent PCMB. All these drugs have the same effect on the respiratory response of intact macrophages. (5) A direct correlation is 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.