Diffusion of extracellular hydrogen peroxide into intracellular compartments of human neutrophils. Studies utilizing the inactivation of myeloperoxidase by hydrogen peroxide and azide.
It is well known that catalase is transformed to nitric oxide-Fe2+-catalase by hydrogen peroxide (H2O2) plus azide. In this report, we show that myeloperoxidase is also inactivated by H2O2 plus azide. Utilizing this system, we studied the presence and source of intracellular H2O2 generated by activated neutrophils. Stimulation of neutrophils with phorbol myristate acetate (PMA, 100 ng/ml) plus azide (5 mM) for 30 min completely inactivated intragranular myeloperoxidase and reduced cytosolic catalase to 35% of resting cells. This intracellular inactivation of heme enzymes did not occur in normal neutrophils incubated with either PMA or azide alone or in neutrophils from patients with chronic granulomatous disease (CDG) which cannot produce H2O2 in response to PMA. Incubation of neutrophils with azide and a H2O2 generating system (glucose-glucose oxidase) inactivated 41% of neutrophil myeloperoxidase. Glutathione-glutathione peroxidase (GSH-GSH peroxidase), an extracellular H2O2 scavenger, totally protected neutrophil myeloperoxidase from inactivation by azide plus glucose-glucose oxidase. In addition, when a mixture of normal and CGD cells was stimulated with PMA in the presence of azide, 90% of the myeloperoxidase in CGD neutrophils was inactivated. Therefore, H2O2 released extracellularly from activated neutrophils can diffuse into cells. In contrast, myeloperoxidase in normal polymorphonuclear leukocytes stimulated with PMA in the presence of azide and GSH-GSH peroxidase was 75% inactivated. Thus, the results indicate that a GSH-GSH peroxidase-insensitive pool of H2O2 is also generated, presumably at the plasma membrane, and this pool of H2O2 can undergo direct internal diffusion to inactivate myeloperoxidase.
Ohno
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Y
Gallin
J I
JI
D000818:Animals; D001386:Azides / Q000494:pharmacology*; D001826:Body Fluids / Q000378:metabolism*; D002374:Catalase / Q000378:metabolism; D004285:Dogs; D005110:Extracellular Space / Q000378:metabolism*; D005949:Glucose Oxidase / Q000378:metabolism; D005966:Glucuronidase / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007424:Intracellular Fluid / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects; D009195:Peroxidase / Q000037:antagonists & inhibitors*; D010544:Peroxidases / Q000037:antagonists & inhibitors*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001386:Azides; D006418:Heme; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D010544:Peroxidases; D002374:Catalase; D009195:Peroxidase; D005966:Glucuronidase; D005978:Glutathione; D013755:Tetradecanoylphorbol Acetate