Production of myeloid cell cytosols functionally and immunochemically deficient in the 47 kDa or 67 kDa NADPH oxidase cytosolic factors.
Professional phagocytes contain a unique NADPH oxidase responsible for the production of microbicidal oxidants. Activation of this oxidase requires participation of cytosolic and membrane proteins, but the interactions of these components are incompletely understood. Patients with autosomal recessive Chronic Granulomatous Diseases (CGD) are characterized by functional defects in phagocyte oxidase activity resulting from a deficiency of either a 47 kDa (p47) or a 67 kDa (p67) cytosolic oxidase component. Cytosols from such patients are valuable for biochemical studies of the oxidase, but are not generally available because CGD is a rare disorder. The present study illustrates means of producing cytosols functionally and immunochemically deficient in either p47 or p67. Cytosol from monocytes cultured for 6 days is immunochemically deficient in p47 but not p67, while cytosol from HL-60 cells induced with retinoic acid for 3 days is deficient in p67 but not p47. Each of these cytosols fail to generate superoxide when added to neutrophil membranes in a cell-free assay but complement each other when combined. Complementation studies in which these cytosols were mixed in the cell-free assay with p47- or p67- deficient CGD cytosol established the functional characteristics of the experimentally produced cytosols.
Levy
R
R
Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, N.I.H., Bethesda, Maryland 20092.
Malech
H L
HL
Rotrosen
D
D
D002474:Cell-Free System; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation; D006105:Granulomatous Disease, Chronic / Q000201:enzymology; D006801:Humans; D015151:Immunoblotting; D009000:Monocytes / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000172:deficiency* / Q000378:metabolism; D019255:NADPH Oxidases; D013481:Superoxides / Q000378:metabolism
D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases