Activation of neutrophil NADPH oxidase in a cell-free system. Partial purification of components and characterization of the activation process.
The superoxide-generating enzyme of human neutrophils, NADPH oxidase, is converted from an inactive to an active form upon stimulation of the neutrophil. This activation process was examined using a recently developed cell-free system in which dormant oxidase is activated by arachidonic acid in the presence of a soluble factor from the neutrophil (Curnutte, J. T. (1985) J. Clin. Invest. 75, 1740-1743). NADPH oxidase from unstimulated human neutrophils was detected only in the membrane fraction. The soluble activation factor was localized entirely to the cytosolic fraction and exhibited two peaks of activity when partially purified under nondenaturing conditions: a major peak with a molecular mass of approximately 250 kDa and a variable minor peak with a mass of approximately 40 kDa. Both forms activated NADPH oxidase in a similar manner and did not exhibit synergy when combined. The cytosolic factor is not protein kinase C (or another kinase) as both peaks of factor activity could be resolved from the protein kinase C peak and neither required calcium or ATP to activate the oxidase. Activation of NADPH oxidase did require the simultaneous presence of the membrane fraction, the cytosolic factor, arachidonic acid, and magnesium. Following activation, however, only the membrane fraction was then required for O2- production. Cytosolic factor levels were normal in five patients with either X-linked or autosomal recessive cytochrome b-negative chronic granulomatous disease. In contrast, the membrane fractions from each failed to generate O2-, indicating that the defects in these two genetic forms of chronic granulomatous disease reside either in the oxidase itself or in a membrane component required for activation.
D002474:Cell-Free System; D003573:Cytochrome b Group / Q000097:blood; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D007700:Kinetics; D008297:Male; D009247:NADH, NADPH Oxidoreductases / Q000097:blood* / Q000302:isolation & purification; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010101:Oxygen Consumption
D003573:Cytochrome b Group; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases