Activation of the respiratory burst enzyme from human neutrophils in a cell-free system. Evidence for a soluble cofactor.
Activation of the respiratory burst in phagocytic cells, an important host defense process, is not yet well understood. We now report the development of a cell-free system for activation of NADPH oxidase, the respiratory burst enzyme, in human neutrophils. Activation was achieved by the addition of arachidonic acid to a postnuclear supernatant (500 g) from disrupted unstimulated cells (no arachidonate, 0.2; with arachidonate, 3.4 nmol superoxide anion/min per mg) and was dependent on both the concentration of arachidonate and on the amount of cellular material present. Activity stimulated by arachidonate appeared to be NADPH oxidase based on a Michaelis constant for NADPH of 32 microM and a pH optimum of 7.0-7.5. Separation of the 500-g supernatant by high speed centrifugation revealed a requirement for both soluble and particulate cofactors. Activation of NADPH oxidase by arachidonate did not occur in the high speed pellet fraction from unstimulated cells but could be restored by the addition of the high speed supernatant. In addition, priming of intact neutrophils with low concentrations of the chemoattractant N-formyl-methionyl-leucyl-phenylalanine or the tumor promoter phorbol myristate acetate replaced the soluble factor requirement for NADPH oxidase activation by arachidonate in the high speed pellet. This cell-free system can now be used to provide further insight into the biochemical basis of priming and the terminal mechanisms involved in the activation of NADPH oxidase.
McPhail
L C
LC
Shirley
P S
PS
Clayton
C C
CC
Snyderman
R
R
D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000494:pharmacology; D002474:Cell-Free System; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D013481:Superoxides / Q000097:blood
D001095:Arachidonic Acids; D013481:Superoxides; D016718:Arachidonic Acid; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases