Evidence that activation of the respiratory burst oxidase in a cell-free system from human neutrophils is accomplished in part through an alteration of the oxidase-related 67-kDa cytosolic protein.
Sodium dodecyl sulfate (SDS) is able to activate the respiratory burst oxidase in a system containing cytosol and solubilized membranes from human neutrophils. When SDS was used to treat cytosol in an otherwise identical system in which the solubilized membrane solution was omitted, the ability of the SDS-treated cytosol to support O2- production was lost in a first-order reaction whose rate constant was virtually identical to the rate constant for the first-order activation of the oxidase in the complete system. Studies with chronic granulomatous disease cytosols showed that the component whose activity was lost was the oxidase-related 67-kDa cytosolic protein. The similarity in the rates of oxidase activation and p67 inactivation suggested that the activation of the respiratory burst oxidase in the cell-free system could involve an SDS-mediated alteration in p67. Further support for this idea was provided by kinetic experiments demonstrating that, although the yield of oxidase showed a 2.5-order dependence on cytosol concentration, oxidase activation was nevertheless kinetically irreversible. These two findings, incompatible in general, can be reconciled by a mechanism in which SDS acts specifically on a single oxidase component (i.e. p67), but with an effect that depends on circumstances: oxidase activation, if the SDS-sensitive component is part of a completely assembled oxidase precursor; loss of p67 activity, if not.
Fujimoto
S
S
Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, California 92037.
Smith
R M
RM
Curnutte
J T
JT
Babior
B M
BM
D000328:Adult; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010088:Oxidoreductases / Q000097:blood*; D013481:Superoxides / Q000097:blood
D013481:Superoxides; D010088:Oxidoreductases; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme