The respiratory burst oxidase of human neutrophils. Guanine nucleotides and arachidonate regulate the assembly of a multicomponent complex in a semirecombinant cell-free system.
We recently characterized a "semirecombinant" cell-free NADPH-oxidase system, comprised of plasma membrane plus the recombinant cytosolic proteins p47-phox and p67-phox, wherein superoxide generation was activated by an anionic amphiphile plus guanosine 5'-O-(2-thiotriphosphate) (GTP gamma S) (Uhlinger, D. J., Inge, K. L., Kreck, M. L., Tyagi, S. R., Neckelmann, N., and Lambeth, J. D. (1992) Biochem. Biophys. Res. Commun. 186, 509-516). Based on preincubation with guanine nucleotides, we show that plasma membrane contains G protein(s) that support oxidase activation at submaximal rates. By varying p47-phox and p67-phox concentrations, kinetic parameters (EC50 and Vmax) for each were determined. For both, GTP gamma S increased the Vmax and decreased the EC50, whereas guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) produced the opposite effect, consistent with the participation of a G protein in an activation complex containing p47-phox and p67-phox. Using [35S]methionine-labeled p47-phox and p67-phox, we investigated the association of these components with both normal plasma membranes and chronic granulomatous disease membranes lacking cytochrome b558. p47-phox translocation was stimulated by arachidonate but not GTP gamma S, was about 50% cytochrome-dependent, and occurred independently of p67-phox. Arachidonate-stimulated translocation of p67-phox required both cytochrome and p47-phox and was enhanced by GTP gamma S. The mass of p47-phox and p67-phox which assembled with cytochrome b558 indicated a ternary complex with a 1:1:1 stoichiometry.
Uhlinger
D J
DJ
Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322.
Tyagi
S R
SR
Inge
K L
KL
Lambeth
J D
JD
D000328:Adult; D001095:Arachidonic Acids / Q000378:metabolism*; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D004247:DNA; D019204:GTP-Binding Proteins / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010750:Phosphoproteins / Q000378:metabolism; D016897:Respiratory Burst; D013481:Superoxides / Q000378:metabolism
D001095:Arachidonic Acids; D006150:Guanine Nucleotides; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004247:DNA; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase; D019204:GTP-Binding Proteins