A neutrophil GTP-binding protein that regulates cell free NADPH oxidase activation is located in the cytosolic fraction.
The dormant O2(-)-generating oxidase in plasma membranes from unstimulated neutrophils becomes activated in the presence of arachidonate and a multicomponent cytosolic fraction. This process is stimulated by nonhydrolyzable GTP analogues and may involve a pertussis toxin insensitive GTP-binding protein. Our studies were designed to characterize the putative GTP-binding protein, localizing it to either membrane or cytosolic fraction in this system. Exposure of the isolated membrane fraction to guanosine-5'-(3-O-thio)triphosphate (GTP gamma S), with or without arachidonate, had no effect on subsequent NADPH oxidase activation by the cytosolic fraction. Preexposure of the cytosolic fraction to GTP gamma S alone did not enhance activation of the membrane oxidase. However, preexposure of the cytosol to GTP gamma S then arachidonate caused a four-fold enhancement of its ability to activate the membrane oxidase. This enhancement was evident after removal of unbound GTP gamma S and arachidonate, and was not augmented by additional GTP gamma S during membrane activation. A reconstitution assay was developed for cytosolic component(s) responsible for the GTP gamma S effect. Cytosol preincubated with GTP gamma 35S then arachidonate was fractionated by anion exchange chromatography. A single peak of protein-bound GTP gamma 35S was recovered that had reconstitutive activity. Cytosol preincubated with GTP gamma 35S alone was similarly fractionated and the same peak of protein-bound GTP gamma 35S was observed. However, this peak had no reconstitutive activity. We conclude that the GTP-binding protein regulating this cellfree system is located in the cytosolic fraction. The GTP gamma S-liganded form of this protein may be activated or stabilized by arachidonate.
Gabig
T G
TG
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
Eklund
E A
EA
Potter
G B
GB
Dykes
J R
JR
D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000032:analysis*; D004789:Enzyme Activation; D005227:Fatty Acids / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000032:analysis*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000032:analysis*; D013873:Thionucleotides / Q000494:pharmacology
D001095:Arachidonic Acids; D005227:Fatty Acids; D013873:Thionucleotides; D016718:Arachidonic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D019204:GTP-Binding Proteins