A neutrophil gtp-binding protein that regulates cellfree nadph oxidase activation is located in the cytosolic fraction

Theodore G. Gabig*, Elizabeth A. Eklund, G. Bruce Potter, John R. Dykes

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

19 Scopus citations


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 (GTPγS), with or without arachidonate, had no effect on subsequent NADPH oxidase activation by the cytosolic fraction. Preexposure of the cytosolic fraction to GTPγS alone did not enhance activation of the membrane oxidase. However, preexposure of the cytosol to GTPγS then arachidonate caused a four-fold enhancement of its ability to activate the membrane oxidase. This enhancement was evident after removal of unbound GTPγS and arachidonate, and was not augmented by additional GTPγS during membrane activation. A reconstitution assay was developed for cytosolic component(s) responsible for the GTPγS effect. Cytosol preincubated with GTPγ35S then arachidonate was fractionated by anion exchange chromatography. A single peak of protein-bound GTPγ35S was recovered that had reconstitutive activity. Cytosol preincubated with GTPγ35S alone was similarly fractionated and the same peak of protein-bound GTPγ35S 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 GTPγS-liganded form of this protein may be activated or stabilized by arachidonate.

Original languageEnglish (US)
Pages (from-to)945-951
Number of pages7
JournalJournal of Immunology
Issue number3
StatePublished - Aug 1 1990

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology


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