Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride

T. G. Gabig, D. English, L. P. Akard, M. J. Schell

Research output: Contribution to journalArticle

Abstract

Guanine nucleotide-binding regulatory proteins (G proteins) transduce a remarkably diverse group of extracellular signals to a relatively limited number of intracellular target enzymes. In the neutrophil, transduction of the signal following fMet-Leu-Phe receptor-ligand interaction is mediated by a pertussis toxin substrate (G(i)) that activates inositol-specific phospholipase C. We have utilized a plasma membrane-containing fraction from unstimulated human neutrophils as the target enzyme to explore the role of G proteins in arachidonate and cytosolic cofactor-dependent activation of the NADPH-dependent O2--generating oxidase. When certain guanine nucleotides or their nonhydrolyzable analogues were present during arachidonate and cytosolic cofactor-dependent activation, they exerted substantial dose-dependent effects. The GTP analogue, GTPγS, caused a 2-fold increase in NADPH oxidase activation (half-maximal stimulation, 1.1 μM). Either GDP or its nonhydrolyzable analogue, GDPβS, inhibited up to 80% of the basal NADPH oxidase activation (K(i)GDP=0.12 mM, GDPβS=0.23 mM). GTP caused only slight and variable stimulation, whereas F-, an agent known to promote the active conformation of G proteins, caused a 1.6-fold stimulation of NADPH oxidase activation. NADPH oxidase activation in the cell-free system was absolutely and specifically dependent on Mg2+. Although O2- production in response to fMet-Leu-Phe was inhibited >90% in neutrophils pretreated with pertussis toxin, cytosolic cofactor and target oxidase membranes from neutrophils treated with pertussis toxin showed no change in basal- or GTPγS-stimulated NADPH oxidase activation. Cholera toxin treatment of neutrophils also had no effect on the cell-free activation system. Our results suggest a role for a G protein that is distinct from G(s) or G(i) in the arachidonate and cytosolic cofactor-dependent NADPH oxidase cell-free activation system.

Original languageEnglish (US)
Pages (from-to)1685-1690
Number of pages6
JournalJournal of Biological Chemistry
Volume262
Issue number4
StatePublished - Jan 1 1987

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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