TY - JOUR
T1 - Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride
AU - Gabig, T. G.
AU - English, D.
AU - Akard, L. P.
AU - Schell, M. J.
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1987
Y1 - 1987
N2 - 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.
AB - 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.
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M3 - Article
C2 - 3027097
AN - SCOPUS:0023190159
SN - 0021-9258
VL - 262
SP - 1685
EP - 1690
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 4
ER -