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Resolution of a low molecular weight G protein in neutrophil cytosol required for NADPH oxidase activation and reconstitution by recombinant Krev-1 protein
Ist Teil von
The Journal of biological chemistry, 1991-07, Vol.266 (21), p.13964-13970
Ort / Verlag
Bethesda, MD: American Society for Biochemistry and Molecular Biology
Erscheinungsjahr
1991
Quelle
MEDLINE
Beschreibungen/Notizen
Activation of the membrane-associated NADPH oxidase in intact human neutrophils requires a receptor-associated heterotrimeric
GTP-binding protein that is sensitive to pertussis toxin. Activation of this NADPH oxidase by arachidonate in a cell-free
system requires an additional downstream pertussis toxin-insensitive G protein (Gabig, T. G., English, D., Akard, L. P., and
Schell, M. J. (1987) (J. Biol. Chem. 262, 1685-1690) that is located in the cytosolic fraction of unstimulated cells (Gabig,
T. G., Eklund, E. A., Potter, G. B., and Dykes, J. R. (1990) J. Immunol. 145, 945-951). In the present study, immunodepletion
of G proteins from the cytosolic fraction of unstimulated neutrophils resulted in a loss of the ability to activate NADPH
oxidase in the membrane fraction. The activity in immunodepleted cytosol was fully reconstituted by a partially purified fraction
from neutrophil cytosol that contained a 21-kDa GTP-binding protein. Purified human recombinant Krev-1 p21 also completely
reconstituted immunodepleted cytosol whereas recombinant human H-ras p21 or yeast RAS GTP-binding proteins had no reconstitutive
activity. Rabbit antisera raised against a synthetic peptide corresponding to the effector region of Krev-1 (amino acids 31-43)
completely inhibited cell-free NADPH oxidase activation, and this inhibition was blocked by the synthetic 31-43 peptide. An
inhibitory monoclonal antibody specific for ras p21 amino acids 60-77 (Y13-259) had no effect on cell-free NADPH oxidase activation.
Activation of the NADPH oxidase in intact neutrophils by stimulation with phorbol myristate acetate caused a marked increase
in the amount of membrane-associated antigen recognized by 151 antiserum on Western blot. Thus a G protein in the cytosol
of unstimulated neutrophils antigenically and functionally related to Krev-1 may be the downstream effector G protein for
NADPH oxidase activation. This system represents a unique model to study molecular interactions of a ras-like G protein.