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Reconstitution of GTP gamma S-induced NADPH oxidase activity in streptolysin-O-permeabilized neutrophils by specific cytosol fractions

Leino, L; Forbes, L; Segal, A; Cockcroft, S; (1999) Reconstitution of GTP gamma S-induced NADPH oxidase activity in streptolysin-O-permeabilized neutrophils by specific cytosol fractions. BIOCHEM BIOPH RES CO , 265 (1) 29 - 37.

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Abstract

GTP gamma S activates the NADPH oxidase and this activity declines rapidly with time after preexposure to streptolysin O. This wars not due to loss of p47(phox), p67(phox), or Rac. To identify the component(s) leaking out of the permeabilized cell responsible for loss of activity, a GTP gamma S-dependent reconstitution assay was established. Neutrophil cytosol was subjected to chromatographic fractionation steps for purification of the minimum fraction required to restore activity. The reconstitution of the GTP gamma S-stimulated activity was dependent on ATP. The inhibitors staurosporine and calphostin C greatly reduced the activity in the reconstitution assay, implicating the involvement of a protein kinase C (PKC) pathway. PKC isoforms beta and delta were eliminated as the active factors in the most pure reconstitution fraction. With this novel cell-based reconstitution assay, we have identified the requirement for a protein kinase, or its substrate, for the restoration of GTP gamma S activation. of the NADPH oxidase. (C) 1999 Academic Press.

Type: Article
Title: Reconstitution of GTP gamma S-induced NADPH oxidase activity in streptolysin-O-permeabilized neutrophils by specific cytosol fractions
Keywords: PROTEIN-KINASE-C, ADP-RIBOSYLATION FACTOR, PHOSPHOLIPASE-D, SIGNAL-TRANSDUCTION, ACTIVATION, PHOSPHORYLATION, TRANSLOCATION, P47(PHOX), RECEPTORS, CELLS
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Neuro, Physiology and Pharmacology
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine > Internal Medicine
URI: http://discovery.ucl.ac.uk/id/eprint/76243
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