L. Leino et al., Reconstitution of GTP gamma S-induced NADPH oxidase activity in streptolysin-O-permeabilized neutrophils by specific cytosol fractions, BIOC BIOP R, 265(1), 1999, pp. 29-37
Citations number
28
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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-depe
ndent reconstitution assay was established. Neutrophil cytosol was subjecte
d to chromatographic fractionation steps for purification of the minimum fr
action 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, impl
icating the involvement of a protein kinase C (PKC) pathway. PKC isoforms b
eta and delta were eliminated as the active factors in the most pure recons
titution 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 Acad
emic Press.