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

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.