CHARACTERIZATION OF THE GTP-DEPENDENT ACTIVATION OF THE SUPEROXIDE-PRODUCING NADPH OXIDASE IN A CELL-FREE SYSTEM OF PIG NEUTROPHILS

We characterized the cell-free activating system of the superoxide (O2 -)-producing NADPH oxidase of pig neutrophils. Activation of the oxida se required both the membrane and cytosolic fractions in the presence of sodium dodecyl sulfate. Chromatography on 2',5'-ADP-Sepharose resul ted in separation of the cytosolic fraction into two fractions, the fl ow-through and bound fractions, which synergistically supported the O2 - production with the membrane fraction in the absence of guanosine 5' -O-(3-thiotriphosphate) (GTPgammaS), whereas only the bound fraction b esides the membrane fraction was required for the activation in the pr esence of GTPgammaS. The effective factors in the bound fraction were further purified by gel filtration on Superdex G-200 and anion exchang e chromatography on Mono Q and found to be p47-phox and p63-phox. The purified recombinant p47-phox and p65-phox replaced corresponding nati ve factors for the activation. These results suggest that the membrane fraction from pig neutrophils contains the GTP-binding protein respon sible for the activation. Furthermore, the presence of the GTP-binding protein for the activation in the flow-through fraction from 2',5'-AD P-Sepharose was also shown on the basis of the findings that extensive dialysis of the flow-through fraction resulted in complete loss of th e ability to activate the oxidase with the recombinant factors and the washed membrane of human neutrophils which contained no GTP-binding p rotein for the activation and the lost ability was recovered by the ad dition of GTPgammaS. Thus, activation of the oxidase in the cell-free system of pig neutrophils absolutely requires the GTP-binding protein which localizes in the membrane fraction or in the cytosolic fraction.