THE RESPIRATORY BURST OXIDASE OF HUMAN NEUTROPHILS - GUANINE-NUCLEOTIDES AND ARACHIDONATE REGULATE THE ASSEMBLY OF A MULTICOMPONENT COMPLEXIN A SEMIRECOMBINANT CELL-FREE SYSTEM

We recently characterized a ''semirecombinant'' cell-free NADPH-oxidas e system, comprised of plasma membrane plus the recombinant cytosolic proteins p47-phox and p67-phox, wherein superoxide generation was acti vated by an anionic amphiphile plus guanosine 5'-O-(2-thiotriphosphate ) (GTPgammaS) (Uhlinger, D. J., Inge, K. L., Kreck, M. L., Tyagi, S. R ., Neckelmann, N., and Lambeth, J. D. (1992) Biochem. Biophys. Res. Co mmun. 186, 509-516). Based on preincubation with guanine nucleotides, we show that plasma membrane contains G protein(s) that support oxidas e activation at submaximal rates. By varying p47-phox and p67-phox con centrations, kinetic parameters (EC50 and V(max)) for each were determ ined. For both, GTPgammaS increased the V(max) and decreased the EC50, whereas guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS) produced the op posite effect, consistent with the participation of a G protein in an activation complex containing p47-phox and p67-phox. Using [S-35]methi onine-labeled p47-phox and p67-phox, we investigated the association o f these components with both normal plasma membranes and chronic granu lomatous disease membranes lacking cytochrome b558. p47-phox transloca tion was stimulated by arachidonate but not GTPgammaS, was about 50% c ytochrome-dependent, and occurred independently of p67-phox. Arachidon ate-stimulated translocation of p67-phox required both cytochrome and p47-phox and was enhanced by GTPgammaS. The mass of p47-phox and p67-p hox which assembled with cytochrome b558 indicated a ternary complex w ith a 1:1:1 stoichiometry.