NUCLEOTIDE-BINDING COMPONENT OF THE RESPIRATORY BURST OXIDASE OF HUMAN NEUTROPHILS

The respiratory burst oxidase of neutrophils is a multicomponent enzyme, dormant in resting cells, that catalyzes the reduction of oxygen to O-2(-) at the expense of NADPH. In the resting neutrophil, some of the components of the oxidase, including proteins p47 and p67, are in the cytosol, while the rest are in the plasma membrane. Recent evidence has suggested that at least some of the cytosolic oxidase components exist as a complex The cytosolic complex with a molecular weight of similar to 240 kDa was found to bind to blue-agarose and 2',5'-ADP-agarose, which recognize nucleotide requiring enzymes. In order to identify the nucleotide binding component of the cytosolic complex we purified recombinant p47 and p67 fusion proteins using the pGEX system. Pure recombinant p47 was retained completely on 2',5'-ADP-agarose, whereas pure recombinant p67 did not bind to these affinity beads. On the basis of these results, we infer that p47 may contain the nucleotide binding site.