NUCLEOSIDE-TRIPHOSPHATE BINDING OF THE 2 CYTOSOLIC COMPONENTS OF THE RESPIRATORY BURST OXIDASE SYSTEM - EVIDENCE FOR ITS INHIBITION BY THE 2',3'-DIALDEHYDE DERIVATIVE OF NADPH AND DESENSITIZATION IN THEIR TRANSLOCATED STATES

Affinity labeling of the two cytosolic components of the respiratory burst oxidase system, p49-phox and p63-phox, from resting porcine neutrophils was carried out with [P-32]NADPH dialdehyde (oNADPH), [P-32]oGTP and [P-32]oATP. p49-phox and p63-phox showed 10-times higher affinities for both oGTP and oATP than for oNADPH, suggesting that they are nucleoside triphosphate (NTP)-binding proteins, rather than the NADPH-binding site of the oxidase. In addition, oNADPH markedly inhibited the affinity labeling of p49-phox with [P-32]oGTP and [P-32]oATP, well reflecting its inhibitory effect on the oxidase activity in the cell-free system, which was previously reported to propose the NADPH-binding site in a cytosolic component. Stimulation of porcine neutrophils with either myristic acid or phorbol myristate acetate resulted in great enhancement of the oxidase activity, and in considerable translocation of p49-phox and p63-phox. Nevertheless, the affinity labeling of the stimulated cell membranes in both cases revealed no labeled bands corresponding to molecular masses of 49 kDa and 63 kDa. p49-phox derived from the stimulated membranes had lost its [P-32]oGTP binding ability in contrast with that from resting cytosol, suggesting that the NTP-binding sites of the two cytosolic components may be desensitized on NTP binding in their translocated states.