ANALYSIS OF ACTIVATION-INDUCED CONFORMATIONAL-CHANGES IN P47(PHOX) USING TRYPTOPHAN FLUORESCENCE SPECTROSCOPY

Activation of the neutrophil NADPH oxidase requires translocation of c ytosolic proteins p47(phox), P67(phox), and Pac to the plasma membrane or phagosomal membrane, where they assemble with membrane-bound flavo cytochrome b, During this process, it appears that p47(phox) undergoes conformational changes, resulting in the ex exposure of binding sites involved in assembly and activation of the oxidase, In the present st udy, we have directly evaluated activation-induced conformational chan ges in p47(phox) using tryptophan fluorescence and circular dichroism spectroscopy. Treatment of p47(phox) with amphiphilic agents known to activate the NADPH oxidase (SDS and arachidonic acid) caused a dose de pendent quenching in the intrinsic tryptophan fluorescence of p47(phox ), whereas treatment with a number of other amphiphilic agents that fa iled to activate the oxidase had no effect on p47(phox) fluorescence. In addition, the concentration range of activating agents required to induce changes in fluorescence correlated with the concentration range of these agents that induced maximal NADPH oxidase activity in a cell -free assay system. We next determined if activation by phosphorylatio n caused the same type of conformational changes in p47(phox). Protein kinase C phosphorylation of p47(phox) in vitro resulted in comparable quenching of fluorescence, which also correlated directly with NADPH oxidase activity, Finally, the circular dichroism (CD) spectrum of p47 (phox) was significantly changed by the addition of SDS, whereas treat ment with a non-activating detergent had no effect on the CD spectrum, These results support the conclusion that activation by amphiphilic a gents results in changes in the secondary structure of p47(phox). Thus , our studies provide direct evidence linking conformational changes i n p47(phox) to the NADPH oxidase activation/assembly process and also further support the hypothesis that amphiphile-mediated activation of the NADPH oxidase induces changes in p47(phox) that are similar to tho se mediated by phosphorylation in rico.