Abstract
Activation of the neutrophil NADPH oxidase requires translocation of cytosolic proteins p47(phox), p67(phox), and Rac to the plasma membrane or phagosomal membrane, where they assemble with membrane-bound flavocytochrome b. During this process, it appears that p47(phox) undergoes conformational changes, resulting in the exposure of binding sites involved in assembly and activation of the oxidase. In the present study, we have directly evaluated activation-induced conformational changes 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-dependent quenching in the intrinsic tryptophan fluorescence of p47(phox), whereas treatment with a number of other amphiphilic agents that failed 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 phosphorylation 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 treatment with a non-activating detergent had no effect on the CD spectrum. These results support the conclusion that activation by amphiphilic agents results in changes in the secondary structure of p47(phox). Thus, our studies provide direct evidence linking conformational changes in 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 those mediated by phosphorylation in vivo.
Highlights
Human neutrophils play an essential role in host defense and are key participants in the inflammatory response to foreign pathogens
We present data showing that both amphiphiles (SDS and arachidonic acid) and protein kinase C (PKC) phosphorylation cause similar changes in the inherent tryptophan fluorescence of p47phox, and these changes in fluorescence correlated directly with NADPH oxidase activity
Effects of Amphiphile Treatment on p47phox Fluorescence and NADPH Oxidase Activity—To analyze potential activation-induced conformational changes in p47phox, we measured its intrinsic tryptophan fluorescence before and after exposure to agents that are used as activators of the NADPH oxidase in the cell-free assay system
Summary
Human neutrophils play an essential role in host defense and are key participants in the inflammatory response to foreign pathogens (for reviews, see Refs. 1 and 2). In the cell-free assay system does not require phosphorylation of p47phox [20, 21], there is a requirement for anionic amphiphilic detergents such as sodium dodecyl sulfate (SDS) or arachidonic acid [22,23,24] It is still not clear whether amphiphile activation in vitro is mimicking actual physiological events in vivo, but it has been suggested that activation with amphiphiles may share mechanistic similarities with activation by phosphorylation [5, 23]. Generation in the cell-free system without added amphiphiles, mimicking the phosphorylation-dependent activation process occurring in vivo [25, 26] It has been proposed by several investigators that phosphorylation causes a conformational change in p47phox and/or neutralizes the cationic region of the protein in vivo so that it may interact with the membrane [15, 20]. We hypothesized that if activation of the NADPH oxidase induced unmasking of this region of p47phox (28 –31), significant and measurable changes would occur in the micro-environment of these tryptophan residues
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