Cell-free translocation of recombinant p47-phox, a component of the neutrophil NADPH oxidase: effects of guanosine 5'-O-(3-thiotriphosphate), diacylglycerol, and an anionic amphiphile

100

Activation of the neutrophil NADPH oxidase requires translocation of cytosolic proteins p47phox , 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.

Section: Effects Of Amphiphile Treatment On P47 Phox Fluorescence Andmentioning

confidence: 99%

Analysis of Activation-induced Conformational Changes in p47 Using Tryptophan Fluorescence Spectroscopy

Swain

Helgerson

Davis

et al. 1997

100

“…In resting cells, p47 phox and p67 phox reside in the cytosol in a 240-kDa complex with a third component, p40 phox (9 -11). Upon cell activation, p47 phox and p67 phox associate with the membrane, where they interact with flavocytochrome b 558 (12)(13)(14)(15). The small GTP-binding protein Rac is complexed in the cytosol with an inhibitory protein, RhoGDI (Rho GDP dissociation inhibitor), and translocates to the plasma membrane independently of the other cytosolic components (16 -18).…”

mentioning

confidence: 99%

Regulation of the Neutrophil Respiratory Burst Oxidase

Han

Freeman²,

Lee

et al. 1998

Self Cite

203

Superoxide generation by the neutrophil respiratory burst oxidase (NADPH oxidase) can be reconstituted in a cell-free system using flavocytochrome b 558 phox that is essential for NADPH oxidase activity. A series of C-terminal truncation mutants of p67 phox showed that residues 211 to the C terminus (residue 526) are not needed for cell-free activity. However, shorter truncations were inactive, pointing to an activation domain within the region spanning residues 199 -210. p67 phox mutated at single amino acid residues within this region showed diminished activity, and p67 phox V204A was completely inactive. The effects of mutations on activity were independent of p47 phox , and mutations did not affect the binding of p67 phox to Rac. In the presence of wild-type p67 phox , the V204A mutant was a potent inhibitor of superoxide generation, and inhibition was partially reversed by high concentrations of p67 phox , but not by p47 phox or Rac. The V204A mutant competed with native p67 phox for translocation to neutrophil plasma membrane, indicating that p67 phox V204A assembles to form an inactive complex. The data imply a direct activation of flavocytochrome b 558 by an activation domain in p67 phox .During the respiratory burst, neutrophils generate superoxide and secondarily generate reactive oxygen species (H 2 O 2 , O 2 . , and HOCl) that together participate in killing invading microorganisms (1-4). Superoxide generation is catalyzed by a multicomponent enzyme, the respiratory burst oxidase (NADPH oxidase). The enzyme consists of a plasma membrane-associated flavocytochrome b 558 composed of two subunits, gp91 phox (where phox is phagocytic oxidase) and p22 phox . The former contains all of the electron-carrying groups (one FAD and two hemes) needed to transfer electrons from NADPH to molecular oxygen as well as a candidate NADPH-binding site (5-8). In addition, three cytosolic factors, p47 phox , p67 phox , and Rac, are needed for optimal activity. In resting cells, p47 phox and p67 phox

“…The enzyme is dormant in resting cells but becomes activated when the cells are exposed to appropriate stimuli. Upon activation, a cytosolic complex consisting of the oxidase components p47 PHOX , p67 PHOX , and p40 PHOX associates with the membrane-bound cytochrome b 558 to assemble the active oxidase (2)(3)(4)(5)(6)(7).…”

mentioning

confidence: 99%

Activation of the Leukocyte NADPH Oxidase by Protein Kinase C in a Partially Recombinant Cell-free System

Lopes¹,

Hoyal²,

Knaus³

et al. 1999

The leukocyte NADPH oxidase is an enzyme present in phagocytes and B lymphocytes that when activated catalyzes the production of O 2. from oxygen at the expense of NADPH. A correlation between the activation of the oxidase and the phosphorylation of p47 PHOX , a cytosolic oxidase component, is well recognized in whole cells, and direct evidence for a relationship between the phosphorylation of this oxidase component and the activation of the oxidase has been obtained in a number of cell-free systems containing neutrophil membrane and cytosol. Using superoxide dismutase-inhibitable cytochrome c reduction to quantify O 2. production, we now show that p47 PHOX phosphorylated by protein kinase C activates the NADPH oxidase not only in a cell-free system containing neutrophil membrane and cytosol, but also in a system in which the cytosol is replaced by the recombinant proteins p67 PHOX , Rac2, and phosphorylated p47 PHOX , suggesting that neutrophil plasma membrane plus those three cytosolic proteins are both necessary and sufficient for oxidase activation. In both the cytosol-containing and recombinant cell-free systems, however, activation by SDS yielded greater rates of O 2 . production than activation by protein kinase C-phosphorylated p47 PHOX , indicating that a system that employs protein kinase C-phosphorylated p47 PHOX as the sole activating agent, although more physiological than the SDS-activated system, is nevertheless incomplete.