Respiratory burst oxidase and three of four oxidase-related polypeptides are associated with the cytoskeleton of human neutrophils.

Abstract

Resting and phorbol-activated human neutrophils were separated by treatment with Triton X-100 into detergent-extractable and cytoskeleton fractions. Respiratory burst oxidase activity was restricted entirely to the cytoskeleton. The cytoskeleton also contained 15% of the neutrophil cytochrome b5!, an oxidase-associated heme protein, as well as most of the oxidase-related cytosolic polypeptide p67x. In contrast, the components of the oxidase-associated phosphoprotein family p47'" were found almost exclusively in the detergent extract, suggesting that p47' is needed for oxidase activation but not for 02 production by the activated oxidase. Activation of the oxidase had no apparent effect on the distribution of any of these species between the cytoskeleton and the detergent extract. Our results support earlier studies implying that the cytoskeleton participates in an important way in regulating the activity of the 02-forming respiratory burst oxidase of neutrophils.

Role of the actin cytoskeleton during respiratory burst in chemoattractant‐stimulated neutrophils

“…It has been shown that actin can directly interact with components of the oxidase. For example p47 phox , p21rac and p67 phox have been localized in the detergent-insoluble cytoskeletal fraction of stimulated neutrophils (Knaus et al, 1992;Nauseef et al, 1991;Wientjes et al, 2001;Woodman et al, 1991).…”

Intracellular Localization and Preassembly of the NADPH Oxidase Complex in Cultured Endothelial Cells

“…Separate rabbit polyclonal antibodies against recombinant p47 phox (R360) and p67 phox (R1469), p22 phox holoprotein (R3179), and a 12-amino acid C-terminal fragment of gp91 phox (R2085) were kindly provided by Dr. M. T. Quinn (Montana State University) (22,23). Anti-p47 phox and anti-p67 phox monoclonal antibodies were purchased from Transduction Laboratories.…”

“…Fractions-Extracting cell protein into Triton X-100-soluble and -insoluble (cytoskeleton) fractions was performed as described previously (23,30) with some modifications. Confluent PIEC were washed twice in ice-cold PBS and detached by scratching.…”

“…Fractions-It has been reported in neutrophils that NADPH oxidase activity is associated with the cytoskeleton (23,30). In view of the data presented so far indicating an intracellular co-location of NADPH oxidase subunits as functional complexes, we examined the possibility that the NADPH oxidase subunits may be associated with the cytoskeleton.…”

Deficiency of p67phox, p47phox or gp91phox in chronic granulomatous disease does not impair leucocyte chemotaxis or motility

“…Superoxide generation is confined to the phagocytic vacuole when a particle is engulfed by the phagocyte, a localization that could be affected by the reorganization of the cytoskeleton that accompanies formation of the vacuole. At least a proportion of p67 phox , but very little p47 phox , remains associated with the detergent-insoluble material after the addition of Triton X-100 to neutrophils, suggesting a possible cytoskeletal attachment (Nauseef et al, 1991;Woodman et al, 1991). In addition, activation of the respiratory burst by a chemotactic peptide or by phorbol myristate acetate (PMA) is associated with changes in the shape of the neutrophils (Wymann et al, 1989), also probably a reflection of cytoskeletal involvement (Keller et al, 1995).…”