A point mutation in gp91-phox of cytochrome b558 of the human NADPH oxidase leading to defective translocation of the cytosolic proteins p47-phox and p67-phox.

The superoxide-forming NADPH oxidase of human phagocytes is composed of membrane-bound and cytosolic proteins which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients suffering from chronic granulomatous disease (CGD) are defective in one of the following components: p47-phox and p67-phox, residing in the cytosol of resting phagocytes, and gp91-phox and p22-phox, constituting the membrane-bound cytochrome b558. In an X-linked CGD patient we identified a novel missense mutation predicting an Asp-->Gly substitution at residue 500 of gp91-phox, associated with normal amounts of nonfunctional cytochrome b558 in the patient's neutrophils. In PMA-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient was strongly disturbed. Furthermore, a synthetic peptide mimicking domain 491-504 of gp91-phox inhibited NADPH oxidase activity in the cell-free assay (IC50 about 10 microM), and the translocation of p47-phox and p67-phox in the cell-free translocation assay. We conclude that residue 500 of gp91-phox resides in a region critical for stable binding of p47-phox and p67-phox.     

Inhibitory effect of porcine surfactant on the respiratory burst oxidase in human neutrophils. Attenuation of p47phox and p67phox membrane translocation as the mechanism.

Surfactant has been shown to inhibit the production of reactive oxygen intermediates by various cells including alveolar macrophages and peripheral blood neutrophils. Superoxide O2-. production by the respiratory burst oxidase in isolated plasma membranes prepared from PMA-treated human neutrophils was significantly attenuated by prior treatment with native porcine surfactant. The effect was concentration dependent with half-maximal inhibition seen at approximately 0.050 mg surfactant phospholipid/ml. Kinetic analyses of the membrane-bound enzyme prepared from neutrophils stimulated by PMA in the presence or absence of surfactant demonstrated that surfactant treatment led to a decrease in the maximal velocity of O2-. production when NADPH was used as substrate, but there was no effect on enzyme substrate affinity. Immunoblotting studies demonstrated that surfactant treatment induced a decrease in the association of two oxidase components, p47phox and p67phox, with the isolated plasma membrane. In contrast, surfactant treatment of the cells did not alter the phosphorylation of p47phox. A mixture of phospholipids (phosphatidylcholine and phosphatidylglycerol in a 7:3 ratio) showed similar inhibition of the PMA-induced O2-. generation. Taken together, these data suggest the mechanism of surfactant-induced inhibition of O2-. production by human neutrophils involves attenuation of translocation of cytosolic components of the respiratory burst oxidase to the plasma membrane. The phospholipid components of surfactant appear to play a significant role in this mechanism.     

156Pro-->Gln substitution in the light chain of cytochrome b558 of the human NADPH oxidase (p22-phox) leads to defective translocation of the cytosolic proteins p47-phox and p67-phox

Src homology 3 (SH3) domains have been suggested to play an important role in the assembly of the superoxide-forming nicotinamide adenine dinucleotide phosphate (NADPH) oxidase upon activation of phagocytes, which involves the association of membrane-bound and cytosolic components. We studied the translocation of the cytosolic proteins to the plasma membrane in neutrophils of a patient with a point mutation in the gene encoding the light chain of cytochrome b558. This mutation leads to a substitution at residue 156 of a proline into a glutamine in a putative SH3 binding domain of p22-phox (Dinauer, M., E. A. Pierce, R. W. Erickson, T. Muhlebach, H. Messner, R. A. Seger, S. H. Orkin, and J. T. Curnutte. 1991. Proc. Natl. Acad. Sci. 88:11231). In PMA- stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient's neutrophils was virtually absent. In contrast, when solubilized membranes of the patient's neutrophils were activated with phospholipids in the absence of cytosol (Koshkin, V., and E. Pick. 1993. FEBS [Fed. Eur. Biochem. Soc.] Lett. 327:57), the rate of NADPH- dependent oxygen uptake was observed at a rate similar to that of control membranes. We suggest that the binding of an SH3 domain of p47- phox to p22-phox, and thus activation of the oxidase, does not occur in the neutrophils of this patient, although under artificial conditions, electron flow from NADPH to oxygen in cytochrome b558 is possible.     

Neutrophil nicotinamide adenine dinucleotide phosphate oxidase assembly. Translocation of p47-phox and p67-phox requires interaction between p47-phox and cytochrome b558.

Two of the cytosolic NADPH oxidase components, p47-phox and p67-phox, translocate to the plasma membrane in normal neutrophils stimulated with phorbol myristate acetate (PMA). We have now studied the translocation process in neutrophils of patients with chronic granulomatous disease (CGD), an inherited syndrome in which the oxidase system fails to produce superoxide due to lesions affecting any one of its four known components: the gp91-phox and p22-phox subunits of cytochrome b558 (the membrane-bound terminal electron transporter of the oxidase), p47-phox, and p67-phox. In contrast to normal cells, neither p47-phox nor p67-phox translocated to the membrane in PMA-stimulated CGD neutrophils which lack cytochrome b558. In one patient with a rare X-linked form of CGD caused by a Pro----His substitution in gp91-phox, but whose neutrophils have normal levels of this mutant cytochrome b558, translocation was normal. In two patients with p47-phox deficiency, p67-phox failed to translocate, whereas p47-phox was detected in the particulate fraction of PMA-stimulated neutrophils from two patients deficient in p67-phox. Our data suggest that cytochrome b558 or a closely linked factor provides an essential membrane docking site for the cytosolic oxidase components and that it is p47-phox that mediates the assembly of these components on the membrane.     

Identification of a thermolabile component of the human neutrophil NADPH oxidase. A model for chronic granulomatous disease caused by deficiency of the p67-phox cytosolic component.

Mild heating of human neutrophils inactivates the respiratory burst oxidase, producing a defect in superoxide production and bacterial killing comparable to that seen in patients afflicted with chronic granulomatous disease (CGD). We have now investigated the mechanism and specificity of this inactivation by examining the effect of mild heating on the known oxidase components: the membrane-bound subunits of the cytochrome b558 (gp91-phox and p22-phox) and the two cytosolic oxidase factors (p47-phox and p67-phox). Heating (46 degrees C for 7.5 min) caused intact neutrophils to lose greater than 85% of their capacity to produce superoxide, a defect which was localized to the cytosolic, but not the membrane, fraction. Complementation studies with CGD cytosols deficient in either p47-phox or p67-phox suggested that the defective component of heat-inactivated cytosol was p67-phox. This was confirmed by experiments showing that recombinant p67-phox, but not p47-phox, exhibited lability at 46 degrees C and completely reconstituted oxidase activity of heat-treated cytosol. These studies indicate that mild heating of either intact neutrophils or normal neutrophil cytosol results in a selective inactivation of p67-phox, providing a model oxidase system for the extremely rare p67-phox-deficient form of CGD.     

Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation.

The superoxide-forming respiratory burst oxidase of human neutrophils is composed of membrane-associated catalytic components and cytosolic constituents required for oxidase activation. This study concerns the hypothesis that cytosolic oxidase components translocate to a membrane fraction when neutrophils are stimulated and the oxidase is activated. A polyclonal antiserum that recognizes two discrete cytosolic oxidase components of 47 and 67 kD was used to probe transfer blots of electrophoresed membrane and cytosol fractions of resting and stimulated neutrophils. In contrast to their strictly cytosolic localization in unstimulated cells, both proteins were detected in membrane fractions of neutrophils activated by phorbol esters and other stimuli. This translocation event was a function of stimulus concentration as well as time and temperature of exposure to the stimulus. It was inhibited by concentrations of N-ethylmaleimide that blocked superoxide formation but was unaffected by 2-deoxyglucose. There was a correlation between translocation of the cytosolic proteins and activation of the oxidase as determined by superoxide formation. Quantitative analyses suggested that approximately 10% of total cellular p47 and p67 became membrane-associated during phorbol ester activation of the oxidase. Analysis of Percoll density gradient fractions indicated that the target membrane for translocation of both proteins was the plasma membrane rather than membranes of either specific or azurophilic granules. In the cell-free oxidase system arachidonate-dependent but membrane-independent precipitation of the cytosolic oxidase proteins was demonstrated. The data show that activation of the respiratory burst oxidase in stimulated human neutrophils is closely associated with translocation of the 47- and 67-kD cytosolic oxidase components to the plasma membrane. We suggest that this translocation event is important in oxidase activation.     

Fluoride-mediated activation of the respiratory burst in human neutrophils. A reversible process.

Fluoride ion (F-) is an effective activator of the respiratory burst in neutrophils, as indicated by its ability to induce O2- production by these cells. Other halide ions did not activate the burst, Cl-, in particular appeared to antagonize the effect of F- on O2- production. F- stimulated O2- production showed a requirement for Ca++, but was independent of other exogenous cations. Neither phagocytosis nor degranulation were necessary for respiratory burst activation by F-. The effect of F- on the respiratory burst was reversible. Washing the cells after treatment with F-, while they were still producing large amounts of O2-, returned them to the resting state. They could then be stimulated again to produce O2- in amounts equivalent to those originally produced. Our experiments indicated that restimulation did not represent the activation of a population of cells that had not been activated during the initial exposure to F-, nor did it represent serial activation of different subpopulation of the O2- forming enzyme molecules present in a given cell. Rather, our data suggest that the entire population of O2- forming enzyme molecules was activated in a reversible fashion by F-.     

The particulate superoxide-forming system from human neutrophils. Properties of the system and further evidence supporting its participation in the respiratory burst.

Studies were performed to characterize the previously reported particulate O2--forming system from human neutrophils. Of eight reducing agents examined, including glutathione, ascorbic acid, and intermediates of the glycolytic and hexose monophosphate shunt pathways, only the pyridine nucleotides could serve as electron donors. At 0.1 mM pyridine nucleotide, O2- production was relatively independent of pH. The Km for NADH was approximately 0.7 mM regardless of pH, while with NADPH the Km varied from 0.02 mM at pH 6.0 to 0.3 mM at pH 7.5. The molar ratio of NADPH oxidized to O2- produced was consistent with the reaction: NADPH + 2 O2- leads to NADP+ H+; the product nucleotide was shown enzymatically to be NADP. O2- production was not inhibited by CN-, Na-, EDTA, or 1,10-phenanthroline. Particulate O2- production accounted for 35% of the oxygen taken up during the respiratory burst by an equivalent number of intact neutrophils. Greatly diminished O2- production was seen with particles prepared from cells obtained from three patients with chronic granulomatous disease, with 2.5 mM NADPH as electron donor. With 5.0 mM NADH similar observations were made with particles from two of the patients, but with this nucelotide, O2- production was only slightly reduced in the third case. The evidence available suggests that this particulate O2- -forming system is the one responsible for the respiratory burst in activated neutrophils. The relationship between this system and other O2- -forming system found in human neutrophils is discussed.     

The cytosolic subunit p67phox contains an NADPH-binding site that participates in catalysis by the leukocyte NADPH oxidase.

The NADPH-dependent respiratory burst oxidase of human neutrophils catalyzes the reduction of oxygen to superoxide using NADPH as the electron donor and is essential for normal host defenses. To gain insight into the function of the various oxidase subunits that are required for the full expression of catalytic activity, we studied the interactions between the 2',3'-dialdehyde derivative of NADPH (NADPH dialdehyde) and neutrophil cytosol. NADPH dialdehyde treatment of cytosol resulted in the loss of the ability of the cytosol to participate in cell-free oxidase activation; this inactivation was blocked by NADPH but not by NAD, NADP, or GTP. Partial purification of neutrophil cytosol yielded a single peak which could restore the activity lost in cytosol treated with NADPH dialdehyde. This peak contained p67phox but not p47phox or Rac2. Purified recombinant p67phox was similarly able to restore the activity lost in NADPH dialdehyde-treated cytosol and bound [32P]NADPH dialdehyde in a specific fashion. The activity of recombinant p67phox in cell-free oxidase assays was lost on treatment with NADPH dialdehyde. Together, these data suggest p67phox contains the catalytic NADPH-binding site of the leukocyte NADPH oxidase.     

Activation of the respiratory burst enzyme from human neutrophils in a cell-free system. Evidence for a soluble cofactor.

Activation of the respiratory burst in phagocytic cells, an important host defense process, is not yet well understood. We now report the development of a cell-free system for activation of NADPH oxidase, the respiratory burst enzyme, in human neutrophils. Activation was achieved by the addition of arachidonic acid to a postnuclear supernatant (500 g) from disrupted unstimulated cells (no arachidonate, 0.2; with arachidonate, 3.4 nmol superoxide anion/min per mg) and was dependent on both the concentration of arachidonate and on the amount of cellular material present. Activity stimulated by arachidonate appeared to be NADPH oxidase based on a Michaelis constant for NADPH of 32 microM and a pH optimum of 7.0-7.5. Separation of the 500-g supernatant by high speed centrifugation revealed a requirement for both soluble and particulate cofactors. Activation of NADPH oxidase by arachidonate did not occur in the high speed pellet fraction from unstimulated cells but could be restored by the addition of the high speed supernatant. In addition, priming of intact neutrophils with low concentrations of the chemoattractant N-formyl-methionyl-leucyl-phenylalanine or the tumor promoter phorbol myristate acetate replaced the soluble factor requirement for NADPH oxidase activation by arachidonate in the high speed pellet. This cell-free system can now be used to provide further insight into the biochemical basis of priming and the terminal mechanisms involved in the activation of NADPH oxidase.     

GM-CSF increases in vitro the respiratory burst of human neutrophils after liver transplantation

Objective: Superoxide production by polymorphonuclear neutrophils (PMNs) under cyclosporin A (CsA) therapy following kidney transplantation is impaired. We investigated if the respiratory burst of PMNs is similarly depressed in patients undergoing CsA treatment following orthotopic liver transplantation (OLTx). Additionally, the in vitro influence of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the superoxide anion production was examined during the respiratory burst. Patients: 10 patients after OLTx and 10 healthy blood donors (control group). Measurements and results: PMNs were stimulated with bacteria (Escherichia coli) or a combination of tumour necrosis factor alpha (TNFα) and N-formyl-methionyl-leucyl-phenylalanine (FMLP). The respiratory burst was measured by oxidation of non-fluorescent dihydrorhodamine to the fluorescent rhodamine by means of flow cytometry. No differences in respiratory bursts from OLTx patients compared to those from healthy blood donors could be seen. Under TNFα/FMLP stimulation, the respiratory burst was significantly increased after in vitro incubation with GM-CSF (500 U ml^–1) in patients following OLTx (from 58.2 to 74.5 %) as well as in the control group (from 47.4 to 61.9 %). Conclusions: Our results demonstrate that superoxide production is not impaired under CsA treatment following OLTx. The respiratory burst of these patients' PMNs can even be augmented by GM-CSF in vitro. Received: 30 July 1998 Final revision received: 28 March 1999 Accepted: 6 April 1999     

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

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 approximately 15% of the neutrophil cytochrome b558, an oxidase-associated heme protein, as well as most of the oxidase-related cytosolic polypeptide p67phox. In contrast, the components of the oxidase-associated phosphoprotein family p47phox were found almost exclusively in the detergent extract, suggesting that p47phox is needed for oxidase activation but not for O2- 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 O2(-)-forming respiratory burst oxidase of neutrophils.     

Lipopolysaccharide priming of human neutrophils for an enhanced respiratory burst. Role of intracellular free calcium.

Lipopolysaccharide (LPS) pretreatment "primes" neutrophils to release increased amounts of superoxide anion (O2-) when stimulated. We investigated the molecular basis of this enhanced activity. Comparison of kinetic parameters of the respiratory burst NADPH oxidase in unstimulated LPS-primed and control neutrophils disclosed a similar Km for NADPH and no difference was seen in the content of cytochrome b. Pertussis toxin, which inhibits some G proteins, did not prevent priming. Change in membrane potential (delta psi) was five-fold greater in LPS-primed cells and paralleled the increased O2- release. Cytofluorographic analysis indicated that the increased change in delta psi was due to the creation of a new population of active cells. Changes in the concentration of intracellular free Ca2+ ([Ca2+]i) are believed to antecede changes in delta psi. There was a consistent increment (67 +/- 8%, n = 12) in resting [Ca2+]i in cells preincubated with LPS compared with control. When stimulated, the peak [Ca2+]i was significantly higher in LPS-primed cells. Ca2+-dependent protein kinase C activity was unaltered in resting and FMLP-stimulated neutrophils preexposed to LPS. Addition to cells of the intracellular Ca2+ chelator MAPTAM before preincubation with LPS blocked the changes in [Ca2+]i and the enhanced respiratory burst that characterize LPS priming. The increased resting [Ca2+]i in LPS-primed cells may enhance stimulus-induced cellular activity by modifying a Ca2+-dependent step in signal transduction.     

Influence of cefodizime on chemotaxis and the respiratory burst in neutrophils from diabetics

The effect of cefodizime on the function in vitro of neutrophils from poorly-controlled, non-insulin-dependent diabetics was demonstrated by the chemotactic index and by chemiluminescence. Without cefodizime treatment, the chemotactic index for diabetic neutrophils was significantly lower than that for healthy controls (1.0 +/- 0.1 vs 3.6 +/- 0.7; mean +/- S.E.M., P less than 0.01). When neutrophils were pretreated with cefodizime at 1 or 10 mg/l, cefodizime restored the chemotactic activity of diabetic neutrophils (1.0 +/- 0.1 vs 2.7 +/- 0.3, P less than 0.01), but did not affect the activity in healthy controls. Conversely, cefodizime at 100 mg/l had no enhancing effect on diabetic neutrophil chemotaxis, but decreased the chemotactic activity of healthy neutrophils (1.1 +/- 0.2 vs 3.6 +/- 0.7, P less than 0.01). A difference in neutrophil chemiluminescence was also found between diabetics and healthy subjects but no influence of cefodizime on chemiluminescence was noted in neutrophils from either diabetics or healthy subjects.     

Priming by grepafloxacin on respiratory burst of human neutrophils: its possible mechanism

Grepafloxacin is a broad-spectrum fluoroquinolone derivative that has good tissue penetration. We demonstrated that grepafloxacin showed a priming effect on neutrophil respiratory burst, triggered by either a chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (fMLP) or leukotriene B4 (LTB4), but not by the phorbol ester phorbol 12-myristate 13-acetate (PMA). The priming effect of grepafloxacin on fMLP-stimulated superoxide generation by human neutrophils correlated with the penetration of grepafloxacin into cells. Removal of extracellular grepafloxacin did not inhibit the priming effect on fMLP-stimulated superoxide generation. Furthermore, grepafloxacin induced the translocation of p47-phox and p67-phox to the membrane fraction of neutrophils, whereas tyrosine phosphorylation was hardly observed in neutrophils exposed to grepafloxacin. The priming effect of grepafloxacin on superoxide generation from neutrophils was not inhibited by treatment with pertussis toxin, a protein-tyrosine kinase inhibitor (ST-638) or a protein kinase C inhibitor (calphostin C), or chelation of extracellular calcium. Grepafloxacin did not change the fMLP receptor-binding properties. Taken together, these findings suggest that grepafloxacin evokes a priming effect on neutrophil superoxide generation intracellularly through the translocation of p47-phox and even p67-phox protein to the membrane fractions. GTP binding protein, protein-tyrosine phosphorylation and protein kinase C activation are not involved in the priming effect.     

Activation of H+ conductance in neutrophils requires assembly of components of the respiratory burst oxidase but not its redox function.

In phagocytes, superoxide generation by the NADPH oxidase is accompanied by metabolic acid production. Cytoplasmic acidification during this metabolic burst is prevented by a combination of H+ extrusion mechanisms, including a unique H+ conductance. NADPH oxidase is deficient in chronic granulomatous disease (CGD) patients. The burst of acid production is absent in CGD patients lacking the 47-kD (p47-phox) or the 91-kD (gp91-phox) subunits of the oxidase. Activation of the H+ conductance is also defective in these patients suggesting that (a) the oxidase itself undertakes H+ translocation or (b) oxidase assembly is required to stimulate a separate H+ conducting entity. To discern between these possibilities, three rare forms of CGD were studied. In neutrophils expressing nonfunctional cytochrome b, the conductance was activated to near-normal levels, implying that functional oxidase is not required to activate H+ extrusion. CGD cells expressing diminished amounts of cytochrome displayed H+ conductance approaching normal levels, suggesting that the oxidase itself does not translocate H+. Finally, the conductance was only partially inhibited in patients lacking the 67-kD subunit, indicating that this component is not essential for stimulation of H+ transport. We propose that normal assembly of the oxidase subunits is required for optimal activation of a closely associated but distinct H+ conducting entity.     

Arrangement of the respiratory burst oxidase in the plasma membrane of the neutrophil.

Evidence was obtained regarding the way the O-2-forming NADPH oxidase of human neutrophils is arranged within the plasma membrane. O-2 production by particles from zymosan-activated human neutrophils rose two- to threefold when the particles were assayed in the presence of appropriate concentrations of Triton X-100. The portion of activity revealed by the detergent was not affected by treating the particles with trypsin or with p-chloromercuribenzene sulfonate, a nonpenetrating sulfhydryl reagent, but the activity detectable in the absence of detergent was abolished by these treatments. O-2 production by phagocytic vesicles was not augmented by detergent, and was almost entirely eliminated by tryptic digestion of the vesicles regardless of whether or not detergent was present during the assay. These results suggest that the O-2-forming oxidase is embedded in the plasma membrane with a portion extending into the cytoplasm and the rest buried in the lipid bilayer. It is proposed that the pyridine nucleotide-binding site is located on the cytoplasmic extension and the oxygen binding site is on the intramembranous portion of the enzyme.     

Defensins. Natural peptide antibiotics of human neutrophils.

We extracted a granule-rich sediment from normal human neutrophils and subjected it to chromatographic, electrophoretic, and functional analysis. The extract contained three small (molecular weight less than 3,500) antibiotic peptides that were named human neutrophil peptide (HNP)-1, HNP-2, and HNP-3, and which will be referred to as "defensins." HNP 1-3, a mixture of the three defensins, killed Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli effectively in vitro when tested in 10 mM phosphate buffer containing certain nutrients, but it had little or no bactericidal activity in nutrient-free buffer. In contrast, the nutrient-free buffer supported a high degree of activity by HNP 1-3 against Cryptococcus neoformans. In addition to its antibacterial and antifungal properties, HNP 1-3 directly inactivated herpes simplex virus, Type 1. Two of the individual purified defensins, HNP-1 and HNP-2, were as microbicidal as the mixture HNP 1-3. HNP-3 was less active than the other defensins against most but not all of the microbes tested. Immunoperoxidase stains revealed HNP 1-3 to have a granular localization in the neutrophil's cytoplasm by light microscopy. Frozen thin section immunogold transmission electron microscopy showed HNP 1-3 to be localized in azurophil granules. These studies define a broad-spectrum antimicrobial system in human neutrophils. The defensin system may operate in conjunction with or independently from oxygen-dependent microbicidal processes to enable human neutrophils to inactivate and destroy potential pathogens.     

The phosphorylation targets of p47phox, a subunit of the respiratory burst oxidase. Functions of the individual target serines as evaluated by site-directed mutagenesis.

The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to O2- at the expense of NADPH. Dormant in resting cells, the oxidase is activated by exposing the cells to appropriate stimuli. During activation, p47phox, a cytosolic oxidase subunit, becomes extensively phosphorylated on a number of serines located between S303 and S379. To determine whether this phosphorylation is necessary for oxidase activation, we examined phorbol-elicited oxidase activity in EBV-transformed B lymphoblasts deficient in p47phox after transfection with plasmids expressing various S-->A mutants of p47phox. The mutant containing S-->A mutations involving all serines between S303 and S379 [S(303-379)A] was not phosphorylated, did not translocate to plasma membrane during activation and was almost devoid of function. As to individual serines, S379 was of special interest because (a) p47 phox S379 was phosphorylated in phorbol-activated lymphoblasts expressing wild-type p47phox, and (b) p47phox S379A failed to translocate to the membrane, and was as functionless as p47phox S(303-379)A; other single S-->A mutations had little effect on oxidase activity. These findings suggest that the phosphorylation of S379 may be important for oxidase activation in whole cells.