Human neutrophil cytosolic activation factor of the NADPH oxidase. Characterization of activation kinetics

The kinetics of sodium dodecyl sulfate-induced activation of respiratory burst oxidase (NADPH oxidase) in a fully soluble cell-free system from resting (control) or phorbol myristate acetate (PMA)-stimulated human neutrophils were investigated. In a cell-free system containing solubilized membranes and cytosol fractions (cytosol) derived from control neutrophils (control cell-free system), the values of Km and Vmax for NADPH of the NADPH oxidase from control neutrophils continuously increased with increasing concentrations of cytosol, but with increasing concentrations of solubilized membranes from the control neutrophils, Km values continuously decreased, suggesting cytosolic activation factor-dependent continuous changes in the affinity of NADPH oxidase to NADPH. In a cell-free system containing solubilized membranes and cytosol prepared from PMA-stimulated neutrophils, NADPH oxidase was not activated after the addition of NADPH. However, cytosol from control neutrophils activated the NADPH oxidase of PMA-stimulated neutrophils in a cell-free system. Cytosol from PMA-stimulated neutrophils did not activate the control neutrophil oxidase, although it contained no inhibitors of NADPH oxidase activation. The results suggest that, in PMA-stimulated neutrophils, cytosolic activation factors may be consumed or exhausted with an increasing period of time after the stimulation of neutrophils, and that the affinity of PMA-stimulated neutrophil NADPH oxidase to NADPH may almost be the same as that of control neutrophil oxidase. It was concluded that the affinity of NADPH oxidase to NADPH was closely associated with interaction between solubilized membranes and cytosolic activation factors, as indicated by the concentration ratio.     

Activation of the NADPH oxidase in a cell-free system from human neutrophils stimulated by phorbol myristate acetate

Kinetics of activation of the NADPH oxidase in a fully soluble cell-free system from phorbol myristate acetate (PMA)-stimulated human neutrophils were investigated. In a cell-free system in which Mg2+ and sodium dodecyl sulfate, an anionic detergent required for the activation of NADPH oxidase are contained, cytosol prepared from PMA-stimulated neutrophils failed to activate PMA-stimulated neutrophil oxidase. However, cytosol prepared from resting (control) neutrophils was capable of activating PMA-stimulated neutrophil oxidase in a cell-free system in which its Km for NADPH was almost similar to that of control neutrophil oxidase. Cytosol from PMA-stimulated neutrophils could not activate control neutrophil oxidase, although it did not contain any inhibitors of NADPH oxidase activation. These results suggest that, in PMA-stimulated neutrophils, cytosolic activation factors may be consumed or exhausted, and that the affinity for NADPH of PMA-stimulated neutrophil oxidase may be the same as that of control neutrophil oxidase.     

Cytosolic factors in bovine neutrophil oxidase activation. Partial purification and demonstration of translocation to a membrane fraction

The O2(.-)-generating oxidase of bovine neutrophils is activated in a cell-free system consisting of a particulate fraction enriched in plasma membrane and containing the dormant oxidase, a high-speed supernatant from neutrophil homogenate (cytosol), Mg ions, GTP gamma S, and arachidonic acid [Ligeti, E., Doussiere, J., & Vignais, P.V. (1988) Biochemistry 27, 193-200]. The cytosolic components participating in the activation of the membrane-bound oxidase have been investigated. These components were resolved into several active peaks by Q Sepharose chromatography. The oxidase-activating potency of these peaks was synergistically enhanced by combining samples from separate peaks, or by supplying them with a threshold amount of crude cytosol. Partial purification of two active fractions containing a limited number of proteins of 65, 56, 53, and 45 kDa was achieved by gel filtration of cytosol on Ultrogel AcA44, followed by chromatography on hydroxylapatite and Mono Q. The specific oxidase-activating potency of these partially purified fractions (activating potency per milligram of soluble protein) was 6-8-fold higher than that of crude cytosol; it was enhanced up to 75-fold by complementation with a minute amount of crude cytosol, which per se had a limited efficiency. These data indicate that oxidase activation requires more than one cytosolic component to be activated. To check whether translocation of cytosolic proteins to the membrane occurred concomitantly with oxidase activation, use was made of radiolabeled cytosolic proteins. Cytosol was treated with phenyl[14C]isothiocyanate ([14C]PITC), such that 60% of its activation potency was still present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of an affinity label to probe the function of the NADPH binding component of the respiratory burst oxidase of human neutrophils

The respiratory burst oxidase of neutrophils can be activated in a cell-free system in which solubilized membranes, cytosol, and Mg2+ are required and in which sodium dodecyl sulfate is used to convert the dormant oxidase to an active form. The 2',3'-dialdehyde analog of NADPH was used as an affinity label for the cytosolic NADPH binding component of the respiratory burst oxidase from human neutrophils. When treated with this affinity label in the presence of sodium cyanoborohydride to reduce Schiff bases, neutrophil cytosol was shown to lose at least 90% of its activity in the cell-free system. In contrast to normal cytosol, treated cytosol had lost its ability to abolish the lag time required for activation of the oxidase, suggesting that the treated cytosol was no longer able to participate in the rate-limiting activation step. Furthermore, the treated cytosol had lost its ability to convert the oxidase from a form with a high Km to a form with a low Km for NADPH. The ability of dialdehyde-treated cytosol to activate the oxidase could be restored by untreated cytosol with a concentration dependence suggesting that only one kinetically active component of the oxidase was inhibited by treatment with the NADPH analog. Like the dialdehyde-treated cytosol, cytosols from patients with chronic granulomatous disease caused by a deficiency in a cytosolic Mr = 47,000 protein (pp47) fail to participate in the rate-limiting activation step (Curnutte, J. T., Scott, P. J., and Babior, B. M. (1989) J. Clin. Invest. 83, 1236-1240). These chronic granulomatous disease cytosols were nevertheless able to restore limited activity to the dialdehyde-inactivated cytosol in a cell-free activation system. These results are consistent with a model in which (a) the NADPH binding subunit of the oxidase exists in a very slowly dissociating complex with one or more additional cytosolic components, including pp47, and (b) the NADPH binding component of the oxidase controls the affinity of the enzyme for NADPH, either directly or through the binding of additional cytosolic factors.     

Purification and characterization of an oxidase activating factor of 63 kilodaltons from bovine neutrophils

A 63-kDa protein, which behaves as an oxidase activating factor in bovine neutrophils, has been purified to electrophoretic homogeneity. The protein was isolated from the cytosol of resting bovine neutrophils after several steps, including ammonium sulfate precipitation and chromatography on AcA44, DE-52 cellulose, Mono Q, and Superose 12 in the presence of dithiothreitol. The oxidase activating potency of the protein was assayed with a cell-free system consisting of neutrophil membranes, GTP gamma S, arachidonic acid, and a complementary cytosolic fraction. The purification factor was 200 and the yield 3%. During the course of gel filtration on calibrated Superose 12, the 63-kDa protein eluted as a dimer. Its isoelectric point was 6.4 +/- 0.1. Antibodies raised in rabbits against the 63-kDa protein reacted with a protein of similar size in human neutrophils and in HL60 promyelocytic cells induced to differentiate into granulocytes. No immune reaction was observed in cytosol from undifferentiated HL60 cells, in extracts from bovine skeletal muscle, liver, and brain, or in cytosol prepared from neutrophils derived from a patient with an autosomal cytochrome b positive form of chronic granulomatous disease lacking the 67-kDa oxidase activating factor. Immunoblotting with the 63-kDa bovine protein antiserum demonstrated that activation of bovine neutrophil oxidase by phorbol myristate acetate induced the translocation of the 63-kDa protein from cytosol to the membrane.     

Localization of the 47 kDa phosphoprotein involved in the respiratory-burst NADPH oxidase of phagocytic cells.

A 47 kDa phosphoprotein is involved in the respiratory-burst oxidase of phagocytic cells. After stimulation of neutrophils with phorbol myristate acetate, this phosphoprotein was identified in both the cytosol and membranes. Peptide mapping of the two forms resulted in identical patterns of phosphopeptides. Dose-response curves for accumulation of phosphoprotein in the two sites were very similar, whereas the detection of the phosphoprotein in the cytosol preceded that in the membranes. The membrane-associated 47 kDa phosphoprotein was absent from the neutrophils of patients with X-chromosome-linked chronic granulomatous disease, which lack cytochrome b-245, and intermediate levels were detected in the membranes of their heterozygote carrier mothers. Activation of the neutrophil oxidase system appears to be dependent upon phosphorylation of the cytosolic 47 kDa protein and its association with cytochrome b-245 in the membranes. It is probably the cytosolic factor required for reconstitution of the active oxidase in cell-free systems.     

Activation of O2.- generating oxidase of bovine neutrophils in a cell-free system. Interaction of a cytosolic factor with the plasma membrane and control by G nucleotides

Activation of the O2.- -generating oxidase of bovine neutrophils was studied in a cell-free system, consisting of a particulate fraction enriched in plasma membrane, cytosol, arachidonic acid, and the non-hydrolyzable nucleotide GTP-gamma-S. Activation of the membrane-bound oxidase was accompanied by the disappearance of the activating factor from the cytosol. Above a cytosol to membrane ratio of 25, the excess of added cytosolic factor remained in active state in the soluble fraction. The process could be partially reversed by serum albumin. Disappearance of the cytosolic factor was promoted by unsaturated long-chain fatty acids, but not by saturated ones, and occurred not only in the presence of GTP-gamma-S but also in the presence of GDP-beta-S or in the absence of Mg ions, although in the latter cases activation of O2.- production was seriously impaired. This suggests that the disappearance of the activating factor from the cytosol and the triggering effect of GTP-gamma-S are related, but distinct, events in the oxidase activation process. The disappearance of the activating factor from cytosol can be explained by translocation of the cytosolic factor to the membrane fraction. Yet under some conditions, including the presence of GDP-beta-S or EDTA, inactivation was prevailing and could be an alternative explanation for the results. Specific binding of radiolabeled GTP-gamma-S could be demonstrated both in the membrane and in the cytosolic fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of O2(-)-generating oxidase in an heterologous cell-free system derived from Epstein-Barr-virus-transformed human B lymphocytes and bovine neutrophils. Application to the study of defects in cytosolic factors in chronic granulomatous disease.

Epstein-Barr-virus-transformed human B lymphocytes (EBV B lymphocytes) stimulated by 4 beta-phorbol 12-myristate 13-acetate exhibit an NADPH-dependent oxidase activity capable of generating the superoxide anion O2-, similar to, but less efficient than that of activated neutrophils. A cell-free system of oxidase activation consisting of a membrane fraction and cytosol from EBV B lymphocyte homogenate supplemented with guanosine 5'-[gamma-thio]triphosphate (GTP[S]), arachidonic acid and Mg2+ was found to be competent in the production of O2-, assessed by the superoxide-dismutase-sensitive reduction of cytochrome c in the presence of NADPH. However, cytochrome c reduction was slow and largely insensitive both to superoxide dismutase, and to iodonium biphenyl, a powerful inhibitor of the oxidase activity in neutrophils. A markedly faster reduction of cytochrome c in the presence of NADPH was obtained with a heterologous system consisting of cytosol from EBV B lymphocytes and bovine neutrophil membranes, GTP[S], arachidonic acid and Mg2+; in this system, reduction of cytochrome c was totally inhibited by superoxide dismutase and iodonium biphenyl. These results show that EBV B lymphocytes contain a substantial amount of cytosolic factors of oxidase activation, and that the limiting factors for O2- production in B lymphocytes are the membrane components of the oxidase complex. The heterologous system of EBV B lymphocyte cytosol and bovine neutrophil membranes provided a rapid and convenient method to diagnose cytosolic defects in autosomal forms of chronic granulomatous disease. In addition, it might be a useful tool to explore the mechanism of action of the cytosolic factors in oxidase activation.     

Purification and properties of a functional 47-kilodalton cytosolic factor required for NADPH-oxidase activation in bovine neutrophils.

A cytosolic factor of 47 kDa required for activation of the NADPH oxidase, and referred to as p47, has been purified in its functional form from the cytosol of resting bovine neutrophils. The purification was monitored by the determination of the activating potency of p47 in a cell-free system of oxidase activation. The recovery was around 10% and the purification factor greater than 1000. P47 was phosphorylated in vitro by protein kinase A and protein kinase C. [32P] labeled p47 was resolved by isoelectric focusing into two major labeled bands of pI 7.0 and 8.5. Polyclonal antibodies were used to demonstrate that p47 is localized specifically in the cytosol of resting neutrophils, and that, upon activation of neutrophils, p47 is translocated from the cytosol to the membrane.     

Resolution of a low molecular weight G protein in neutrophil cytosol required for NADPH oxidase activation and reconstitution by recombinant Krev-1 protein

Activation of the membrane-associated NADPH oxidase in intact human neutrophils requires a receptor-associated heterotrimeric GTP-binding protein that is sensitive to pertussis toxin. Activation of this NADPH oxidase by arachidonate in a cell-free system requires an additional downstream pertussis toxin-insensitive G protein (Gabig, T. G., English, D., Akard, L. P., and Schell, M. J. (1987) (J. Biol. Chem. 262, 1685-1690) that is located in the cytosolic fraction of unstimulated cells (Gabig, T. G., Eklund, E. A., Potter, G. B., and Dykes, J. R. (1990) J. Immunol. 145, 945-951). In the present study, immunodepletion of G proteins from the cytosolic fraction of unstimulated neutrophils resulted in a loss of the ability to activate NADPH oxidase in the membrane fraction. The activity in immunodepleted cytosol was fully reconstituted by a partially purified fraction from neutrophil cytosol that contained a 21-kDa GTP-binding protein. Purified human recombinant Krev-1 p21 also completely reconstituted immunodepleted cytosol whereas recombinant human H-ras p21 or yeast RAS GTP-binding proteins had no reconstitutive activity. Rabbit antisera raised against a synthetic peptide corresponding to the effector region of Krev-1 (amino acids 31-43) completely inhibited cell-free NADPH oxidase activation, and this inhibition was blocked by the synthetic 31-43 peptide. An inhibitory monoclonal antibody specific for ras p21 amino acids 60-77 (Y13-259) had no effect on cell-free NADPH oxidase activation. Activation of the NADPH oxidase in intact neutrophils by stimulation with phorbol myristate acetate caused a marked increase in the amount of membrane-associated antigen recognized by 151 antiserum on Western blot. Thus a G protein in the cytosol of unstimulated neutrophils antigenically and functionally related to Krev-1 may be the downstream effector G protein for NADPH oxidase activation. This system represents a unique model to study molecular interactions of a ras-like G protein.     

Two cytosolic components of the neutrophil NADPH oxidase, P47-phox and P67-phox, are not flavoproteins

Two cytosolic proteins, p47-phox and p67-phox, have been shown to be essential components of the NADPH-dependent oxidase of human neutrophils, although the specific role of each of these proteins in the multicomponent electron transport complex is undetermined. The superoxide-generating activity of this oxidase can be reproduced in a cell-free system, combining cytosol and membranes from unstimulated neutrophils in the presence of fatty acid and NADPH. In the present studies, cytosol was treated with myristic acid, arachidonic acid, or sodium dodecyl sulfate in the absence of membranes and the resultant precipitate collected by centrifugation and analyzed. Both p47-phox and p67-phox precipitated in the presence of fatty acid. However, neither FAD nor FMN was localized in the precipitates, even though substantial amounts of p47-phox and p67-phox precipitated. These results suggest that neither p47-phox nor p67-phox is a flavoprotein and that neither, therefore, is the oxidase component which accepts electrons from NADPH.     

Complementation of NADPH oxidase in p67-phox-deficient CGD patients p67-phox/p40-phox interaction.

Chronic granulomatous disease (CGD) is due to a functional defect of the O2- generating NADPH oxidase of phagocytes. Epstein-Barr-virus-immortalized B lymphocytes express all the constituents of oxidase with activity 100 times less than that of neutrophils. As in neutrophils, oxidase activity of Epstein-Barr-virus-immortalized B lymphocytes was shown to be defective in the different forms of CGD; these cells were used as a model for the complementation studies of two p67-phox-deficient CGD patients. Reconstitution of oxidase activity was performed in vitro by using a heterologous cell-free assay consisting of membrane-suspended or solubilized and purified cytochrome b558 that was associated with cytosol or with the isolated cytosolic-activating factors (p67-phox, p47-phox, p40-phox) from healthy or CGD patients. In p67-phox-deficient CGD patients, two cytosolic factors are deficient or missing: p67-phox and p40-phox. Not more than 20% of oxidase activity was recovered by complementing the cytosol of p67-phox-deficient patients with recombinant p67-phox. On the contrary, a complete restoration of oxidase activity was observed when, instead of cytosol, the cytosolic factors were added in the cell-free assay after isolation in combination with cytochrome b558 purified from neutrophil membrane. Moreover, the simultaneous addition of recombinant p67-phox and recombinant p40-phox reversed the previous complementation in a p40-phox dose-dependent process. These results suggest that in the reconstitution of oxidase activity, p67-phox is the limiting factor; the efficiency of complementation depends on the membrane tissue and the cytosolic environment. In vitro, the transition from the resting to the activated state of oxidase, which results from assembling, requires the dissociation of p40-phox from p67-phox for efficient oxidase activity. In the process, p40-phox could function as a negative regulatory factor and stabilize the resting state.     

Comparison of O2(-)-producing activity of guinea-pig eosinophils and neutrophils in a cell-free system.

1. The NADPH-dependent superoxide (O2-) production in a cell-free system of guinea-pig eosinophils was studied, comparing the eosinophils with neutrophils. 2. Eosinophils produced 2.2-fold more O2- than neutrophils in sonicated and intact cells. 3. The subcellular fractionation experiments showed that the O2- production was dependent on the cooperation between the membrane and cytosol fractions. 4. The cross-mixing experiments indicated that the NADPH oxidase-activating activity of the eosinophil cytosol was about 2-fold greater than that of the neutrophil cytosol. 5. These results suggest that the difference in the O2(-)-producing activity between eosinophils and neutrophils is associated with the difference in cytosolic factors necessary for the activation of NADPH oxidase.     

Purification and characterization of a lipid thiobis ester from human neutrophil cytosol that reversibly deactivates the O2- -generating NADPH oxidase

Intact neutrophils possess a cellular mechanism that efficiently deactivates the microbicidal O2-generating NADPH oxidase during the respiratory burst (Akard, L. P., English, D., and Gabig, T. G. (1988) Blood 72, 322-327). The present studies directed at identifying the molecular mechanism(s) involved in NADPH oxidase deactivation showed that a heat- and trypsin-insensitive species in the cytosolic fraction from normal unstimulated neutrophils was capable of deactivating the membrane-associated NADPH oxidase isolated from opsonized zymosan- or phorbol 12-myristate 13-acetate-stimulated neutrophils. This cytosolic species also deactivated the cell-free-activated oxidase. Deactivation by this cytosolic species occurred in the absence of NADPH-dependent catalytic turnover and was reversible, since NADPH oxidase activity could be subsequently reactivated in the cell-free system. The sedimentable particulate fraction from unstimulated neutrophils did not demonstrate deactivator activity. Deactivator activity was demonstrated in the neutral lipid fraction of neutrophil cytosol extracted with chloroform:methanol. Following complete purification of cytosolic deactivator activity by thin layer chromatography and reversed phase high performance liquid chromatography, the deactivator species was shown to be a lipid thiobis ester compound by mass spectroscopy. Cellular metabolism of this compound in human neutrophils may reveal a unique mechanism for enzymatic control of the NADPH oxidase system and thereby play an important role in regulation of the inflammatory response.     

Evidence that activation of the respiratory burst oxidase in a cell-free system from human neutrophils is accomplished in part through an alteration of the oxidase-related 67-kDa cytosolic protein

Sodium dodecyl sulfate (SDS) is able to activate the respiratory burst oxidase in a system containing cytosol and solubilized membranes from human neutrophils. When SDS was used to treat cytosol in an otherwise identical system in which the solubilized membrane solution was omitted, the ability of the SDS-treated cytosol to support O2- production was lost in a first-order reaction whose rate constant was virtually identical to the rate constant for the first-order activation of the oxidase in the complete system. Studies with chronic granulomatous disease cytosols showed that the component whose activity was lost was the oxidase-related 67-kDa cytosolic protein. The similarity in the rates of oxidase activation and p67 inactivation suggested that the activation of the respiratory burst oxidase in the cell-free system could involve an SDS-mediated alteration in p67. Further support for this idea was provided by kinetic experiments demonstrating that, although the yield of oxidase showed a 2.5-order dependence on cytosol concentration, oxidase activation was nevertheless kinetically irreversible. These two findings, incompatible in general, can be reconciled by a mechanism in which SDS acts specifically on a single oxidase component (i.e. p67), but with an effect that depends on circumstances: oxidase activation, if the SDS-sensitive component is part of a completely assembled oxidase precursor; loss of p67 activity, if not.     

Parameters of activation of the membrane-bound O2- generating oxidase from bovine neutrophils in a cell-free system

Parameters governing the extent of activation of the O2- generating oxidase in a cell-free system derived from bovine neutrophils were examined. The reconstituted system consisted of the following: a particulate fraction enriched in plasma membrane and containing the oxidase, a soluble fraction containing cytosolic factor(s) required for oxidase a soluble fraction containing cytosolic factor(s) required for oxidase activation, a non hydrolyzable analog of GTP, and either arachidonic acid or sodium dodecyl sulfate. When the amount of arachidonic acid or sodium dodecyl sulfate was maintained at a fixed value with respect to the amount of membrane used, a sigmoidal response of oxidase activity to increasing amounts of cytosol added was observed. In contrast, when the concentration of arachidonic acid or sodium dodecyl sulfate was properly adjusted with respect to that of membrane and cytosol, the curve relating oxidase activity to cytosol was hyperbolic, pointing to a simple michaelian relationship for the dependence of oxidase activation on the activating factor(s) of cytosol. Another parameter affecting oxidase activation was the ionic strength of the reconstitution medium, the extent of activation being lower at high ionic strength.     

The translocation of respiratory burst oxidase components from cytosol to plasma membrane is regulated by guanine nucleotides and diacylglycerol.

The respiratory burst oxidase is a multimeric enzyme responsible for O2- production by stimulated neutrophils and a few other cell types. In the resting neutrophil, the oxidase is dormant, and its subunits are distributed between the cytosol, in which they appear to exist in the form of a multisubunit complex, and the plasma membrane; but, when the neutrophil is activated, the cytosolic complex translocates to the membrane to assemble the active enzyme. Using a cell-free system in which oxidase activity was elicited with SDS, we examined the effects of GTP gamma S and dioctanoylglycerol (DiC8) on both the activation of O2- production and the transfer of the cytosolic oxidase components p47phox and p67phox to the plasma membrane. GTP (added as undialyzed cytosol) and GTP gamma S augmented the transfer of the oxidase components to the plasma membrane and was essential for the acquisition of O2- producing activity by the oxidase. DiC8 also supported the SDS-mediated transfer of oxidase components to the membrane, but O2- production did not take place unless GTP or GTP gamma S was present. In the presence of these nucleotides, however, DiC8 augmented both translocation and O2- production. We interpreted these results in terms of a mechanism in which 2 membrane-binding sites are created during the activation of the cytosolic complex, one for diacylglycerol and the other for a second site on the membrane. Development of the second membrane-binding site depends upon the action of a G protein and is essential for the expression of oxidase activity. The results further suggested that the priming of the respiratory burst oxidase in intact neutrophils might be due to an increase in membrane diacylglycerol concentration that occurs in response to the priming stimulus. Because of the increased diacylglycerol content, a larger than usual amount of active respiratory burst oxidase could be assembled on the primed plasma membrane when the neutrophil is fully activated.     

Activation of NADPH-dependent superoxide production in plasma membrane extracts of pig neutrophils by phosphatidic acid

Phosphatidic acid (PA), a molecule that is rapidly produced by the stimulated turnover of phospholipids in a variety of cells including blood neutrophils, elicited NADPH-dependent superoxide anion (O2-) production in detergent extracts from membranes of resting pig neutrophils. The stimulatory effect of PA was independent of cytosolic factors, differing from arachidonic acid and sodium dodecyl sulfate which, on the contrary, absolutely required the presence of cytosol to elicit the same result. The O2(-)-forming activity of the detergent extract activable by PA, as that by sodium dodecyl sulfate and arachidonic acid plus cytosol, was found in the chromatographic fractions containing cytochrome b558 and presented a chromatographic profile identical to that of the activated NADPH oxidase, which was obtained from neutrophils prestimulated with phorbol 12-myristate 13-acetate. The PA-induced NADPH-dependent O2(-)-forming activity showed kinetic properties and sensitivity to the inhibitors similar to the classical ones of the activated neutrophil NADPH oxidase. The data suggest that, in this cell-free system, PA may stimulate O2- formation by direct interaction with latent NADPH oxidase of neutrophils or with some of its regulatory components.     

A carboxy-terminal peptide from p47-phox is a substrate for phosphorylation by protein kinase C and by a neutrophil protein kinase.

Agonist-activated phosphorylation of neutrophil proteins including p47-phox, a cytosolic component of the respiratory burst oxidase, has been implicated in the signal transduction cascade which leads to activation of the superoxide generating respiratory burst. We have previously reported (J. Biol. Chem. 265, 17550-59) that in a cell-free activation system consisting of cytosol plus plasma membrane from human neutrophils, diacylglycerol acts synergistically with an anionic amphiphile such as sodium dodecyl sulfate (SDS) to augment superoxide generation and assembly of the oxidase, and that p47 phosphorylation can occur under these conditions. Herein, we show that a peptide corresponding to a carboxy terminal sequence of p47-phox is a substrate for phosphorylation both by purified protein kinase C (a mixture of alpha, beta, and gamma forms) and by a distinct kinase or kinases present in neutrophil cytosol. Based on its activator requirements, the neutrophil kinase differs from classical protein kinase C, but may be a protein kinase C variant, based on inhibition by a protein kinase C peptide. Although in the cell-free system phosphorylation occurs under conditions which are similar to those for activation of superoxide generation, phosphorylation is not required for activation (1). Rather, protein assembly or aggregation which occurs under activation conditions may also promote phosphorylation.     

Activation of the leukocyte NADPH oxidase by protein kinase C in a partially recombinant cell-free system.

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.