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.     

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.     

A microtechnique for neutrophil respiratory burst oxidase in a cell-free system--characterization of oxidase activation system

1. A microtechnique for quantitating human neutrophil NADPH oxidase in a cell-free system is described. 2. This spectrophotometric discontinuous (fixed time) method is less material-consuming than existing methods and is more useful for experiments in which superoxide production by neutrophils must be measured in a large number of samples. 3. Measurement of NADPH oxidase using the new method can be accomplished in a final vol of 0.15 ml. 4. In the assay, neutrophil membranes solubilized with deoxycholate were incubated for 3 min with cytosolic fractions, magnesium, sodium dodecyl sulfate, and cytochrome c in the absence of NADPH to preincubate the oxidase before the addition of the reducing agent. 5. The reaction was started by adding NADPH and 2 min later terminated by adding superoxide dismutase. 6. The apparent Km for NADPH obtained by the new method was almost the same as that by the authorized method (39.2 +/- 3.1 SD vs 36.8 +/- 1.6). Activation of neutrophil NADPH oxidase was characterized using the new assay method.     

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.     

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.     

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.     

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.     

Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride. Evidence for participation of a pertussis and cholera toxin-insensitive G protein

Guanine nucleotide-binding regulatory proteins (G proteins) transduce a remarkably diverse group of extracellular signals to a relatively limited number of intracellular target enzymes. In the neutrophil, transduction of the signal following fMet-Leu-Phe receptor-ligand interaction is mediated by a pertussis toxin substrate (Gi) that activates inositol-specific phospholipase C. We have utilized a plasma membrane-containing fraction from unstimulated human neutrophils as the target enzyme to explore the role of G proteins in arachidonate and cytosolic cofactor-dependent activation of the NADPH-dependent O-2-generating oxidase. When certain guanine nucleotides or their nonhydrolyzable analogues were present during arachidonate and cytosolic cofactor-dependent activation, they exerted substantial dose-dependent effects. The GTP analogue, GTP gamma S, caused a 2-fold increase in NADPH oxidase activation (half-maximal stimulation, 1.1 microM). Either GDP or its nonhydrolyzable analogue, GDP beta S, inhibited up to 80% of the basal NADPH oxidase activation (Ki GDP = 0.12 mM, GDP beta S = 0.23 mM). GTP caused only slight and variable stimulation, whereas F-, an agent known to promote the active conformation of G proteins, caused a 1.6-fold stimulation of NADPH oxidase activation. NADPH oxidase activation in the cell-free system was absolutely and specifically dependent on Mg2+. Although O2- production in response to fMet-Leu-Phe was inhibited greater than 90% in neutrophils pretreated with pertussis toxin, cytosolic cofactor and target oxidase membranes from neutrophils treated with pertussis toxin showed no change in basal- or GTP gamma S-stimulated NADPH oxidase activation. Cholera toxin treatment of neutrophils also had no effect on the cell-free activation system. Our results suggest a role for a G protein that is distinct from Gs or Gi in the arachidonate and cytosolic cofactor-dependent NADPH oxidase cell-free activation system.     

Activation of the respiratory burst oxidase in a fully soluble system from human neutrophils

The O2(-)-forming respiratory burst oxidase is present in a dormant state in a fully soluble system containing both cytosol and a deoxycholate extract of membranes from resting human neutrophils. Sodium dodecyl sulfate at low concentrations converts this soluble dormant oxidase into its catalytically active form. The Vmax for the activated oxidase was 2.1 mumol of O2-/min/mg of membrane protein. Michaelis constants for NADPH and NADH (38 microM and 1.7 mM, respectively) were similar to those measured previously in other systems. Oxidase activity was not detected after sodium dodecyl sulfate treatment of systems containing solubilized neutrophil membranes obtained from patients with X-linked chronic granulomatous disease. These results suggest that the deoxycholate extract contains both the resting oxidase and those membrane-associated components needed for its activation, all in functioning states.     

Kinetics of cell-free activation of neutrophil NADPH oxidase. Effects of neomycin and guanine nucleotides.

The effects of neomycin, fluoride and the non-hydrolysable guanine nucleotide analogue GTP gamma S on the kinetics of cell-free activation of NADPH oxidase in membranes of resting human neutrophils were investigated. Arachidonate-mediated activation of the oxidase followed a first-order reaction course (kobs. = 0.39 min-1 at 26 degrees C). In the presence of NaF during the activation process, activity was enhanced while the activation rate was slightly reduced (kobs. = 0.25 min-1 at 26 degrees C). Neomycin blocked activation (half-maximal effect at 25 microM) without affecting rates of superoxide release by preactivated enzyme in vitro or in vivo. In spite of reduced specific activity neither the first-order rate constant of the activation nor the Km of the oxidase were altered by neomycin. Oxidase activated in the presence of GTP gamma S exhibited increased specific activity and unchanged Km; the course of the reaction deviated from first-order kinetics. Kinetic evidence is presented for two separate activation reactions: a GTP gamma S-independent, basal, first-order process and a GTP gamma S-dependent sigmoid activation process. The results are compatible with the existence in neutrophil membranes of two separate pools of dormant oxidase. An alternative scheme of the formation of two active forms of NADPH oxidase is also presented.     

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.     

Activation of neutrophil NADPH oxidase in a cell-free system. Partial purification of components and characterization of the activation process

The superoxide-generating enzyme of human neutrophils, NADPH oxidase, is converted from an inactive to an active form upon stimulation of the neutrophil. This activation process was examined using a recently developed cell-free system in which dormant oxidase is activated by arachidonic acid in the presence of a soluble factor from the neutrophil (Curnutte, J. T. (1985) J. Clin. Invest. 75, 1740-1743). NADPH oxidase from unstimulated human neutrophils was detected only in the membrane fraction. The soluble activation factor was localized entirely to the cytosolic fraction and exhibited two peaks of activity when partially purified under nondenaturing conditions: a major peak with a molecular mass of approximately 250 kDa and a variable minor peak with a mass of approximately 40 kDa. Both forms activated NADPH oxidase in a similar manner and did not exhibit synergy when combined. The cytosolic factor is not protein kinase C (or another kinase) as both peaks of factor activity could be resolved from the protein kinase C peak and neither required calcium or ATP to activate the oxidase. Activation of NADPH oxidase did require the simultaneous presence of the membrane fraction, the cytosolic factor, arachidonic acid, and magnesium. Following activation, however, only the membrane fraction was then required for O2- production. Cytosolic factor levels were normal in five patients with either X-linked or autosomal recessive cytochrome b-negative chronic granulomatous disease. In contrast, the membrane fractions from each failed to generate O2-, indicating that the defects in these two genetic forms of chronic granulomatous disease reside either in the oxidase itself or in a membrane component required for activation.     

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)     

Kinetic study of the activation of the neutrophil NADPH oxidase by arachidonic acid. Antagonistic effects of arachidonic acid and phenylarsine oxide

The O(2)(-) generating NADPH oxidase complex of neutrophils comprises two sets of components, namely a membrane-bound heterodimeric flavocytochrome b which contains the redox centers of the oxidase and water-soluble proteins of cytosolic origin which act as activating factors of the flavocytochrome. The NADPH oxidase can be activated in a cell-free system consisting of plasma membranes and cytosol from resting neutrophils in the presence of GTPgammaS and arachidonic acid. NADPH oxidase activation is inhibited by phenylarsine oxide (PAO), a sulfhydryl reagent for vicinal or proximal thiol groups. The site of action of PAO was localized by photolabeling in the beta-subunit of flavocytochrome b [Doussière, J., Poinas, A, Blais, C., and Vignais, P. V. (1998) Eur. J. Biochem. 251, 649-658]. Moreover, the spin state of heme b is controlled by interaction of arachidonic acid with the flavocytochrome b [Doussière, J., Gaillard, J., and Vignais, P. V. (1996) Biochemistry 35, 13400-13410]. Here we report that the promoting effect of arachidonic acid on the activation of NADPH oxidase is due to specific binding of arachidonic acid to flavocytochrome b. Elicitation of NADPH oxidase activity by arachidonic acid is in part associated with an increased affinity of flavocytochrome b for O(2), an effect that was counteracted by the methyl ester of arachidonic acid. On the other hand, the affinity for NADPH was not affected by arachidonic acid. We further demonstrate that PAO antagonizes the effect of arachidonic acid on oxidase activation by decreasing the affinity of the oxidase for O(2), but not for NADPH. PAO induced a change in the spin state of heme b, as arachidonic acid does, with, however, some differences in the constraints imposed to the heme. It is concluded that the opposite effects of arachidonic acid and PAO are exerted on the beta-subunit of flavocytochrome b at two different interacting sites.     

Activation of NADPH oxidase in human neutrophils permeabilized with Staphylococcus aureus alpha-toxin. A lower Km when the enzyme is activated in situ.

The NADPH oxidase is a multicomponent enzyme system that produces the reduced oxygen species essential for bacterial killing by polymorphonuclear leukocytes (PMN). Study of the oxidase has typically been carried out in cell-free systems in which Km values of 20-150 microM NADPH have been reported. However, when compared with affinities reported for other flavoprotein dehydrogenases and when considering the cellular concentration of NADPH/NADP+ of approximately 35 microM, the reported affinity of the oxidase for NADPH appears low. To investigate this apparent discrepancy we have studied the kinetics of NADPH oxidase activation in situ in human PMN permeabilized with Staphylococcus aureus alpha-toxin. alpha-Toxin permeabilization of human PMN did not initiate NADPH oxidase activation at physiologic concentrations of NADPH. If permeabilized cells were stimulated with 1 microM formyl-methionyl-leucyl-phenylalanine, 10 microM guanosine 5'-O-(3-thiotriphosphate), 0.5 mM Ca2+, 5 micrograms/ml cytochalasin B in the presence of varying concentrations of NADPH, we were able to demonstrate activation of the oxidase complex as shown by superoxide dismutase-inhibitable reduction of cytochrome c. In this system we determined that the Km for oxidase activation was 4-7 microM NADPH, a 4-10-fold decrease from reported values. The oxidase was the enzyme being studied as shown by the absence of enzymatic activity in patients with chronic granulomatous disease. In addition, if the enzyme was initially activated in permeabilized cells, the cells homogenized, and the Km for the oxidase determined in a cell-free system, the observed Km reverted to previously reported values (36 microM). These results indicate that NADPH oxidase, studied in situ, has a significantly higher substrate affinity than that observed in isolated membranes and, moreover, indicate that substrate affinity is optimal for catalysis at reported concentrations of cytosolic NADPH.     

Inositol lipids and phosphatidic acid inhibit cell-free activation of neutrophil NADPH oxidase

The effect of inositol lipids on the SDS-initiated cell-free activation of NADPH oxidase in membranes of human neutrophils was investigated. In a system consisting of low density membranes, cytosol and SDS, low doses of phosphatidylinositol, phosphatidylinositol mono- and biphosphates and phosphatidic acid interfered with activation of the oxidase. The inhibition was relieved by increasing concentrations of the cytosol. Conversely, preincubation of multilamellar phosphoinositide vesicles with cytosol reduced its ability to support activation of the oxidase.     

Involvement of membrane charges in constituting the active form of NADPH oxidase in guinea pig polymorphonuclear leukocytes

NADPH oxidase activity in a membrane fraction prepared from phorbol 12-myristate 13-acetate (PMA)-stimulated guinea pig polymorphonuclear leukocytes (PMNL) was inhibited by positively charged myristylamine. The inhibitory effect of myristylamine was significantly suppressed by simultaneous addition of a negatively charged fatty acid, such as myristic acid. However, the suppression by myristylamine was not sufficiently restored when myristic acid was added later. On the other hand, pretreatment of PMA-stimulated PMNL with glutaraldehyde, a protein crosslinking reagent, stabilized NADPH oxidase activity against inhibition by myristylamine, but not against that by p-chloromercuribenzenesulfonic acid. In a cell-free system of reconstituted plasma membrane and cytosolic fractions prepared from unstimulated PMNL, arachidonic acid-stimulated NADPH oxidase activity was also inhibited by myristylamine. During the activation of NADPH oxidase by PMA in intact PMNL and by arachidonic acid in the cell-free system, cytosolic activation factor(s) translocated to plasma membranes. The bound cytosolic activation factor(s) was released from the membranes by myristylamine, accompanied by a loss of NADPH oxidase activity. It is plausible from these results that the inhibitory effect of alkylamine on NADPH oxidase is due to induction of the decoupling and/or dissociation of the cytosolic activation component(s) from the activated NADPH oxidase complex by increments of positive charges in the membranes, and that the glutaraldehyde treatment prevents the dissociation of component(s).     

Reconstitution and characterization of the human neutrophil respiratory burst oxidase using recombinant p47-phox, p67-phox and plasma membrane.

Human neutrophil respiratory burst oxidase (NADPH-oxidase) activity can be reconstituted in a cell-free system consisting of plasma membrane, cytosol and an anionic amphiphile [e.g., sodium dodecyl sulfate (SDS) or arachidonate]. Herein, we report reconstitution of oxidase activity using isolated neutrophil plasma membrane together with purified recombinant p47-phox and p67-phox which had been produced using a baculovirus expression system. Activity required an anionic amphiphile (SDS or arachidonate) and was potentiated by diacylglycerol and GTP gamma S. Serial washes of the plasma membrane failed to affect its ability to reconstitute activity, indicating that a dissociable membrane component was not present. The Km for NADPH, 43 microM, was the same as that determined using cytosol in place of recombinant factors. The EC50 values for p47-phox and p67-phox under optimal activation conditions were 220 nM and 80 nM, respectively, indicating a relatively high affinity of these components in an activation complex. Since neither cytosolic component contains a nucleotide binding consensus sequence, these data indicate that the NADPH binding component of the oxidase resides in the plasma membrane.     

Translocation of a small cytosolic calcium-binding protein (MRP-8) to plasma membrane correlates with human neutrophil activation.

To further understand the mechanisms involved in phagocyte activation in general and in NADPH oxidase activation in particular, a polyclonal antibody was raised in rabbit against a partially purified oxidase preparation. The enzyme was solubilized from zymosan-activated human neutrophils and resting cells and separated by preparative isoelectric focusing electrophoresis. A polyclonal antibody was raised in rabbit against the pI 5.0 fraction, which had the maximum superoxide-producing capacity. Analysis of the polyclonal antibody revealed marked differences between activated and resting neutrophils. The antibody recognized in particular an 8-kDa protein (p8) in resting human neutrophil cytosol and in the membrane of zymosan-activated cells. A polyclonal antibody (anti-p8) was raised against the pure cytosolic p8 protein. This anti-p8 reacted not only with p8, but also with cytosolic proteins of 14 kDa and 6 kDa. N-terminal amino acid sequence analysis of p8 revealed homology with the calcium-binding myeloid related protein (MRP-8). Upon neutrophil activation, translocation of the 8- and 14-kDa proteins to the membrane was observed with stimuli known to depend on extracellular calcium. In calcium-depleted medium, the absence of translocation correlated with a depression of superoxide production, supporting a role for the calcium-binding protein in cellular activation.     

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.