Proteinase 3 (PR3) is a serine protease of neutrophil granules released to the medium or into the... more Proteinase 3 (PR3) is a serine protease of neutrophil granules released to the medium or into the phagocytic vesicle upon neutrophil stimulation. A fraction of the enzyme is thought to associate with the cell membrane yielding membrane PR3 (mPR3). In autoimmune disorders characterized by the presence of antineutrophil cytoplasmic antibodies (ANCA), the reaction of the latter with their target antigen mPR3 activates the cell inflicting injuries on the surrounding tissues. In a previous communication we provided evidence for the presence of mPR3 in lipid rafts obtained by lysis of neutrophils in Triton X-100 and for the mediation of PR3 binding to the membrane by a glycosylphosphatidylinositol (GPI)-anchored neutrophil protein, possibly FcgammaRIIIb. In the current study we employed the mild detergent Brij 58 to isolate high molecular weight (HMW) protein complexes in the void volume of a Sepharose 4B gel filtration minicolumn. HMW complexes of unstimulated neutrophils comprised PR3, FcgammaRIIIb, the beta2 integrin CD11b/CD18 as well as the membrane and cytosolic subunits of the NADPH oxidase, p22phox and p47phox/p67phox. Treatment of neutrophils with phosphatidylinositol-specific phospholipase C (PI-PLC) reduced amounts of PR3 and FcgammaRIIIb in HMW complexes isolated from the treated cells, supporting our previous suggestion that FcgammaRIIIb acts as a membrane adaptor for PR3. FcgammaRIIIb of HMW fractions co-immunoprecipitated with PR3, indicating their presence in the same protein complex. Since HMW fractions contained also the majority of biotinylated proteins obtained by the reaction of neutrophils with a membrane impermeable biotinylating agent Sulfo-NHS-biotin, it was concluded that HMW proteins were derived from cell membranes. Lipid rafts isolated from Brij 58-lysed neutrophils were similar in their protein composition to the HMW complexes but not identical.
Proteinase 3 (PR3), the target autoantigen of antineutrophil cytoplasmic antibodies in the autoim... more Proteinase 3 (PR3), the target autoantigen of antineutrophil cytoplasmic antibodies in the autoimmune vasculitis, Wegener's granulomatosis, is a serine proteinase stored in granules of human neutrophils. PR3 is expressed also on the plasma membrane of unactivated neutrophils, and this expression increases in primed or stimulated cells. In the current study, we demonstrate the presence of PR3, FcgammaRIIIb, and cytochrome b558 of the NADPH oxidase in neutrophil lipid rafts. Activation of neutrophils with PMA, fmet-leu-phe, or TNFalpha known to increase the membrane expression of PR3 did not affect the amount of PR3 in rafts. Unexpectedly, the cytosolic subunits of the NADPH oxidase, p67phox and p47phox, the recruitment of which to the membrane requires cell stimulation, were detected in the rafts of unstimulated neutrophils. Treatment of neutrophils with the cholesterol-sequestering agent methyl-beta-cyclodextrin (MbetaCD) reduced raft p22phox and PR3. MbetaCD diminished membrane FcgammaRIIIb upregulating membrane PR3 (mPR3) and CD11b/CD18. In addition, MbetaCD significantly reduced PMA-induced activity of the NADPH oxidase without altering fmet-leu-phe-elicited activity. Antibody-mediated cross-linking of membrane PR3 caused activation of ERK and JNK kinases and their translocation to rafts. Confocal analysis revealed colocalization of mPR3, FcgammaRIIIb, and p22phox in the membrane, confirmed by their coimmunoprecipitation. Cleavage of neutrophil GPI-anchors by PI-PLC reduced mPR3 and FcgammaRIIIb, implicating a GPI-protein, possibly FcgammaRIIIb, in the attachment of PR3 to the membrane.
Cytochrome c-557 from Crithidia oncopelti and cytochrome c-558 from Euglena gracilis are mitochon... more Cytochrome c-557 from Crithidia oncopelti and cytochrome c-558 from Euglena gracilis are mitochondrial cytochromes c that have an atypical haem-binding site. It was of interest to know whether the loss of one thioether bond affected the physicochemical properties of these cytochromes. The thermodynamic parameters of the redox potential were measured. The reaction with imidazole, the kinetics and thermodynamics of the alkaline isomerization and the effect of heating on the visible spectrum are described for the ferricytochromes. The kinetics of the loss of cyanide, the spectral changes occurring on reduction with dithionite at alkaline pH values and the reactivity with CO are described for the ferrocytochromes. In many respects the cytochromes of the two protozoans are very similar to the cytochromes of horse and yeast. The ferricytochromes do, however, undergo a reversible transition to high-spin species on heating, which may be due to the more flexible attachment of the prosthetic ...
Abstract On incubation of ferricytochrome c with bromoacetate in the presence of cyanide, the met... more Abstract On incubation of ferricytochrome c with bromoacetate in the presence of cyanide, the methionyl residues at positions 65 and 80 are alkylated. A similar product can be obtained by alkylating cytochrome c dimer in the absence of cyanide. In the oxidized form of the reaction product, three different spectrophotometric types can be observed between pH values of 2.8 and 7.0. At neutral pH, the alkylated cytochrome c cannot be reduced with H2, ascorbate, or cysteine. The extinction coefficient of the reduced product at 520 and 550 mµ increases up to pH 10, at which it finally reaches the value for the native unsubstituted protein. The modified hemoprotein in both oxidation states readily forms complexes with metal ligands. In the reduced state, it forms a complex with molecular oxygen. Carboxymethylation of the methionyl residues renders cytochrome c inactive in restoring the respiration of cytochrome c-depleted rat liver mitochondria and in the succinic oxidase enzyme system.
The effects of neomycin, fluoride and the non-hydrolysable guanine nucleotide analogue GTP gamma ... more 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 present...
NADPH oxidase is a superoxide-generating, membrane-bound complex activated in stimulated phagocyt... more NADPH oxidase is a superoxide-generating, membrane-bound complex activated in stimulated phagocytes or in a reconstituted system consisting of membranes, cytosolic components and arachidonate or SDS. To delineate mechanism of oxidase activation in the cell-free system, hydrolysis of phosphoinositides in the combined membrane-cytosol oxidase mixture was investigated. Arachidonate promoted hydrolysis of membrane-[3H]-phosphatidylinositol by cytosolic phospholipase C. PI hydrolysis was similarly supported by other unsaturated fatty acids and by SDS. Unlike activation of the NADPH oxidase, PI hydrolysis required the presence of calcium ions. Implications of these findings to the mechanism of NADPH oxidase activation are discussed.
Abstract The influence of the sodium salts of various anions on the spectral properties of acid f... more Abstract The influence of the sodium salts of various anions on the spectral properties of acid ferricytochrome c was investigated. It was found that chaotropic anions convert the high spin form of the enzyme into a mixed spin spectral type. The following order of efficiency was established: TBA- g TCA- g ClO4- g SCN- g NO3- g Br- g Cl- (where TBA- and TCA- represent tribromoacetate and trichloroacetate ions, respectively). A correlation was shown between the action of the anions and their partial molar entropies.
The superoxide-generating NADPH oxidase of neutrophils can be activated in a cell-free system con... more The superoxide-generating NADPH oxidase of neutrophils can be activated in a cell-free system consisting of cell membranes, cytosol and an activating detergent (e.g. arachidonate or SDS). It has previously been reported [Aviram and Sharabani (1989) Biochem. Biophys. Res. Commun. 161, 712-719] that a mixture of phosphoinositides (PPIs), as well as the individual inositol lipids, interfere with the activation process. In the present study it is shown that exposure of the cytosol to PPI results in a progressive (t1/2 = 30 s) loss of its oxidase-supporting activity and that Mg2+ ions eliminate this inactivation. Neomycin, previously described as an inhibitor of cell-free activation, counteracted the effect of PPI and vice versa. Fractionation experiments implicated the p67-phox cytosolic component of the oxidase in the association with PPI. PPI blocked activity of recombinant p67-phox also and quenched the fluorescence intensity of its tryptophan residues. It is suggested that PPIs may ...
Human neutrophils treated with low concentrations of the homobifunctional cross-linking reagents ... more Human neutrophils treated with low concentrations of the homobifunctional cross-linking reagents disuccinimidyl suberate and dithiobis(succinimidylpropionate) failed to generate superoxide in response to any of several stimuli, including phorbol myristate acetate, fMet-Leu-Phe, A23187, fluoride, and opsonized zymosan. The cross-linking reagent interfered with the activation of NADPH oxidase, but not with its activity. Cells treated with succinimidyl butyrate, a monovalent analog of the cross-linkers, underwent a normal respiratory burst. Cross-linking also inhibited degranulation, phagocytosis, and fluorescence responses of potential-sensitive dyes but had little effect on lactate production, sugar transport, the binding of fMet-Leu-Phe, or the activity of various enzymes in the cross-linked neutrophils. Most of the cellular functions inhibited through the reaction of neutrophils with the cleavable cross-linker dithiobis(succinimidylpropionate) could be restored by reduction of the disulfide bonds of the cell-bound cross-linker with dithiothreitol.
Proteinase 3 (PR3) is a serine protease of neutrophil granules released to the medium or into the... more Proteinase 3 (PR3) is a serine protease of neutrophil granules released to the medium or into the phagocytic vesicle upon neutrophil stimulation. A fraction of the enzyme is thought to associate with the cell membrane yielding membrane PR3 (mPR3). In autoimmune disorders characterized by the presence of antineutrophil cytoplasmic antibodies (ANCA), the reaction of the latter with their target antigen mPR3 activates the cell inflicting injuries on the surrounding tissues. In a previous communication we provided evidence for the presence of mPR3 in lipid rafts obtained by lysis of neutrophils in Triton X-100 and for the mediation of PR3 binding to the membrane by a glycosylphosphatidylinositol (GPI)-anchored neutrophil protein, possibly FcgammaRIIIb. In the current study we employed the mild detergent Brij 58 to isolate high molecular weight (HMW) protein complexes in the void volume of a Sepharose 4B gel filtration minicolumn. HMW complexes of unstimulated neutrophils comprised PR3, FcgammaRIIIb, the beta2 integrin CD11b/CD18 as well as the membrane and cytosolic subunits of the NADPH oxidase, p22phox and p47phox/p67phox. Treatment of neutrophils with phosphatidylinositol-specific phospholipase C (PI-PLC) reduced amounts of PR3 and FcgammaRIIIb in HMW complexes isolated from the treated cells, supporting our previous suggestion that FcgammaRIIIb acts as a membrane adaptor for PR3. FcgammaRIIIb of HMW fractions co-immunoprecipitated with PR3, indicating their presence in the same protein complex. Since HMW fractions contained also the majority of biotinylated proteins obtained by the reaction of neutrophils with a membrane impermeable biotinylating agent Sulfo-NHS-biotin, it was concluded that HMW proteins were derived from cell membranes. Lipid rafts isolated from Brij 58-lysed neutrophils were similar in their protein composition to the HMW complexes but not identical.
Proteinase 3 (PR3), the target autoantigen of antineutrophil cytoplasmic antibodies in the autoim... more Proteinase 3 (PR3), the target autoantigen of antineutrophil cytoplasmic antibodies in the autoimmune vasculitis, Wegener's granulomatosis, is a serine proteinase stored in granules of human neutrophils. PR3 is expressed also on the plasma membrane of unactivated neutrophils, and this expression increases in primed or stimulated cells. In the current study, we demonstrate the presence of PR3, FcgammaRIIIb, and cytochrome b558 of the NADPH oxidase in neutrophil lipid rafts. Activation of neutrophils with PMA, fmet-leu-phe, or TNFalpha known to increase the membrane expression of PR3 did not affect the amount of PR3 in rafts. Unexpectedly, the cytosolic subunits of the NADPH oxidase, p67phox and p47phox, the recruitment of which to the membrane requires cell stimulation, were detected in the rafts of unstimulated neutrophils. Treatment of neutrophils with the cholesterol-sequestering agent methyl-beta-cyclodextrin (MbetaCD) reduced raft p22phox and PR3. MbetaCD diminished membrane FcgammaRIIIb upregulating membrane PR3 (mPR3) and CD11b/CD18. In addition, MbetaCD significantly reduced PMA-induced activity of the NADPH oxidase without altering fmet-leu-phe-elicited activity. Antibody-mediated cross-linking of membrane PR3 caused activation of ERK and JNK kinases and their translocation to rafts. Confocal analysis revealed colocalization of mPR3, FcgammaRIIIb, and p22phox in the membrane, confirmed by their coimmunoprecipitation. Cleavage of neutrophil GPI-anchors by PI-PLC reduced mPR3 and FcgammaRIIIb, implicating a GPI-protein, possibly FcgammaRIIIb, in the attachment of PR3 to the membrane.
Cytochrome c-557 from Crithidia oncopelti and cytochrome c-558 from Euglena gracilis are mitochon... more Cytochrome c-557 from Crithidia oncopelti and cytochrome c-558 from Euglena gracilis are mitochondrial cytochromes c that have an atypical haem-binding site. It was of interest to know whether the loss of one thioether bond affected the physicochemical properties of these cytochromes. The thermodynamic parameters of the redox potential were measured. The reaction with imidazole, the kinetics and thermodynamics of the alkaline isomerization and the effect of heating on the visible spectrum are described for the ferricytochromes. The kinetics of the loss of cyanide, the spectral changes occurring on reduction with dithionite at alkaline pH values and the reactivity with CO are described for the ferrocytochromes. In many respects the cytochromes of the two protozoans are very similar to the cytochromes of horse and yeast. The ferricytochromes do, however, undergo a reversible transition to high-spin species on heating, which may be due to the more flexible attachment of the prosthetic ...
Abstract On incubation of ferricytochrome c with bromoacetate in the presence of cyanide, the met... more Abstract On incubation of ferricytochrome c with bromoacetate in the presence of cyanide, the methionyl residues at positions 65 and 80 are alkylated. A similar product can be obtained by alkylating cytochrome c dimer in the absence of cyanide. In the oxidized form of the reaction product, three different spectrophotometric types can be observed between pH values of 2.8 and 7.0. At neutral pH, the alkylated cytochrome c cannot be reduced with H2, ascorbate, or cysteine. The extinction coefficient of the reduced product at 520 and 550 mµ increases up to pH 10, at which it finally reaches the value for the native unsubstituted protein. The modified hemoprotein in both oxidation states readily forms complexes with metal ligands. In the reduced state, it forms a complex with molecular oxygen. Carboxymethylation of the methionyl residues renders cytochrome c inactive in restoring the respiration of cytochrome c-depleted rat liver mitochondria and in the succinic oxidase enzyme system.
The effects of neomycin, fluoride and the non-hydrolysable guanine nucleotide analogue GTP gamma ... more 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 present...
NADPH oxidase is a superoxide-generating, membrane-bound complex activated in stimulated phagocyt... more NADPH oxidase is a superoxide-generating, membrane-bound complex activated in stimulated phagocytes or in a reconstituted system consisting of membranes, cytosolic components and arachidonate or SDS. To delineate mechanism of oxidase activation in the cell-free system, hydrolysis of phosphoinositides in the combined membrane-cytosol oxidase mixture was investigated. Arachidonate promoted hydrolysis of membrane-[3H]-phosphatidylinositol by cytosolic phospholipase C. PI hydrolysis was similarly supported by other unsaturated fatty acids and by SDS. Unlike activation of the NADPH oxidase, PI hydrolysis required the presence of calcium ions. Implications of these findings to the mechanism of NADPH oxidase activation are discussed.
Abstract The influence of the sodium salts of various anions on the spectral properties of acid f... more Abstract The influence of the sodium salts of various anions on the spectral properties of acid ferricytochrome c was investigated. It was found that chaotropic anions convert the high spin form of the enzyme into a mixed spin spectral type. The following order of efficiency was established: TBA- g TCA- g ClO4- g SCN- g NO3- g Br- g Cl- (where TBA- and TCA- represent tribromoacetate and trichloroacetate ions, respectively). A correlation was shown between the action of the anions and their partial molar entropies.
The superoxide-generating NADPH oxidase of neutrophils can be activated in a cell-free system con... more The superoxide-generating NADPH oxidase of neutrophils can be activated in a cell-free system consisting of cell membranes, cytosol and an activating detergent (e.g. arachidonate or SDS). It has previously been reported [Aviram and Sharabani (1989) Biochem. Biophys. Res. Commun. 161, 712-719] that a mixture of phosphoinositides (PPIs), as well as the individual inositol lipids, interfere with the activation process. In the present study it is shown that exposure of the cytosol to PPI results in a progressive (t1/2 = 30 s) loss of its oxidase-supporting activity and that Mg2+ ions eliminate this inactivation. Neomycin, previously described as an inhibitor of cell-free activation, counteracted the effect of PPI and vice versa. Fractionation experiments implicated the p67-phox cytosolic component of the oxidase in the association with PPI. PPI blocked activity of recombinant p67-phox also and quenched the fluorescence intensity of its tryptophan residues. It is suggested that PPIs may ...
Human neutrophils treated with low concentrations of the homobifunctional cross-linking reagents ... more Human neutrophils treated with low concentrations of the homobifunctional cross-linking reagents disuccinimidyl suberate and dithiobis(succinimidylpropionate) failed to generate superoxide in response to any of several stimuli, including phorbol myristate acetate, fMet-Leu-Phe, A23187, fluoride, and opsonized zymosan. The cross-linking reagent interfered with the activation of NADPH oxidase, but not with its activity. Cells treated with succinimidyl butyrate, a monovalent analog of the cross-linkers, underwent a normal respiratory burst. Cross-linking also inhibited degranulation, phagocytosis, and fluorescence responses of potential-sensitive dyes but had little effect on lactate production, sugar transport, the binding of fMet-Leu-Phe, or the activity of various enzymes in the cross-linked neutrophils. Most of the cellular functions inhibited through the reaction of neutrophils with the cleavable cross-linker dithiobis(succinimidylpropionate) could be restored by reduction of the disulfide bonds of the cell-bound cross-linker with dithiothreitol.
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