Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.3.1 (NADPH oxidase)
11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We determined the effects of okadaic acid (OA), a specific inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), on protein phosphorylation and on the activation of the NADPH oxidase in human neutrophils. In otherwise unstimulated cells, OA induced phosphoprotein accumulation, revealing the presence of constitutively active protein kinases. Pulse-chase experiments in electropermeabilized cells confirmed that this effect was due, at least in part, to inhibition of dephosphorylation. OA potentiated phosphoprotein accumulation induced by phorbol esters and by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). In phorbol ester-stimulated cells, OA prolonged the respiratory response after inhibition of protein kinase C (PKC) with staurosporine, consistent with a reduced rate of dephosphorylation of active phosphorylated components. Similarly, OA delayed the inactivation of the burst after displacement of FMLP from its receptor by a competitive antagonist. This suggests that the substrates of the protein kinases activated by FMLP are dephosphorylated by PP1 and/or PP2A. That phosphatases control the intensity and duration of the respiratory response is suggested by the finding that OA magnified and prolonged the oxidative burst elicited by FMLP. In contrast, pretreatment with OA produced a time-dependent inhibition of the phorbol ester-induced respiratory burst. Under conditions where inhibition of the phorbol ester response was nearly complete, activation by the chemoattractant peptide not only persisted but was in fact accentuated. These findings provide strong evidence that receptor-mediated stimulation of the NADPH oxidase can occur by pathways not involving PKC.
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PMID:Modulation of neutrophil activation by okadaic acid, a protein phosphatase inhibitor. 131 Feb 15

A cDNA expression library from pig blood neutrophils was immunoscreened with a rabbit antiserum raised against a 32 kDa neutrophil membrane phosphoprotein. Previous work indicated this protein as a component of the superoxide-forming NADPH oxidase enzyme complex (1,2). Only one cDNA clone (B+) was highly positive. The B+ clone contained a 1109 bp insert, with an open reading frame encoding for 284 amino acids. The deduced B+ amino acid sequence contained a 72 amino acid domain with proline and glutamine repeats and two domains extremely enriched with serine residues. The isolated cDNA hybridizes with a 3.1 kb mRNA expressed in pig and human leukocytes.
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PMID:Isolation and characterization of a cDNA clone for a novel serine-rich neutrophil protein. 169 79

Neutrophil NADPH:O2 oxidoreductase activity, essential in the killing of bacteria by neutrophils, can be elicited in a cell-free system that requires plasma membranes, cytosol and sodium dodecyl sulfate. In addition, GTP or its nonhydrolyzable analog guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) enhances NADPH oxidase activity. We investigated the mechanism of this effect of GTP gamma S in the cell-free system. Cytosol from human neutrophils was separated in three different soluble oxidase components (SOC I, SOC II, and SOC III). Previously we (Bolscher, B. G. J. M., Van Zwieten, R., Kramer, I. J. M., Weening, R. S., Verhoeven, A. J., and Roos, D. (1989) J. Clin. Invest. 83, 757-763) reported that the cytosol contains two components which act synergistically. We now report that one component (previously labeled SOC II) contains two different components that can be separated by ion exchange chromatography. Immunoblotting with antiserum B-1 (Volpp, B. D., Nauseef, W. M., and Clark, R. A. (1988) Science 242, 1295-1297), directed against a cytosolic complex capable of activating latent membranes in the cell-free system, showed a 47-kDa protein in SOC II and a 67-kDa protein in SOC III. SOC II also contains the 47-kDa phosphoprotein, which indicates that this phosphoprotein and the protein recognized by the antiserum are identical. Low rates of NADPH-dependent O2 consumption can be elicited by SOC II and SOC III in the absence of SOC I. This activity is independent of GTP gamma S. Addition of SOC I increases this activity 3-4-fold, only when GTP gamma S is present. Plasma membranes, incubated with SOC I plus GTP gamma S and re-isolated, showed a similar 3-4-fold enhanced O2 consumption with SOC II and SOC III. The GTP gamma S effect is exerted primarily at the level of the plasma membrane. The concentration of GTP gamma S that causes a half-maximal stimulation was 0.4 mu M. It is concluded that SOC I is a functional component of the NADPH oxidase.
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PMID:The activity of one soluble component of the cell-free NADPH:O2 oxidoreductase of human neutrophils depends on guanosine 5'-O-(3-thio)triphosphate. 220 87

Activation of the phagocytic cell superoxide-generating NADPH oxidase requires interaction of cytosolic and membrane-associated components. With most stimuli activation of the oxidase is accompanied by multisite phosphorylation of the 47-kDa cytosolic oxidase factor (p47) which translocates from cytosol to membranes. Native p47 is a highly basic protein that undergoes stepwise charge shifts with successive phosphorylation events. Phosphorylation of p47 was studied by immunoprecipitation from neutrophil cytosol and membrane fractions followed by two-dimensional gel electrophoresis and autoradiography. In the resting cell p47 was not phosphorylated. In the cytosol of phorbol myristate acetate-activated neutrophils eight distinct p47 phosphoproteins were present. The membrane fraction from these activated cells contained a family of p47 phosphoproteins of electrophoretic mobilities identical to those seen in cytosol plus an additional, more acidic p47 phosphoprotein not present in cytosol. Very early after activation (30 s) only the four most acidic p47 phosphoproteins were present in the membrane fraction. Only at later times (5-15 min) was the full spectrum of p47 phosphoproteins present in the membrane fraction. In contrast, the full spectrum of p47 phosphoproteins was present in the cytosol over the entire time course we studied. In neutrophils from patients with cytochrome b558-deficient chronic granulomatous disease p47 phosphorylation was incomplete and p47 translocation to membrane did not occur. These studies demonstrated that the cytochrome was essential for formation of the three most acidic p47 phosphoproteins and greatly augmented formation of the fourth most acidic p47 phosphoprotein found in normal neutrophils. The temporal correlation between specific p47 phosphorylation events and p47 translocation to membrane is consistent with a model of oxidase activation in which a series of p47 phosphorylation events which occurs in cytosol precedes and may be required for p47 interaction with membrane.
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PMID:Phosphorylation of neutrophil 47-kDa cytosolic oxidase factor. Translocation to membrane is associated with distinct phosphorylation events. 224 68

The NADPH:O2 oxidoreductase (NADPH oxidase) of human neutrophils is converted from a dormant to an active state upon stimulation of the cells. We have studied the soluble fraction that is required for NADPH oxidase activation in a cell-free system. Human neutrophils were separated in a membrane-containing and a soluble fraction. The soluble fraction was separated on carboxymethyl (CM) Sepharose in 10 mM 4-morpholino-ethanesulfonic acid buffer of pH 6.8. Reconstitution of the NADPH oxidase activity, measured as O2 consumption, was only found when the membrane fraction was combined with the flowthrough of the CM Sepharose column as well as with a fraction that eluted at 125 mM NaCl. This result indicates that at least two soluble components are necessary for reconstitution of the NADPH oxidase activity: one that does not bind to CM Sepharose and one that does bind. These components were designated soluble oxidase component (SOC) I and SOC II, respectively. Boiling destroyed the activity in both fractions. In the soluble fraction of human lymphocytes and thrombocytes neither SOC I nor SOC II activity was found. SOC II copurified with a 47-kD phosphoprotein, previously found defective in patients with the autosomal form of chronic granulomatous disease (CGD). Inactive soluble fractions of cells from autosomal CGD patients were reconstituted with a SOC II fraction from control cells. The result of this experiment indicates that autosomal CGD patients are normal in SOC I but defective in SOC II.
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PMID:A phosphoprotein of Mr 47,000, defective in autosomal chronic granulomatous disease, copurifies with one of two soluble components required for NADPH:O2 oxidoreductase activity in human neutrophils. 253 48

Electrically permeabilized human neutrophils were used to study the mechanism of activation of the NADPH oxidase by chemotactic factors. The respiratory burst elicited by formyl-methionyl-leucyl-phenylalanine (fMLP) was strictly dependent on the addition of ATP. The response was also supported by adenosine 5'-[gamma-thio]triphosphate (ATP[S]), but not by the non-hydrolysable analogue (p[NH]ppA). In the presence of ATP, displacement of fMLP from its receptor by antagonist peptides resulted in the abrupt termination of the O2-consumption burst. In contrast, the response persisted after displacement of fMLP when ATP[S] was present. This finding is consistent with the formation of biologically active thiophosphoproteins which are resistant to cleavage by cellular phosphatases. Accordingly, lower concentrations of ATP[S], as compared with ATP, were required to support the fMLP response. The data indicate that protein phosphatases control the extent and duration of the response in cells stimulated with chemoattractants. Unlike ATP, sub-millimolar concentrations of ATP[S] elicited a spontaneous respiratory burst in the absence of fMLP or other stimuli. This effect was inhibited by p[NH]ppA and was not observed in intact (non-permeabilized) cells, indicating interaction of ATP[S] with an intracellular adenine-nucleotide-binding site, possibly a protein kinase. These results suggest that protein kinases are active in neutrophils in the absence of exogenous stimuli, but that accumulation of the essential phosphoprotein(s) is normally prevented by the ongoing vigorous phosphatase activity. It is conceivable that control of the respiratory burst is exerted by inhibition of phosphatase activity, instead of or in addition to the more commonly postulated activation of protein kinases.
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PMID:Activation of electropermeabilized neutrophils by adenosine 5'-[gamma-thio]triphosphate (ATP[S]). Role of phosphatases in stimulus-response coupling. 255 91

The activation of O2- -formation by neutrophil NADPH oxidase is associated with phosphorylation of several membrane and cytosolic proteins. In the membranes a phosphoprotein of 32 kDa belonging to the NADPH oxidase-cytochrome b-245 system (P. Bellavite et al., Free Rad. Res. Commun., 1, 11 (1985] showed the highest relative increase of 32Pi incorporation. Concomitant with the phosphorylation, a shift of the apparent molecular mass of the protein from 31 to 32 kDa occurred. The time-course, the sensitivity to trifluoperazine and the dose-dependence of phosphorylation were similar to those of O2- forming activity, except that the latter showed a longer lag-time than the former. The increase of the 32 kDa phosphoprotein was also comparable to the kinetics of cytochrome b-245 reduction by anaerobically activated neutrophils. The phosphorylation and the NADPH oxidase were triggered by various stimulants including phorbol myristate acetate, opsonized zymosan, arachidonic acid and sodium fluoride. With arachidonic acid the O2- formation was highly active but the phosphorylation was low. With fluoride the enzyme activity was reversible upon removal of the stimulant but the phosphorylation of the 32 kDa peptide was not reversible. Neutrophils treated with PMA at 17 degrees C showed phosphorylation but not activation. The results indicate that phosphorylation of a component of NADPH oxidase is a fundamental but probably not sufficient event in the activation mechanism of the enzyme.
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PMID:Studies on the nature and activation of O2- -forming NADPH oxidase of leukocytes. II. Relationships between phosphorylation of a component of the enzyme and oxidase activity. 285 3

Activation of the NADPH oxidase was examined in electrically permeabilized human neutrophils exposed to non-hydrolysable guanine nucleotides. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) induced a marked increase in the rate of O2 consumption, which was partially resistant to staurosporine, an inhibitor of protein kinase C, under conditions where the response to diacylglycerol was virtually abolished. The respiratory burst elicited by GTP[S] was dependent on the presence of ATP and Mg2+, suggesting involvement of phosphorylation reactions. Accordingly, phosphoprotein formation was greatly stimulated by the guanine nucleotide. The polypeptide phosphorylation pattern induced by GTP[S] was similar to, but not identical with, that observed with diacylglycerol, indicating the activation of kinases other than protein kinase C by the guanine nucleotide. The possible involvement of tyrosine kinases was assessed by immunoblotting using anti-phosphotyrosine antibodies. Treatment of electroporated cells with GTP[S] stimulated the accumulation of tyrosine-phosphorylated proteins. This effect was not induced by diacylglycerol, indicating that tyrosine phosphorylation is not secondary to stimulation of protein kinase C. The results indicate that, in neutrophils, activated G-proteins can stimulate tyrosine kinase and/or inhibit tyrosine phosphatase activity. Changes in the amounts of tyrosine-phosphorylated proteins may signal activation of the respiratory burst.
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PMID:Guanine nucleotides induce tyrosine phosphorylation and activation of the respiratory burst in neutrophils. 293 Apr 92

NADPH oxidase is an enzyme in the plasma membrane of the neutrophil that catalyzes the production of O2-, a species central to the oxygen-dependent killing mechanisms of this cell. The oxidase is dormant in resting cells and is activated upon the addition of a stimulus. Neutrophils of patients with chronic granulomatous disease (CGD) manifest no oxidase activity when stimulated. The possible role of protein phosphorylation in the activation of NADPH oxidase was examined in normal and CGD neutrophils by measuring the incorporation of 32Pi into proteins as determined by gel electrophoresis followed by autoradiography. Resting neutrophils from normal subjects exhibit at least 40 distinct phosphoprotein bands. The level of phosphorylation of these bands was examined after the addition of phorbol myristate acetate (PMA), opsonized zymosan, digitonin, N-formyl-methionyl-phenylalanine (FMLP), or NaF. PMA and opsonized zymosan increased the phosphorylation of a set of 6 protein bands. Digitonin and FMLP consistently caused the phosphorylation of 4 of these protein bands, while NaF failed to induce increased phosphorylation of any protein band. All activators tested caused the dephosphorylation of one specific protein band. The time course of phosphorylation (dephosphorylation) was examined using PMA as the activating agent. Increased phosphorylation of one protein band was evident by 12 sec after the addition of PMA. The most slowly phosphorylated protein band did not slow evidence of change until 5 min after the addition of PMA. Three of the phosphoproteins examined were phosphorylated either earlier than or concomitant with the activation of NADPH oxidase. CGD neutrophils were compared with normal cells for their ability to phosphorylate proteins in response to PMA. The phosphoprotein banding patterns of CGD neutrophils were identical with those of normal neutrophils in both the resting and activated states. The evidence presented shows that the phosphorylation of proteins is a prominent feature of neutrophil metabolism. The striking similarity of phosphorylation changes induced by the various activators tested suggests that protein phosphorylation may play a role in some aspects of neutrophil activation. Evidence was not obtained, however, regarding a link between protein phosphorylation and activation of NADPH oxidase.
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PMID:Endogenous protein phosphorylation by resting and activated human neutrophils. 682 99

The phosphorylation-dependent mechanisms regulating activation of the human neutrophil respiratory-burst enzyme, NADPH oxidase, have not been elucidated. We have shown that phosphatidic acid (PA) and diacylglycerol (DG), products of phospholipase activation, synergize to activate NADPH oxidase in a cell-free system. We now report that activation by PA plus DG involves protein kinase activity, unlike other cell-free system activators. NADPH oxidase activation by PA plus DG is reduced approximately 70% by several protein kinase inhibitors [1-(5-isoquinolinesulfonyl)piperazine, staurosporine, GF-109203X]. Similarly, depletion of ATP by dialysis reduces PA plus DG-mediated NADPH oxidase activation by approximately 70%. Addition of ATP, but not a nonhydrolyzable ATP analog, to the dialyzed system restores activation levels to normal. In contrast, these treatments have little effect on NADPH oxidase activation by arachidonic acid or SDS plus DG. PA plus DG induces the phosphorylation of a number of endogenous proteins. Phosphorylation is largely mediated by PA, not DG. A predominant substrate is p47-phox, a phosphoprotein component of NADPH oxidase. Phosphorylation of p47-phox precedes activation of NADPH oxidase and is markedly reduced by the protein kinase inhibitors. In contrast, arachidonic acid alone or SDS plus DG is a poor activator of protein phosphorylation in the cell-free system. Thus, PA induces activation of one or more protein kinases that regulate NADPH oxidase activation in a cell-free system. This cell-free system will be useful for identifying a functionally important PA-activated protein kinase(s) and for dissecting the phosphorylation-dependent mechanisms responsible for NADPH oxidase activation.
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PMID:Cell-free activation of neutrophil NADPH oxidase by a phosphatidic acid-regulated protein kinase. 764 15


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