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Further evidence for the involvement of a phosphoprotein in the respiratory burst oxidase of human neutrophils.

ABSTRACT: Phosphorylation of a 47 kDa protein in human neutrophils is induced by phorbol 12-myristate 13-acetate (PMA), opsonized latex beads, fMet-Leu-Phe, calcium ionophore A23187 and fluoride. All of these stimuli activate the specialized microbicidal respiratory burst of neutrophils, and in each case the kinetics of activation correspond with the kinetics of phosphorylation of the 47 kDa protein. Trifluoperazine (50 microM) and chlorpromazine (100 microM), inhibitors of calmodulin and protein kinase C, abolish the increase in oxygen consumption and selectively prevent phosphorylation of the 47 kDa protein after PMA stimulation. Treatment of neutrophils with pertussis toxin totally inhibits both superoxide production and phosphorylation of this protein in response to fMet-Leu-Phe, but not in response to PMA, indicating that a GTP-binding protein modulates the fMet-Leu-Phe receptor signal. Phosphorylation of the 47 kDa protein, a phenomenon absent from the neutrophils of subjects with autosomal recessive chronic granulomatous disease, which lack the respiratory burst, appears to be the common trigger for activation of the burst in normal neutrophils.

SUBMITTER: Heyworth PG

PROVIDER: S-EPMC1147346 | biostudies-other | 1986 Nov

REPOSITORIES: biostudies-other

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

Project description: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.

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Respiratory burst oxidase homologues D and F in catalase2 deficient plants

Project description:Hydrogen peroxide (H2O2) can act as a signaling molecule that influences various aspects of plant growth and development, including stress signaling and cell death. Catalase deficient plants are pioneering systems which accumulate hydrogen peroxide (H2O2) from peroxisomal origin during photorespiratory challenges. Respiratory burst oxidase homologues D and F are known to participate in intracellular oxidative stress response launched in cat2 mutants (Chaouch et al., 2012). We studied the compared the transcriptional response of cat2 rbohD and cat2 rbohF double mutants versus the cat2 background to further adress their role during photorespiratory stress.

2016-06-10 | GSE81516 | GEO

Hypoxia selectively inhibits respiratory burst activity and killing of Staphylococcus aureus in human neutrophils.

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Standardization of the antibody-dependent respiratory burst assay with human neutrophils and Plasmodium falciparum malaria.

Project description:The assessment of naturally-acquired and vaccine-induced immunity to blood-stage Plasmodium falciparum malaria is of long-standing interest. However, the field has suffered from a paucity of in vitro assays that reproducibly measure the anti-parasitic activity induced by antibodies in conjunction with immune cells. Here we optimize the antibody-dependent respiratory burst (ADRB) assay, which assesses the ability of antibodies to activate the release of reactive oxygen species from human neutrophils in response to P. falciparum blood-stage parasites. We focus particularly on assay parameters affecting serum preparation and concentration, and importantly assess reproducibility. Our standardized protocol involves testing each serum sample in singlicate with three independent neutrophil donors, and indexing responses against a standard positive control of pooled hyper-immune Kenyan sera. The protocol can be used to quickly screen large cohorts of samples from individuals enrolled in immuno-epidemiological studies or clinical vaccine trials, and requires only 6 μL of serum per sample. Using a cohort of 86 samples, we show that malaria-exposed individuals induce higher ADRB activity than malaria-naïve individuals. The development of the ADRB assay complements the use of cell-independent assays in blood-stage malaria, such as the assay of growth inhibitory activity, and provides an important standardized cell-based assay in the field.

| S-EPMC4571651 | biostudies-literature

Regulation of superoxide generation by myeloperoxidase during the respiratory burst of human neutrophils.

Project description:The role of myeloperoxidase in the regulation of the respiratory burst of human neutrophils activated by the chemotactic peptide (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) plus cytochalasin B was determined by using anti-(human myeloperoxidase) antibody. The respiratory burst activated under these conditions consisted of an initial (1-2 min) phase with high rates of O2 uptake, luminol-dependent chemiluminescence and superoxide radical (O2-.) generation and a second, more sustained, phase of lower magnitude of chemiluminescence and O2 uptake: O2-. generation did not occur during this second phase. In cell suspensions stimulated in the presence of anti-(human myeloperoxidase) antibody, the magnitude of the initial phase of both O2 uptake and O2-. generation was unaffected, but these high rates were maintained over much longer periods than in control suspensions. It is therefore proposed that a product of myeloperoxidase normally regulates the duration of O2-. generation during the respiratory burst, possibly by inhibition of NADPH oxidase.

| S-EPMC1147027 | biostudies-other

Respiratory burst oxidase homologues D and F in catalase2 deficient plants

2016-06-10 | E-GEOD-81516 | biostudies-arrayexpress

Purification of the 47 kDa phosphoprotein associated with the NADPH oxidase of human neutrophils.

Project description:A 47 kDa protein in the cytosol of phagocytic cells becomes heavily phosphorylated and translocates to the cell membrane upon stimulation. This protein was isolated from the cytosol of human neutrophils by chromatography on ion-exchange and hydroxyapatite resins. Polyclonal antibodies to this protein demonstrated that it was present in the neutrophils of two patients with X-linked chronic granulomatous disease (CGD) but not in those of three patients with the autosomal recessive pattern of inheritance. A sequence of amino acids was determined from a tryptic peptide of this protein: Glu-Met-Phe-Pro-Ile-Glu-Ala-Gly-Ala-Ile-Asn-Xaa-Glu. This served to establish that the phosphoprotein isolated here is the same as a protein of a similar molecular mass identified by other workers. These studies confirm the involvement of this 47 kDa phosphoprotein in the molecular pathology of autosomal recessive CGD and describe a method for the purification of the native protein.

| S-EPMC1131315 | biostudies-other

Oxidation-reduction properties of the cytochrome b found in the plasma-membrane fraction of human neutrophils. A possible oxidase in the respiratory burst.

Project description:The oxidation-reduction midpoint potential of the cytochrome b found in the plasma membrane of human neutrophils has been determined at pH 7.0 (Em,7.0) from measurements of absorption spectra at fixed potentials. In both unstimulated and phorbol myristate acetate-stimulated cells Em,7.0 was -245 mV. Changes in pH affected the Em of the cytochrome b, with a slope of approx. 25 mV/pH unit change. The Em,7.0 of the haem group(s) of the membrane-bound myeloperoxidase of human neutrophils was found to be +34 mV. The plasma membranes contained no detectable ubiquinone, and no iron-sulphur compounds were detected by e.p.r. spectroscopy at 5-20 K. No flavins were detected by e.p.r. spectroscopy. The cytochrome b-245 was not reduced by added NADH or NADPH. Dithionite-reduced cytochrome b-245 formed a complex with CO, supplied as a saturated solution, which was dissociated with 26 microseconds illumination from a xenon flash lamp, and the recombination with CO had a half-time of approx. 6 ms. Partly (80%) reduced cytochrome b-245 was oxidized by added air-saturated buffer with a half-time faster than 1 s at 20 degrees C, a resolution limited by mixing time. These results are compatible with cytochrome b-245 acting as an oxidase.

| S-EPMC1162784 | biostudies-other

Unsaturated long-chain fatty acids induce the respiratory burst of human neutrophils and monocytes in whole blood.

Project description:BackgroundIt is increasingly recognized that infectious complications in patients treated with total parenteral nutrition (TPN) may be caused by altered immune responses. Neutrophils and monocytes are the first line of defence against bacterial and fungal infection through superoxide anion production during the respiratory burst. To characterize the impact of three different types of lipid solutions that are applied as part of TPN formulations, we investigated the unstimulated respiratory burst activation of neutrophils and monocytes in whole blood.MethodsWhole blood samples were incubated with LCT (Intralipid(R)), LCT/MCT (Lipofundin(R)) and LCT-MUFA (ClinOleic(R)) in three concentrations (0.06, 0.3 and 0.6 mg ml-1) for time periods up to one hour. Hydrogen peroxide production during the respiratory burst of neutrophils and monocytes was measured by flow cytometry.ResultsLCT and LCT-MUFA induced a hydrogen peroxide production in neutrophils and monocytes without presence of a physiological stimulus in contrast to LCT/MCT.ConclusionWe concluded that parenteral nutrition containing unsaturated oleic (C18:1) and linoleic (C18:2) acid can induce respiratory burst of neutrophils and monocytes, resulting in an elevated risk of tissue damage by the uncontrolled production of reactive oxygen species. Contradictory observations reported in previous studies may in part be the result of different methods used to determine hydrogen peroxide production.

| S-EPMC2483276 | biostudies-literature

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