Components Of Respiratory Burst Oxidase Research Articles

RbohA, a rice homologue of the mammalian gp91phox respiratory burst oxidase gene.

It has been hypothesized that plants contain respiratory burst oxidase which, upon activation, oxidize NADPH and generate extracellular superoxide, O2.-. These proteins are proposed to play a central role in defence against pathogens. However, plant DNA sequences that encode proteins with similarity to components of respiratory burst oxidase have not previously been reported. This paper describes the complete cDNA and genomic DNA sequence of the rice rbohA (for respiratory burst oxidase homologue) gene. The predicted RbohA product is most similar to the main catalytic subunit, gp91phox, of the respiratory burst oxidase of neutrophils. Reverse transcriptase PCR detects rbohA transcripts in both roots and shoots of healthy rice plants.

メシル酸ガベキセート（ＧＭ）およびその分解産物（ＧＣＡ）・分解類似物（ＰＨＢＡＭＥ）の好中球ＮＡＤＰＨ ｏｘｉｄａｓｅ ｓｙｓｔｅｍに与える影響

We investigated the mechanism by which the synthetic protease inhibitor, gabexate mesilate, inhibits the production of the superoxide anion by human neutrophils and its active site of gabexate mesilate. We found that gabexate mesilate suppressed lucigenin-dependent chemiluminescence in intact neutrophils activated with PMA, but its degraded analog, methylparaben and another degraded product, guanidinocaproic acid did not.Dose-dependent suppression of SOD-inhabitable cytochrome c reduction was also detected in gabexate mesilate-treated neutrophils activated with phorbol ester. Gabexate mesilate slightly scavenged the superoxide anion in the pyrogallol assay. Gabexate mesilate also inhibited superoxide anion production in a dose-dependent manner in a cell-free oxidase-activating system. Translocation of the cytosolic respiratory burst oxidase components, the 47- and 65-kDa proteins, to membranes was suppressed by gabexate mesilate in intact cells stimulated with phorbol ester. Gabexate mesilate also reduced arachidonic acid-induced translocation of the components to the membrane fraction in the cell-free system.These results demonstrate that gabexate mesilate suppresses superoxide anion production by inhibiting the translocation of the 47- and 65-kDa proteins to the plasma membrane and suppression of superoxide anion production requires whole molecule of gabexate mesilate.

Inhibitory effect of a synthetic protease inhibitor (gabexate mesilate) on the respiratory burst oxidase in human neutrophils.

We investigated the mechanism by which the synthetic protease inhibitor gabexate mesilate inhibits the production of the superoxide anion by human neutrophils. We found that gabexate mesilate suppressed SOD-inhibitable cytochrome c reduction in a dose-dependent manner in intact neutrophils activated with phorbol ester. Gabexate mesilate slightly scavenged the superoxide anion in the pyrogallol assay. The reagent also inhibited superoxide anion production in a dose-dependent manner in a cell-free oxidase-activating system. Translocation of the cytosolic respiratory burst oxidase components, the 47- and 65-kDa proteins, to membranes was suppressed by the reagent in intact cells stimulated with phorbol ester. Gabexate mesilate also reduced arachidonic acid-induced translocation of the components to the membrane fraction in the cell-free system. These results demonstrate that gabexate mesilate suppresses superoxide anion production by reducing the translocation of the 47- and 65-kDa proteins to the plasma membrane.

Isolation of a complex of respiratory burst oxidase components from resting neutrophil cytosol.

The respiratory burst oxidase of neutrophils is a multicomponent enzyme, dormant in resting cells, that catalyzes the reduction of oxygen to O2- at the expense of NADPH. In the resting neutrophil, some of the components of the oxidase, including proteins p47phox and p67phox, are in the cytosol, while the rest are in a fraction that usually copurifies with plasma membrane. Recent evidence has suggested that at least some of the cytosolic oxidase components exist as a complex. We have now purified such a complex from the cytoplasm of resting neutrophils using an affinity column prepared with an antibody that recognizes the C-terminal decapeptide of p47phox. Immunoblotting showed that the complex contained both p47phox and p67phox. When supplemented with recombinant p67phox, the complex displayed considerable activity in a cell-free oxidase-activating system, and even without added p67phox, the complex could more than double O2- production in an oxidase-activating system supplemented with suboptimal amounts of cytosol. Isolation of the complex was blocked by preincubating the affinity column with CFSTKRKLASAV, the peptide against which the antibody was raised. On gel filtration, the complex migrated with a molecular weight of 240-300K, similar to that observed with whole neutrophil cytosol. The p47phox/p67phox ratio in the gel-filtered complex was approximately 1 to 1. These results indicate that in resting neutrophil cytosol, p47phox and p67phox exist as a complex.

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.

Isolation of the respiratory burst oxidase: the role of a flavoprotein component.

The article reviews the enzymatic and electron transfer properties of a low-potential FAD-dependent flavoprotein that is a component of the NADPH-dependent O2-.-generating respiratory burst oxidase of phagocytes. Current methods available for isolation of the respiratory burst oxidase and the flavoprotein component of the complex are also reviewed. These studies and data obtained from affinity-labeling of respiratory burst oxidase components, suggest that the flavoprotein has a molecular weight of 65-67 kD. The prevailing evidence suggests that the flavoprotein functions as a dehydrogenase/electron transferase and can directly catalyse NADPH-dependent O2-.formation when isolated. However, in neutrophil plasma membranes, the prevailing evidence suggests that the flavoprotein functions primarily to transfer electrons from NADPH to cytochrome b-245 and that this latter redox component is the catalytic side of O2-.formation. A working model for the arrangement of the flavoprotein and cytochrome b-245 components of the respiratory burst oxidase in neutrophil membranes is proposed.