Purification of the 260 kDa cytosolic complex involved in the Superoxide production of guinea pig neutrophils

Someya, Akimasa; Nagaoka, Isao; Yamashita, Tatsuhisa

doi:10.1016/0014-5793(93)80276-z

Cited by 78 publications

(76 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Lane 2 in Fig. 7 shows the p67 phox counterpart in guinea pig neutrophil with a lower size of 63 kDa, consistent with the size reported for p67 phox in guinea pig 35) and which will be called p63 phox herein. GMC fractions showed two bands cross-reacting with the antibody: one parallel to p63 phox (lower band in lanes 3-8) appearing mainly in the cytosol, and another band above 67 kDa.…”

Section: Substrate Preference Of Nox1 and Conditions For Optimal Insupporting

confidence: 80%

Superoxide Generation by Nox1 in Guinea Pig Gastric Mucosal Cells Involves a Component with p67phox-Ability

Yoshida

Nishida

Shimoyama

et al. 2004

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

Section: Substrate Preference Of Nox1 and Conditions For Optimal Insupporting

confidence: 80%

Superoxide Generation by Nox1 in Guinea Pig Gastric Mucosal Cells Involves a Component with p67phox-Ability

Yoshida

Nishida

Shimoyama

et al. 2004

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

“…Comparative western blots carried out with LPS-primed DCs and unprimed DCs did not reveal any significant difference in the amounts of p60 and the cytosolic factors of oxidase activation (data not shown); an increased synthesis of these proteins in LPS-primed DCs is therefore unlikely. In neutrophils p67phox, p47phox and p40phox are associated to form a complex in the cytosol (Someya et al, 1993). A similar complex was recovered from DC cytosol by immunoprecipitation, using p67phox antibodies (data not shown).…”

Section: Resultsmentioning

confidence: 80%

Cryptic O2–-generating NADPH oxidase in dendritic cells

Elsen¹,

Doussière²,

Villiers

et al. 2004

Journal of Cell Science

View full text Add to dashboard Cite

All the components of the O2–-generating NADPH oxidase typically found in neutrophils, namely a membrane-bound low potential flavocytochrome b and oxidase activation factors of cytosolic origin, are immunodetectable in murine dendritic cells (DCs). However, in contrast to neutrophils, DCs challenged with phorbol myristate acetate (PMA) can barely mount a significant respiratory burst. Nevertheless, DCs generate a substantial amount of O2– in the presence of PMA following preincubation with pro-inflammatory ligands such as lipopolysaccharide and pansorbin, and to a lesser extent with anti-CD40 or polyinosinic polycytidylic acid. We found that the virtual lack of the oxidase response to PMA alone is specifically controlled in DCs. Through the use of homologous and heterologous cell-free systems of oxidase activation, we showed the following: (1) a NADPH oxidase inhibitory factor is located in DC membranes; it exerts its effect on oxidase activation and not on the activated oxidase. (2) The inhibition is relieved by pretreatment of DC membranes with β-octylglucoside (β-OG). (3) The β-OG-extracted inhibitory factor prevents the activation of neutrophil oxidase. (4) The inhibitory activity is lost after treatment of DC membranes with proteinase K or heating, which points to the protein nature of the inhibitory factor. Overall, these data indicate that the O2–-generating oxidase in DCs is cryptic, owing to the presence of a membrane-bound inhibitor of protein nature that prevents oxidase activation. The inhibition is relieved under specific conditions, including a prolonged contact of DCs with pro-inflammatory ligands from microbial origin, allowing a substantial production of O2–, which may contribute to the response of DCs to a microbial exposure.

show abstract

“…phox , p47 phox , and p67 phox exist as a tight cytosolic complex of undefined stoichiometry that can be purified by gel filtration chromatography with an apparent molecular mass of 250 -300 kDa (5)(6)(7)(8). Activation of the NADPH oxidase is initiated by phosphorylation, which is believed to induce conformational changes that subsequently lead to rearrangements in intra-and intermolecular interactions in the p40-p47-p67…”

mentioning

confidence: 99%

Architecture of the p40-p47-p67 Complex in the Resting State of the NADPH Oxidase

Lapouge¹,

Smith²,

Groemping³

et al. 2002

Journal of Biological Chemistry

137

144

View full text Add to dashboard Cite

The phagocyte NADPH oxidase is a multiprotein enzyme whose subunits are partitioned between the cytosol and plasma membrane in resting cells. Upon exposure to appropriate stimuli multiple phosphorylation events in the cytosolic components take place, which induce rearrangements in a number of protein-protein interactions, ultimately leading to translocation of the cytoplasmic complex to the membrane. To understand the molecular mechanisms that underlie the assembly and activation process we have carried out a detailed study of the protein-protein interactions that occur in the p40-p47-p67 phox complex of the resting oxidase. Here we show that this complex contains one copy of each protein, which assembles to form a heterotrimeric complex. The apparent high molecular weight of this complex, as observed by gel filtration studies, is due to an extended, non-globular shape rather than to the presence of multiple copies of any of the proteins. Isothermal titration calorimetry measurements of the interactions between the individual components of this complex demonstrate that p67 phox is the primary binding partner of p47 phox in the resting state. These findings, in combination with earlier reports, allow us to propose a model for the architecture of the resting complex in which p67 phox acts as the bridging molecule that connects p40 phox and p47 phox .

show abstract

Purification of the 260 kDa cytosolic complex involved in the Superoxide production of guinea pig neutrophils

Cited by 78 publications

References 27 publications

Superoxide Generation by Nox1 in Guinea Pig Gastric Mucosal Cells Involves a Component with p67phox-Ability

Superoxide Generation by Nox1 in Guinea Pig Gastric Mucosal Cells Involves a Component with p67phox-Ability

Cryptic O2–-generating NADPH oxidase in dendritic cells

Architecture of the p40-p47-p67 Complex in the Resting State of the NADPH Oxidase

Contact Info

Product

Resources

About