Inactivation of NADPH oxidase from human neutrophils by affinity labelling with pyridoxal 5′-diphospho-5′-adenosine

Ravel, Pascale; Lederer, Florence

doi:10.1016/0006-291x(91)92074-t

Cited by 4 publications

(1 citation statement)

References 40 publications

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“…The NADPH-and FAD-binding sites of flavocytochrome b 558 are localized in the gp91phox subunit, as demonstrated by photolabeling with photoactivatable azido derivatives of NADPH [96,106] and FAD [95] and also by affinity labeling, using pyridoxal-5¢-diphospho-5-adenosine [107,108]. The crystal structure of several NADPH-dependent flavoproteins is known, and the regions of their polypeptide chains that participate in binding NADPH and FAD have been characterized.…”

Section: Mapping Of Nadph Fad and Heme-binding Sites In The Gp91phoxmentioning

confidence: 98%

The superoxide-generating NADPH oxidase: structural aspects and activation mechanism

Vignais¹

2002

Cellular and Molecular Life Sciences (CMLS)

698

597

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Flavocytochrome b558 is the catalytic core of the respiratory-burst oxidase, an enzyme complex that catalyzes the NADPH-dependent reduction of O2 into the superoxide anion O2 in phagocytic cells. Flavocytochrome b558 is anchored in the plasma membrane. It is a heterodimer that consists of a large glycoprotein gp91phox (phox forphagocyte oxidase) (beta subunit) and a small protein p22phox (alpha subunit). The other components of the respiratory-burst oxidase are water-soluble proteins of cytosolic origin, namely p67phox, p47phox, p40phox and Rac. Upon cell stimulation, they assemble with the membrane-bound flavocytochrome b558 which becomes activated and generates O2-. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections, illustrating the role of O2- and the derived metabolites H2O2 and HOCl in host defense against invading microorganisms. The electron carriers, FAD and hemes b, and the binding site for NADPH are confined to the gp91phox subunit of flavocytochrome b558. The p22phox subunit serves as a docking site for the cytoso lic phox proteins. This review provides an overview of current knowledge on the structural organization of the O2(-)-generating flavocytochrome b558, its kinetics, its mechanism of activation and the regulation of its biosynthesis. Homologues of gp91phox, called Nox and Duox, are present in a large variety of non-phagocytic cells. They exhibit modest O2(-)-generating oxidase activity, and some act as proton channels. Their role in various aspects of signal transduction is currently under investigation and is briefly discussed.

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Section: Mapping Of Nadph Fad and Heme-binding Sites In The Gp91phoxmentioning

confidence: 98%

The superoxide-generating NADPH oxidase: structural aspects and activation mechanism

Vignais¹

2002

Cellular and Molecular Life Sciences (CMLS)

698

597

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Perfluoroalkylphosphocholines are poor protein-solubilizing surfactants, as tested with neutrophil plasma membranes

Mardirossian¹,

Krafft²,

Gulik-Krzywicki³

et al. 1998

Biochimie

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Critical assessment of the presence of an NADPH binding site on neutrophil cytochrome b558 by photoaffinity and immunochemical labeling

Doussière¹,

Brandolin²,

Derrien³

et al. 1993

Biochemistry

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The presumed NADPH dehydrogenase function of the heterodimeric cytochrome b558 in the neutrophil oxidase complex has been investigated by combined photoaffinity labeling and immunoblot analysis of membrane proteins from bovine neutrophils. The photoaffinity probe was a radiolabeled analog of NADPH, [4-[N-(4-azido-2-nitrophenyl)[3H]amino]butyryl]NADPH ([3H]azido-NADPH), and the antibodies were directed against the C-terminal regions of the two subunits of cytochrome b558. Plasma membrane vesicles obtained by differential centrifugation of bovine neutrophil homogenates were routinely used as a source of NADPH oxidase. They were permeabilized by sodium deoxycholate to facilitate the access of NADPH or its azido analog to the totality of the specific binding sites. In the absence of light, azido-NADPH behaved as a competitive inhibitor of NADPH oxidase with a Ki of 6 microM, and was able to bind to high-affinity specific binding sites with a Kd of 5-6 microM, indicating a higher affinity of the oxidase for the photoprobe than for the substrate NADPH (KM = 30-40 microM). Upon photolabeling, the oxidase was fully inactivated. Following resolution of the membrane proteins by SDS-PAGE, a predominant photolabeled protein band of 80-100 kDa was revealed, which coincided with the large subunit (beta) of cytochrome b558 identified by immunoblot in a parallel gel. The enzymatic deglycosylation of photolabeled neutrophil membranes shifted the masses of both the photolabeled band and the immunoreactive beta subunit from 80-100 to 55-65 kDa in accordance with the glycoprotein nature of the beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

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Inactivation of NADPH oxidase from human neutrophils by affinity labelling with pyridoxal 5′-diphospho-5′-adenosine

Cited by 4 publications

References 40 publications

The superoxide-generating NADPH oxidase: structural aspects and activation mechanism

The superoxide-generating NADPH oxidase: structural aspects and activation mechanism

Perfluoroalkylphosphocholines are poor protein-solubilizing surfactants, as tested with neutrophil plasma membranes

Critical assessment of the presence of an NADPH binding site on neutrophil cytochrome b558 by photoaffinity and immunochemical labeling

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