TAILIEUCHUNG - Báo cáo Y học: The S100A8/A9 protein as a partner for the cytosolic factors of NADPH oxidase activation in neutrophils

In a previous study, the S100A8/A9 protein, a Ca2+- and arachidonic acid-binding protein, abundant in neutrophil cytosol, was found to potentiate the activation of the redox component of the O2– generating oxidase in neutrophils, namely the membrane-bound flavocytochrome b, by the cytosolic phox proteins p67phox, p47phox and Rac ` (Doussiere J., Bouzidi F. and Vignais . (2001) Biochem. Biophys. Res. Commun. 285, 1317–1320). This led us to check by immunoprecipitation and protein fractionation whether the cytosolic phox proteins could bind to S100A8/A9. Following incubation of a cytosolic extract from nonactivated bovine neutrophil with protein A–Sepharose bound to antip67phox antibodies, the. | Eur. J. Biochem. 269 3246-3255 2002 FEBS 2002 doi The S100A8 A9 protein as a partner for the cytosolic factors of NADPH oxidase activation in neutrophils Jacques Doussiere Farid Bouzidi and Pierre V. Vignais Laboratoire de Biochimie et Biophysique des Systemes Integres UMR 5092 CEA-CNRS-UJF Departement Reponse et Dynamique Cellulaires CEA-Grenoble France In a previous study the S100A8 A9 protein a Ca2 - and arachidonic acid-binding protein abundant in neutrophil cytosol was found to potentiate the activation of the redox component of the O2- generating oxidase in neutrophils namely the membrane-bound flavocytochrome b by the cytosolic phox proteins p67phox p47phox and Rac Doussiere J. Bouzidi F. and Vignais . 2001 Biochem. Biophys. Res. Commun. 285 1317-1320 . This led us to check by immunoprecipitation and protein fractionation whether the cytosolic phox proteins could bind to S100A8 A9. Following incubation of a cytosolic extract from nonactivated bovine neutrophil with protein A-Sepharose bound to anti-p67phox antibodies the recovered immunoprecipitate contained the S100 protein p47phox and p67phox. Cytosolic protein fractionation comprised two successive chromatographic steps on hydroxyapatite and DEAE cellulose followed by isoelectric focusing. The S100A8 A9 heterodimeric protein comigrated with the cytosolic phox proteins and more particularly with p67phox and Rac2 whereas the isolated S100A8 protein displayed a tendancy to bind to p47phox. Using a semirecombinant cell-free system of oxidase activation consisting of recombinant p67phox p47phox and Rac2 neutrophil membranes and arachidonic acid we found that the S100A8 A9-dependent increase in the elicited oxidase activity corresponded to an increase in the turnover of the membrane-bound flavocytochrome b but not to a change of affinity for NADPH or O2. In the absence of S100A8 A9 oxidase activation departed from michaelian kinetics above a critical threshold concentration .

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