Regulation of phagocyte NADPH oxidase activity: identification of two cytochrome b558 activation states.
Activation of the phagocyte NADPH oxidase (phox) requires the association of cytosolic proteins (p67-phox, p47-phox, p40-phox, and Rac1/2) with the membrane cytochrome b558, leading to a hemoprotein conformation change. To clarify this mechanism, the phagocyte NADPH oxidase complex was isolated through cytochrome b558 purification after three chromatographic steps. The purified neutrophil complex was constitutively active in the absence of an amphiphile agent with a maximum turnover (125 mol O2(-) x s(-1) x mol heme b(-1)), indicating that cytochrome b558 has been activated by cytosolic proteins and is in an "open conformation," able to transfer a maximum rate of electrons. In contrast, the phox complex prepared with B lymphocyte cytosol shows a lower constitutive turnover (approximately 50 mol O2(-) x s(-1) x mol heme b(-1)). Analysis of phox complex components by Western blot and mass spectrometry showed the presence of cytosolic factors (especially p67-phox) and structural proteins (moesin, ezrin). To investigate the difference in activity of phox complexes, we evaluated the effect of MRP8 and MRP14, specifically expressed in neutrophils, on the activity of the B lymphocyte complex. MRPs induce the switch between the partially and the fully "open" cytochrome b558 conformation. Moreover, their effect was independent of p67-phox. Data point out two potential cytochrome b558 activation states.
Paclet
Marie-Hélène
MH
GREPI EA 2938, Laboratory Enzymologie/DBPC, CHU Grenoble BP 217, 38043, Grenoble Cedex 9, France. mhpaclet@chu-grenoble.fr
Berthier
Sylvie
S
Kuhn
Lauriane
L
Garin
Jérôme
J
Morel
Françoise
F
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D040502:Calgranulin B / Q000378:metabolism; D002478:Cells, Cultured; D003573:Cytochrome b Group / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008956:Models, Chemical; D019255:NADPH Oxidases / Q000096:biosynthesis* / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology; D010100:Oxygen / Q000378:metabolism; D010586:Phagocytes / Q000201:enzymology*; D010750:Phosphoproteins / Q000378:metabolism; D011487:Protein Conformation
C435500:ABCC11 protein, human; D018528:ATP-Binding Cassette Transporters; D040502:Calgranulin B; D003573:Cytochrome b Group; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; C036888:cytochrome b558; D019255:NADPH Oxidases; D010100:Oxygen