Characterization of neutrophil NADPH oxidase activity reconstituted in a cell-free assay using specific monoclonal antibodies raised against cytochrome b558.
The immunochemical characterization of NADPH oxidase activity of cytochrome b558 purified from human neutrophils was determined after reconstitution in a cell-free assay using the native hemoprotein and recombinant purified cytosolic activating factors. The oxidase activity showed a strict dependence on the heme content at each step of the hemoprotein purification process. The immunochemical properties of the reconstituted oxidase made use of monoclonal antibodies raised against membrane-bound and octyl-glucoside-extracted cytochrome b. From nine specific monoclonal antibodies reacting with gp91-phox cytochrome b558, two were selected, both of which were found to bind to the beta subunit of cytochrome b558 and to inhibit superoxide formation in the oxidase reconstituted cell-free assay. The extent of inhibition was dependent on the phospholipid environment. Neutrophil membrane extracts from X-linked chronic granulomatous disease patients did not produce O2- in the reconstituted system and did not bind to the antibodies.
Laboratoire d'Enzymologie, Centre Hospitalier Universitaire, Grenoble, France.
G
G
Batot
C
C
Martel
N
N
Capdeville
F
F
Wientjes
F
F
Morel
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002474:Cell-Free System; D003573:Cytochrome b Group / Q000276:immunology*; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010084:Oxidation-Reduction
D000911:Antibodies, Monoclonal; D003573:Cytochrome b Group; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases