Author(s): Batot G, Martel C, Capdeville N, Wientjes F, Morel F
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Abstract 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.
This article was published in Eur J Biochem
and referenced in Pharmaceutica Analytica Acta