Open AccessCharacterization of Neutrophil NADPH Oxidase Activity Reconstituted in a Cell‐Free Assay Using Specific Monoclonal Antibodies Raised Against Cytochrome b 558
Author(s) -
Batot Géraldine,
Martel Cécile,
Capdeville Nicolas,
Wientjes Frans,
Morel Françoise
Publication year - 1995
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1995.208_c.x
Subject(s) - monoclonal antibody , hemeprotein , chemistry , cytochrome , biochemistry , oxidase test , microbiology and biotechnology , cytochrome c , cytochrome b , cytochrome c oxidase , heme , antibody , biology , enzyme , mitochondrion , gene , immunology , mitochondrial dna
The immunochemical characterization of NADPH oxidase activity of cytochrome b 558 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 b 558 , two were selected, both of which were found to bind to the β subunit of cytochrome b 558 , 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 O 2 − in the reconstituted system and did not bind to the antibodies.