Mutation at Histidine 338 of gp91 Depletes FAD and Affects Expression of Cytochrome b 558 of the Human NADPH Oxidase

Abstract

phox . This was in agreement with the finding that a single base substitution (C1024 to T) changed His-338 to Tyr in gp91 phox in a predicted FAD-binding domain of the flavocytochrome model. The loss of FAD could not be corrected even after addition of reagent FAD or a FADrich dehydrogenase fraction isolated from normal neutrophils to the patient's membranes, in a reconstitution in vitro with normal cytosol. These results indicate that His-338 is a very critical residue for FAD incorporation into the NADPH oxidase system. This is the first such mutation found in CGD.

Superoxide Generation by Nox1 in Guinea Pig Gastric Mucosal Cells Involves a Component with p67phox-Ability

“…We have shown that an antibody against human gp91 phox C-terminus 28) seemed able to recognize guinea pig Nox1. 20) The same antibody, however, did not cross-react with human Nox1 protein, which required the raising of another antibody for detecting Nox1 in Caco-2 cells.…”

“…17) The antibodies against C-terminal peptides of human gp91 phox and p22 phox were the same as in. 28) Deglycosylation of N-linked carbohydrates of the large subunits of cytochrome b 558 from human (gp91 phox ) and guinea pig (ca. 62 kDa) neutrophils was performed with peptide: N-glycosidase F (PNGase F, New England Biolabs), as described.…”

“…62 kDa) neutrophils was performed with peptide: N-glycosidase F (PNGase F, New England Biolabs), as described. 28) Western blotting after resolution of proteins on 10% acrylamide gels was performed as previously described, 17) using an enhanced chemiluminescence detection assay (Amersham Pharmacia Biotech). Bands were visualized after exposing polyvinylidene difluoride sheets to Xomat films (Eastman Kodak) and development by a Fuji FPM 8000A machine.…”

“…25,27) The supernatant from a 1ϫ10 5 -g centrifugation is designated as cytosol, and the twice washed pellets after resuspension in buffer A containing the above protease inhibitors, as membranes. 28) Protein was determined by the method of Lowry, using bovine serum albumin as a standard. Cytochrome b 558 heme contents in human and guinea pig neutrophil membranes were determined from their dithionite-reduced minus oxidized spectra.…”

“…28) Electron Flow Indicators The electron acceptors used herein (cytochrome c, acetylated cytochrome c and INT) do not cross the cell membrane. Native and acetylated cytochrome c capture one electron from O 2 Ϫ released by cells, but differ from each other concerning susceptibility to enzymatic reduction since acetylation of lysyl residues of cytochrome c prevents its interaction with reductases.…”