Affinity-labeling of an NADPH-binding site on the heavy subunit of flavocytochrome b558 in particulate NADPH oxidase from activated human neutrophils.
Cell stimulation of blood phagocytes activates the superoxide-producing NADPH oxidase. Cytochrome b558, one of the two oxidase redox components, comprises a light (alpha) and a heavy glycosylated (beta) subunit. The other redox component, a flavoprotein, is now thought to be the heavy subunit, on the basis of amino acid sequence comparisons and of reconstitution experiments with purified components. We published that pyridoxal-5'-diphospho-5'-adenosine is an inactivating affinity label for the NADPH-binding site of particulate oxidase from activated neutrophils. We have now radiolabeled the inactivated oxidase by reducing with Na[3H]BH4 the Schiff base formed between proteins and the reagent. Upon SDS-PAGE, the NADPH-inhibitable incorporation is found at the same position as the immunodetectable cytochrome heavy subunit, before and after deglycosylation. Membranes from either activated cells of a cytochrome-deficient X-linked granulomatous disease patient or normal resting cells show no incorporation at this position. Our results provide experimental evidence for the existence on the cytochrome b558 heavy chain of an NADPH-binding site which can only be affinity-labeled by PLP-AMP when the oxidase is active. This suggests the occurrence of a conformational change in the cofactor binding site upon enzyme activation.
Ravel
P
P
CNRS URA 1461, Hôpital Necker, Paris, France.
Lederer
F
F
D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D001853:Bone Marrow / Q000378:metabolism / Q000473:pathology; D001894:Borohydrides / Q000378:metabolism; D003573:Cytochrome b Group / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006105:Granulomatous Disease, Chronic / Q000378:metabolism; D006801:Humans; D007938:Leukemia / Q000378:metabolism / Q000473:pathology; D046911:Macromolecular Substances; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000378:metabolism* / Q000502:physiology; D010084:Oxidation-Reduction; D012545:Schiff Bases
D001894:Borohydrides; D003573:Cytochrome b Group; D046911:Macromolecular Substances; D012545:Schiff Bases; D009249:NADP; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases