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Identification of the NADPH‐binding protein of the neutrophil superoxide‐generating oxidase of guinea pigs
Author(s) -
Ge F.,
Guillory RJ
Publication year - 1994
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1994.tb00289.x
Subject(s) - superoxide , nadph oxidase , cytosol , gtp' , chemistry , biochemistry , membrane , oxidase test , reactive oxygen species , enzyme
A cell‐free system prepared from polymorphonuclear neutrophils is capable of NADPH‐dependent generation of superoxide anion, but requires the simultaneous presence of plasma membranes, cytosol, arachidonate and guanosine 5′‐[gamma‐thio]triphosphate (GTP[S]). The isolated membranes from such a preparation are able to catalyse NADPH‐dependent superoxide formation independently of added cytosol and activators. Such activated membranes, activated in the cell‐free system, must consequently contain all of the essential components required by the oxidase for superoxide formation, including the NADPH‐binding component. Arylazido‐beta‐alanyl‐[32P]NADPH (3′‐O‐(3‐[N‐(4‐azido‐2‐nitrophenyl)‐amino] propionyl)‐[32P]NADPH), an NADPH analogue and photoaffinity probe, is shown to act in the dark as a substrate for the oxidase activity in the activated membranes and an irreversible photodependent inhibitor following photoirradiation. The photoaffinity probe has been used to identify the specific NADPH‐binding component of the oxidase in the activated membranes. In contrast with the sensitivity of the activated membranes, photoirradiation of arylazido‐beta‐alanyl‐[32P]‐NADPH under identical conditions, but with non‐activated membranes, did not prevent subsequent activation of the treated membranes by cytosol, arachidonate and GTP[S]. However, photoirradiation of the cytosolic fraction in the presence of arylazido‐beta‐alanyl‐[32P]NADPH resulted in an inhibition of the cytosol's ability to activate superoxide generation upon subsequent incubation with plasma membranes in the presence of arachidonate and GTP[S]. These observations are taken as a strong indication that the NADPH‐binding protein of the oxidase is a cytosolic factor which associates with the plasma membrane upon activation. The superoxide‐generating activity of the activated membranes was inhibited irreversibly in a concentration‐ and photo‐dependent manner by arylazido‐beta‐alanyl‐NADPH. The arylazido‐beta‐alanyl‐NADPH‐photodependent inhibition of superoxide generation in the activated membranes correlated with the photodependent labelling of a protein of 55.6 kDa by the arylazido‐beta‐alanyl‐NADPH. Specificity of labelling was indicated by a lack of labelling of the 55.6 kDa region in the non‐activated membranes and protection of the photodependent inhibition and labelling of the 55.6 kDa protein by NADPH. It is proposed that the arylazido‐beta‐alanyl‐NADPH‐labelled 55.6 kDa protein present on the activated membranes is the NADPH‐binding protein of the neutrophil superoxide‐generating oxidase.