Phagocyte NADPH-oxidase. Studies with flavin analogues as active site probes in triton X-100-solubilized preparations.
NADPH-oxidase of stimulated human neutrophil membranes was solubilized in Triton X-100 and activity reconstituted with FAD, 8-F-FAD, 8-phenyl-S-FAD, and 8-S-FAD. The enzyme had similar affinities for all the flavins with Km values in the 60-80 nM range. Vmax was found to increase 4-fold with increasing redox midpoint potential of the flavin. 8-F-FAD reconstituted with the enzyme was reactive toward thiophenol, suggesting exposure of the 8-position to solvent, a finding supported by unsuccessful attempts to label the enzyme with the photoaffinity probe 8-N3-[32P]FAD. Solubilized oxidase stabilized the red thiolate form of 8-S-FAD, a characteristic of flavoproteins of the dehydrogenase/electron transferase classes which stabilize the blue neutral form of the flavin semiquinone radical.
Parkinson
J F
JF
Indiana Elks Cancer Center, Department of Medicine, Indiana University School of Medicine, Indianapolis 46223.
Gabig
T G
TG
D002462:Cell Membrane / Q000201:enzymology; D003902:Detergents; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000097:blood* / Q000302:isolation & purification; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D017830:Octoxynol; D010586:Phagocytes / Q000201:enzymology; D011092:Polyethylene Glycols; D011485:Protein Binding; D012995:Solubility
D003902:Detergents; D005182:Flavin-Adenine Dinucleotide; D011092:Polyethylene Glycols; D017830:Octoxynol; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases