Uncompetitive inhibition of superoxide generation by a synthetic peptide corresponding to a predicted NADPH binding site in gp91-phox, a component of the phagocyte respiratory oxidase.
The large subunit of cytochrome b558, gp91-phox, is believed to play a key role in superoxide generation in neutrophils by accepting electrons from NADPH and donating them to molecular oxygen. We found that a peptide corresponding to a predicted NADPH binding site in gp91-phox inhibited superoxide generation in a cell-free system consisting of neutrophil membrane and cytosol. Minimum essential sequence for the inhibition was KSVWYK, which corresponded to residues 420-425 (IC50 = 30 microM). Unlike other peptides known to inhibit the reaction, this peptide was effective even when added to the system after activation or to activated membrane from stimulated neutrophils. Furthermore, the peptide inhibited superoxide generation in a membrane system activated without cytosol. Kinetic analysis revealed that the peptide inhibited the reaction uncompetitively. These results suggest that the peptide combines with the activated cytochrome b558-NADPH complex and thereby inhibits electron transfer from NADPH to molecular oxygen.
Tsuchiya
T
T
Institute of Medical Science, University of Tokyo, Tokyo, 108-8639, Japan.
Imajoh-Ohmi
S
S
Nunoi
H
H
Kanegasaki
S
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003573:Cytochrome b Group / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*; D018375:Neutrophil Activation; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D016897:Respiratory Burst / Q000187:drug effects; D017384:Sequence Deletion; D013481:Superoxides / Q000378:metabolism*
D003573:Cytochrome b Group; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D013481:Superoxides; D009249:NADP; C036888:cytochrome b558; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases