Inhibition of the neutrophil NADPH oxidase and associated H+ channel by diethyl pyrocarbonate (DEPC), a histidine-modifying agent: evidence for at least two target sites.
Diethyl pyrocarbonate (DEPC), a histidine-modifying reagent, has been utilized to demonstrate the importance of histidine residues in the functioning of proteins. In previous studies of the NADPH oxidase, histidine residues have been determined to be important in the ability of gp91(phox) to function as an H(+) pathway and in the binding of haem and FAD. We have investigated the ability of DEPC to inhibit H(+) flux and superoxide generation by human neutrophils. Proton flux through the NADPH oxidase-associated H(+) channel was inhibited by DEPC only if applied simultaneously with an activator of the channel. This suggested that the site modified by DEPC is not accessible in the closed channel. Superoxide generation by the NADPH oxidase was also inhibited by DEPC when applied after or simultaneously with the activator. Translocation of the NADPH oxidase cytosolic components, p67(phox) and p47(phox), to the membrane was unaffected by DEPC. In a cell-free system, DEPC-treated membranes failed to support superoxide generation or the reduction of Iodonitrotetrazolium Violet and showed a loss of the characteristic cytochrome b(558) spectrum. Superoxide generation by DEPC-treated cytosol was inhibited slightly. Therefore it can be concluded that there are two sites within the NADPH oxidase that interact with DEPC, one in the H(+) pathway, only accessible in the activated oxidase, and a second accessible prior to activation of the NADPH oxidase. The latter non-proton pathway DEPC site is located within the membrane components of the NADPH oxidase and is associated with the binding of haem in the enzyme complex.
Mankelow
T J
TJ
Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.
Henderson
L M
LM
D003573:Cytochrome b Group / Q000737:chemistry; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D006418:Heme; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000737:chemistry; D007473:Ion Channels / Q000037:antagonists & inhibitors*; D017136:Ion Transport; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010084:Oxidation-Reduction; D011522:Protons; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D013481:Superoxides / Q000378:metabolism; D013778:Tetrazolium Salts / Q000737:chemistry
D003573:Cytochrome b Group; D007202:Indicators and Reagents; D007473:Ion Channels; D011522:Protons; D013778:Tetrazolium Salts; D013481:Superoxides; C013574:iodonitrotetrazolium; D006418:Heme; D006639:Histidine; C036888:cytochrome b558; D019255:NADPH Oxidases; D004047:Diethyl Pyrocarbonate