Iron-mediated inhibition of H+-ATPase in plasma membrane vesicles isolated from wheat roots.
The mechanisms of iron-mediated inhibition of the H(+)-ATPase activity of plasma membrane (PM) vesicles isolated from wheat roots were investigated. Both FeSO(4) and FeCl(3) significantly inhibited PM H(+)-ATPase activity, and the inhibition could be reversed by the addition of the metal ion chelator EDTA-Na(2) or a specific Fe(2+) chelator, indicating that the inhibitory effect was due to specific action of Fe(2+) or Fe(3+). Measurement of the extent of lipid peroxidation showed that oxidative damage on the PM caused by Fe(2+) or Fe(3+) seemed to be correlated with the inhibition of PM H(+)-ATPase activity. However, prevention of lipid peroxidation with butylated hydroxytoluene did not affect iron-mediated inhibition in the PM H(+)-ATPase, suggesting that the inhibition of the PM H(+)-ATPase was not a consequence of lipid peroxidation caused by iron. Investigation of the effects of various reactive oxygen species scavengers on the iron-mediated inhibition of H(+)-ATPase activity indicated that hydroxyl radicals (*OH) and hydrogen peroxide (H(2)O(2)) might be involved in the Fe(2+)-mediated decrease in PM H(+)-ATPase activity. Moreover, iron caused a decrease in plasma protein thiol (P-SH), and Fe(3+) brought a higher degree of oxidation in thiol groups than Fe(2+) at the same concentration. Modification of the thiol redox state in the PM suggested that reducing thiol groups were essential to maintain PM H(+)-ATPase activity. Incubation of the specific thiol modification reagent 5,5-dithio-bis(2-nitrobenzoic acid) with the rightside-out and inside-out PM revealed that thiol oxidation occurred at the apoplast side of the PM. Western blotting analysis revealed a decrease in H(+)-ATPase content caused by iron. Taken together, these results suggested that thiol oxidation might account for the inhibition of PM H(+)-ATPase caused by iron, and that *OH and H(2)O(2) were also involved in Fe(2+)-mediated inhibition.
Yang
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Key Laboratory of Desert and Desertification, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou 730000, China.
Zhang
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He
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Wang
X M
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Zhang
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LX
D002462:Cell Membrane / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism; D007501:Iron / Q000494:pharmacology*; D007700:Kinetics; D015227:Lipid Peroxidation / Q000187:drug effects; D010084:Oxidation-Reduction; D018517:Plant Roots / Q000201:enzymology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014908:Triticum / Q000201:enzymology*
D004791:Enzyme Inhibitors; D013438:Sulfhydryl Compounds; D013481:Superoxides; D006861:Hydrogen Peroxide; D007501:Iron; D006180:Proton-Translocating ATPases