Iron-induced oxidative damage of corn root plasma membrane H+-ATPase

The effect of iron on the activity of the plasma membrane H+-ATPase (PMA) from corn root microsomal fraction (CRMF) was investigated. In the presence of either Fe2+ or Fe3+ (100-200 muM of FeSO4 or FeCl3, respectively), 80-90% inhibition of ATP hydrolysis by PMA was observed. Half-maximal inhibition was attained at 25 muM and 50 muM for Fe2+ and Fe3+, respectively. Inhibition of the ATPase activity was prevented in the presence of metal ion chelators such as EDTA, deferoxamine or o-phenanthroline in the incubation medium. However, preincubation of CRMF in the presence of 100 muM Fe2+, but not with 100 muM Fe3+, rendered the ATPase activity (measured in the presence of excess EDTA) irreversibly inhibited. Inhibition was also observed using a preparation further enriched in plasma membranes by gradient centrifugation. Addition of 0.5 mM ATP to the preincubation medium, either in the presence or in the absence of 5 mM MgCl2, reduced the extent of irreversible inhibition of the H+-ATPase. Addition of 40 muM butylated hydroxytoluene and/or 5 mM dithiothreitol, or deoxygenation of the incubation medium by bubbling a stream of argon in the solution, also caused significant protection of the ATPase activity against irreversible inhibition by iron. Western blots of CRMF probed with a polyclonal antiserum against the yeast plasma membrane H+-ATPase showed a 100 kDa cross-reactive band, which disappeared in samples previously exposed to 500 muM Fe2+. Interestingly, preservation of the 100 kDa band was observed when CRMF were exposed to Fe2+ in the presence of either 5 mM dithiothreitol or 40 muM butylated hydroxytoluene. These results indicate that iron causes irreversible inhibition of the corn root plasma membrane H+-ATPase by oxidation of sulfhydryl groups of the enzyme following lipid peroxidation. (C) 2001 Published by Elsevier Science B.V.