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

摘要

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 Fe~(2+) or Fe~(3+) (100-200 μM of FeSO_4 or FeCl_3, respectively), 80-90% inhibition of ATP hydrolysis by PMA was observed. Half-maximal inhibition was attained at 25 μM and 50 μM for Fe~(2+) and Fe~(3+), 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 μM Fe~(2+), but not with 100 μM Fe~(3+), 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 MgCl_2, reduced the extent of irreversible inhibition of the H~+-ATPase. Addition of 40 μM 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 μM Fe~(2+). Interestingly,preservation of the 100 kDa band was observed when CRMF were exposed to Fe~(2+) in the presence of either 5 mM dithiothreitol or 40 μM 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.