| 11406113 |
Souza-Santos P, Ramos RS, Ferreira ST, Carvalho-Alves PC: Iron-induced oxidative damage of corn root plasma membrane H (+)-ATPase. . Biochim Biophys Acta. 2001 Jun 6;1512(2):357-66.
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 microM of FeSO (4) or FeCl (3), respectively), 80-90% inhibition of ATP hydrolysis by PMA was observed. Half-maximal inhibition was attained at 25 microM and 50 microM 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 microM Fe (2+), but not with 100 microM 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 microM 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 microM 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 microM 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. |
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