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

2001; Elsevier BV; Volume: 1512; Issue: 2 Linguagem: Inglês

10.1016/s0005-2736(01)00341-8

1879-2642

Patrı́cia Souza-Santos, Renata S Ramos, Sérgio T. Ferreira, Paulo C. Carvalho-Alves,

Plant Stress Responses and Tolerance

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 μM of FeSO4 or FeCl3, respectively), 80–90% inhibition of ATP hydrolysis by PMA was observed. Half-maximal inhibition was attained at 25 μM and 50 μM 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 μM Fe2+, but not with 100 μM 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 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 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 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.