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 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.
Souza-Santos
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Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, 21941-590, Rio de Janeiro, Brazil.
Ramos
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Ferreira
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Carvalho-Alves
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D002084:Butylated Hydroxytoluene / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology*; D002712:Chlorides; D004229:Dithiothreitol / Q000494:pharmacology; D005290:Ferric Compounds / Q000494:pharmacology*; D005296:Ferrous Compounds / Q000494:pharmacology*; D007700:Kinetics; D015227:Lipid Peroxidation / Q000187:drug effects*; D008861:Microsomes / Q000201:enzymology*; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000494:pharmacology; D018517:Plant Roots / Q000201:enzymology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis; D003313:Zea mays / Q000201:enzymology*
D002712:Chlorides; D005290:Ferric Compounds; D005296:Ferrous Compounds; D010618:Phenanthrolines; D017392:Thiobarbituric Acid Reactive Substances; D002084:Butylated Hydroxytoluene; C020748:ferrous sulfate; D006180:Proton-Translocating ATPases; D004229:Dithiothreitol; C024555:ferric chloride; C025205:1,10-phenanthroline