Oxidative damage to sarcoplasmic reticulum Ca(2+)-pump induced by Fe2+/H2O2/ascorbate is not mediated by lipid peroxidation or thiol oxidation and leads to protein fragmentation.
The major protein in the sarcoplasmic reticulum (SR) membrane is the Ca2+ transporting ATPase which carries out active Ca2+ pumping at the expense of ATP hydrolysis. The aim of this work was to elucidate the mechanisms by which oxidative stress induced by Fenton's reaction (Fe(2+)+H2O2-->HO.+OH-+Fe3+) alters the function of SR. ATP hydrolysis by both SR vesicles (SRV) and purified ATPase was inhibited in a dose-dependent manner in the presence of 0-1.5 mM H2O2 plus 50 microM Fe2+ and 6 mM ascorbate. Ca2+ uptake carried out by the Ca(2+)-ATPase in SRV was also inhibited in parallel. The inhibition of hydrolysis and Ca2+ uptake was not prevented by butylhydroxytoluene (BHT) at concentrations which significantly blocked formation of thiobarbituric acid-reactive substances (TBARS), suggesting that inhibition of the ATPase was not due to lipid peroxidation of the SR membrane. In addition, dithiothreitol (DTT) did not prevent inhibition of either ATPase activity or Ca2+ uptake, suggesting that inhibition was not related to oxidation of ATPase thiols. The passive efflux of 45Ca2+ from pre-loaded SR vesicles was greatly increased by oxidative stress and this effect could be only partially prevented (ca 20%) by addition of BHT or DTT. Trifluoperazine (which specifically binds to the Ca(2+)-ATPase, causing conformational changes in the enzyme) fully protected the ATPase activity against oxidative damage. These results suggest that the alterations in function observed upon oxidation of SRV are mainly due to direct effects on the Ca(2+)-ATPase. Electrophoretic analysis of oxidized Ca(2+)-ATPase revealed a decrease in intensity of the silver-stained 110 kDa Ca(2+)-ATPase band and the appearance of low molecular weight peptides (MW < 100 kDa) and high molecular weight protein aggregates. Presence of DTT during oxidation prevented the appearance of protein aggregates and caused a simultaneous increase in the amount of low molecular weight peptides. We propose that impairment of function of the Ca(2+)-pump may be related to aminoacid oxidation and fragmentation of the protein.
Castilho
R F
RF
Departamento de Bioquimica, Universidade Estadual de Campinas, Rio de Janeiro, Brazil.
Carvalho-Alves
P C
PC
Vercesi
A E
AE
Ferreira
S T
ST
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002084:Butylated Hydroxytoluene / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002458:Cell Fractionation; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007501:Iron / Q000494:pharmacology; D015227:Lipid Peroxidation; D018482:Muscle, Skeletal; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014268:Trifluoperazine / Q000494:pharmacology
D000975:Antioxidants; C045076:Fenton's reagent; D013438:Sulfhydryl Compounds; D002084:Butylated Hydroxytoluene; D014268:Trifluoperazine; D006861:Hydrogen Peroxide; D007501:Iron; D000252:Calcium-Transporting ATPases; D001205:Ascorbic Acid; D002118:Calcium; D004229:Dithiothreitol