caa a22 4500 477119123 CHVBK 20180405111631.0 cr unu---uuuuu 170330e19960901xx s 000 0 eng 10.1007/BF00420912 doi (NATIONALLICENCE)springer-10.1007/BF00420912 Oxidative damage to sarcoplasmic reticulum Ca2+-pump induced by Fe2+/H2O2/ascorbate is not mediated by lipid peroxidation or thiol oxidation and leads to protein fragmentation [Elektronische Daten] [Roger Castilho, Paulo Carvalho-Alves, Anibal Vercesi, Sérgio Ferreira] 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 (Fe2+ + 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 μM Fe2+ and 6 mM ascorbate. Ca2+ uptake carried out by the Ca2+-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 Ca2+-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 Ca2+-ATPase. Electrophoretic analysis of oxidized Ca2+-ATPase revealed a decrease in intensity of the silver-stained 110 kDa Ca2+-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 Ca2+-pump may be related to aminoacid oxidation and fragmentation of the protein. Kluwer Academic Publishers, 1996 sarcoplasmic reticulum nationallicence Ca2+-ATPase nationallicence free radicals nationallicence protein oxidation nationallicence lipid peroxidation nationallicence AcP : acetylphosphate nationallicence BHT : butylhydroxytoluene nationallicence DTT : dithiothreitol nationallicence Hepes : 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid nationallicence SDS : sodium dodecyl sulfate nationallicence SDS-PAGE : polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate nationallicence SR : sarcoplasmic reticulum nationallicence SRV : sarcoplasmic reticulum vesicles nationallicence TBA : thiobarbituric acid nationallicence TBARS : thiobarbituric acid-reactive substances nationallicence TFP : trifluoperazine nationallicence Castilho Roger Departamento de Bioquimíca, Instituto de Biologia, Universidade Estadual de Campinas, SP 13084-100, Campinas aut Carvalho-Alves Paulo Departamento de Bioquimíca Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, RJ 21941-590, Rio de Janeiro, Brazil aut Vercesi Anibal Departamento de Bioquimíca, Instituto de Biologia, Universidade Estadual de Campinas, SP 13084-100, Campinas aut Ferreira Sérgio Departamento de Bioquimíca, Instituto de Biologia, Universidade Estadual de Campinas, SP 13084-100, Campinas aut Molecular and Cellular Biochemistry Kluwer Academic Publishers 159/2(1996-09-01), 105-114 0300-8177 159:2<105 1996 159 11010 https://doi.org/10.1007/BF00420912 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00420912 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Castilho Roger Departamento de Bioquimíca, Instituto de Biologia, Universidade Estadual de Campinas, SP 13084-100, Campinas aut NATIONALLICENCE 700 1- Carvalho-Alves Paulo Departamento de Bioquimíca Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, RJ 21941-590, Rio de Janeiro, Brazil aut NATIONALLICENCE 700 1- Vercesi Anibal Departamento de Bioquimíca, Instituto de Biologia, Universidade Estadual de Campinas, SP 13084-100, Campinas aut NATIONALLICENCE 700 1- Ferreira Sérgio Departamento de Bioquimíca, Instituto de Biologia, Universidade Estadual de Campinas, SP 13084-100, Campinas aut NATIONALLICENCE 773 0- Molecular and Cellular Biochemistry Kluwer Academic Publishers 159/2(1996-09-01), 105-114 0300-8177 159:2<105 1996 159 11010 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer