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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

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Abstract

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.

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Abbreviations

AcP:

acetylphosphate

BHT:

butylhydroxytoluene

DTT:

dithiothreitol

Hepes:

4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid

SDS:

sodium dodecyl sulfate

SDS-PAGE:

polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate

SR:

sarcoplasmic reticulum

SRV:

sarcoplasmic reticulum vesicles

TBA:

thiobarbituric acid

TBARS:

thiobarbituric acid-reactive substances

TFP:

trifluoperazine

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Authors and Affiliations

  1. Departamento de Bioquimíca, Instituto de Biologia, Universidade Estadual de Campinas, SP 13084-100, Campinas

    Roger F. Castilho, Anibal E. Vercesi & Sérgio T. Ferreira

  2. Departamento de Bioquimíca Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, RJ 21941-590, Rio de Janeiro, Brazil

    Paulo C. Carvalho-Alves

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  1. Roger F. Castilho
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  3. Anibal E. Vercesi
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Castilho, R.F., Carvalho-Alves, P.C., Vercesi, A.E. et al. 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. Molecular and Cellular Biochemistry 159, 105–114 (1996). https://doi.org/10.1007/BF00420912

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