Original Contributions
Oxidative damage to sarcoplasmic reticulum Ca2+-ATPase at submicromolar iron concentrations: evidence for metal-catalyzed oxidation

https://doi.org/10.1016/S0891-5849(98)00084-7Get rights and content

Abstract

The sarcoplasmic reticulum (SR) calcium ATPase carries out active Ca2+ pumping at the expense of ATP hydrolysis. We have previously described the inhibition of SR ATPase by oxidative stress induced by the Fenton reaction (Fe2+ + H2O2 → HOradical dot + HO + Fe3+). Inhibition was not related to peroxidation of the SR membrane nor to oxidation of ATPase thiols, and involved fragmentation of the ATPase polypeptide chain. The present study aims at further characterizing the mechanism of inhibition of the Ca2+-ATPase by oxygen reactive species at Fe2+ concentrations possibly found in pathological conditions of iron overload. ATP hydrolysis by SR vesicles was inhibited in a dose-dependent manner by micromolar concentrations of Fe2+, H2O2, and ascorbate. Measuring the rate constants of inactivation (kinact) at different Fe2+ concentrations in the presence of saturating concentrations of H2O2 and ascorbate (100 μM each) revealed a saturation profile with half-maximal inactivation rate at ca. 2 μM Fe2+. Inhibition was not affected by addition of 200 μM Ca2+ to the medium, indicating that it was not related to iron binding to the high affinity Ca2+ binding sites in the ATPase. Furthermore, inhibition was not prevented by the water-soluble hydroxyl radical scavengers mannitol or dimethylsulfoxide, nor by butylated hydroxytoluene (a lipid peroxidation blocker) or dithiothreitol (DTT). However, when Cu2+ was used instead of Fe2+ in the Fenton reaction, ATPase inhibition could be prevented by DTT. We propose that functional impairment of the Ca2+-pump may be related to oxidative protein fragmentation mediated by site-specific Fe2+ binding at submicromolar or low micromolar concentrations, which may occur in pathological conditions of iron overload.

Introduction

Several cellular events are regulated by oscillations of cytosolic Ca2+ concentration around 10−7–10−6 M. Abnormally higher intracellular Ca2+ levels have been associated to mechanisms of cell injury promoted by chemical intoxicants and/or by reactive oxygen species generated under ischemia-reperfusion situations.1, 2 Most of the Ca2+ stored in the cell is accumulated in the endo(sarco)plasmic reticulum; thus, disturbances in cellular Ca2+ homeostasis may result from modifications of the permeability or uptake capacity of the reticulum. The sarcoplasmic reticulum (SR) is one of the best studied Ca2+ transport systems from both structural and functional points of view,3, 4, 5, 6 being a very convenient model for investigation of the mechanism(s) of oxidative damage that lead to cytosolic Ca2+ imbalance.

Exposure of SR vesicles to Fe2+/H2O2/ascorbate (a system that generates radical dotOH radicals by Fenton chemistry: Fe2+ + H2O2radical dotOH + OH + Fe3+) caused inhibition of ATP hydrolysis and Ca2+ uptake, which could not be prevented by blocking lipid peroxidation with BHT or by addition of the thiol reducing agent DTT.7 Inhibition was shown to be related to fragmentation of the ATPase polypeptide chain.7 Our present results indicate that inhibition of the SR ATPase involves Fe2+ binding to a specific site in the enzyme, leading to polypeptide chain cleavage, without involving thiol oxidation. According to this mechanism, radical dotOH radicals generated in situ rapidly react with neighboring aminoacid side-chains in the enzyme; thus, low Fe2+ concentrations are required for extensive ATPase inhibition. This type of mechanism has been described as a metal-catalyzed oxidation.8–10 These results also indicate a possible role of SR oxidative damage in the physiopathology of diseases associated with high iron levels in the organism (siderosis), as damage to endo(sarco)plasmic reticulum may occur at submicromolar or low micromolar Fe2+ concentrations possibly reached under pathological conditions.

Section snippets

Enzyme preparation

SR vesicles were isolated from rabbit skeletal muscle.11 The ATPase content in the vesicles was approximately 80% based on total protein content, as indicated by SDS-PAGE stained with Coomasie Blue.

Oxidative stress

SRV (50 μg/ml) were incubated in the presence of 0.5–10 μM Fe(NH3)2SO4, 20–100 μM ascorbate, 20 mM HEPES-K+, pH 7.4, 80 mM KCl. Reaction was started at room temperature by addition of 0–100 μM H2O2, in a final volume of 100 μl, and stopped by dilution of reaction medium in 700 μl of buffer containing

Results

In a previous study, we showed that the SR Ca2+-ATPase was inactivated when exposed to Fe2+, H2O2, and ascorbate.7 However, the concentrations of oxidants used in that study were too high to be of physiopathological relevance. A more detailed investigation of the dependence on Fe2+ concentration revealed that 80% inhibition of ATPase activity was observed when 50 μg/ml (∼0.5 μM) ATPase was incubated for 7 min in the presence of 1 mM H2O2, 1 mM ascorbate, and micromolar Fe2+ concentrations (Fig.

Discussion

We have previously shown7 that impairment of SR ATPase function may occur under oxidative stress produced by Fe2+/H2O2, a system that has been used to perform hydroxylation reactions.17 Because the ATPase is responsible for uptake of Ca2+ into the reticulum, it represents a good model for understanding pathological processes in which cytosolic Ca2+ homeostasis is disrupted.

Although several studies dealing with the SR ATPase and other Ca2+-ATPases have demonstrated that oxidation of critical

Acknowledgements

This work was supported by grants from Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico, Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro, and Programa de Apoio ao Desenvolvimento Cientı́fico e Tecnológico to S. T. F. V. H. M. is supported by Coordenação de Aperfeiçoamento de Pessoal Docente de Ensino Superior. S. T. F. is a Howard Hughes Medical Institute International Research Scholar.

References (38)

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