THE OXIDATIVE INACTIVATION OF SARCOPLASMIC-RETICULUM CA2-ATPASE BY PEROXYNITRITE()

The oxidative inactivation of rabbit skeletal muscle Ca2+-ATPase in sa rcoplasmic reticulum (SR) vesicles by peroxynitrite (ONOO-) was invest igated. The exposure of SR vesicles (10 mg/ml protein) to low peroxyni trite concentrations (less than or equal to 0.2 mM) resulted in a decr ease of Ca2+-ATPase activity primarily through oxidation of sulfhydryl groups. Most of this deactivation (ca. 70%) could be chemically rever sed by subsequent reduction of the enzyme with either dithiothreitol ( DTT) or sodium borohydride (NaBH4), indicating that free cysteine grou ps were oxidized to disulfides. The initial presence of 5 mM glutathio ne failed to protect the SR Ca2+-ATPase activity. However, as long as peroxynitrite concentrations were kept less than or equal to 0.45 mM, the efficacy of DTT to reverse Ca2+-ATPase inactivation was enhanced f or reaction mixtures which initially contained 5 mM glutathione. At le ast part of the disulfides were formed intermolecularly since gel elec trophoresis revealed protein aggregation which could be reduced under reducing conditions. The application of higher peroxynitrite concentra tions (greater than or equal to 0.45 mM) resulted in Ca2+-ATPase inact ivation which could not be restored by exposure of the modified protei n to reducing agents. On the other hand, treatment of modified protein with NaBH4 recovered all SR protein thiols. This result indicates tha t possibly the oxidation of other amino acids contributes to enzyme in activation, corroborated by amino acid analysis which revealed some ad ditional targets for peroxynitrite or peroxynitrite-induced processes such as Met, Lys, Phe, Thr, Ser, Leu and Tyr. Tyr oxidation was confir med by a significant lower sensitivity of oxidized SR proteins to the Lowry assay. However, neither bityrosine nor nitrotyrosine were formed in significant yields, as monitored by fluorescence spectroscopy and immunodetection, respectively. The Ca2+-ATPase of SR is involved in ce llular Ca2+-homeostasis. Thus, peroxynitrite mediated oxidation of the Ca2+-ATPase might significantly contribute to the loss of Ca2+-homeos tasis observed under biological conditions of oxidative stress.