Sarcoplasmic reticulum Ca2+-ATPase from rabbit skeletal muscle modified by peroxynitrite.

AbstractObjective: Effect of peroxynitrite on SERCA1 activity was studied in correlation with enzyme carbonylation. Kinetic parameters and location of peroxynitrite effect on SERCA1 were determined.Methods: Carbonyls were determined by immunoblotting. FITC, NCD-4 and Trp fluorescence were used to indicate changes in cytosolic and transmembrane regions of SERCA1.Results: Peroxynitrite-concentration-dependent decrease of SERCA1 activity was associated with elevation of protein carbonyls. 4-HNE was not involved in carbonylation of SERCA1. Increased FITC fluorescence in the presence of peroxynitrite correlated with the decrease of the enzyme affinity to ATP.Discussion and conclusion: Peroxynitrite-induced SERCA1 carbonylation that was not accompanied with the formation of 4-HNE-SERCA1 adducts is indicative of direct oxidation of SERCA1. As assessed by FITC fluorescence and decreased affinity of the enzyme to ATP, peroxynitrite impairment was found to occur in the cytosolic ATP-binding region of SERCA1.