Hydroxyl Radical-Mediated Reduction of Ca²⁺-ATPase Activity of Masseter Muscle Sarcoplasmic Reticulum

Abstract

To understand the effect of oxygen free radicals on Ca²⁺-ATPase, we used sarcoplasmic reticulum (SR) microsomes of canine masseter muscle as a model system in which to explore the effects of oxidation on a biological membrane, and we investigated the effect of hydroxyl radicals (·OH) generated from Fenton''s reagent (H₂0₂/FeSO₄). H₂0₂(10 mM) alone had no effect on Ca²⁺ -ATPase activity; in the presence of FeSO₄ (0.2 mM), H₂0₂ inhibited the enzyme activity. Oxygen free radical species generated from H₂0₂/FeSO₄ under the conditions employed in the Ca²⁺ -ATPase assay were verified by highly sensitive electron spin resonance spectroscopy and the spin-trap 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) in the absence of SR vesicles; the 1:2:2:1 quartet (A_N= A_H=1.49 mT), characteristic of the DMPO-OH spin adduct, was observed. The Ca²⁺ -ATPase activity was inversely correlated with the calculated signal intensity of DMPO-OH, which is indicative of the amount of ·OH radical generated. The effect of Fenton''s reagent was effectively inhibited by catalase, dimethylsulfoxide, and dimethylthiourea; the effect was also inhibited by sulfhydryl (SH) group reducing agents, cysteine and dithiothreitol. The SH group modifying agents, p-chloromercuric benzoate and 5, 5'' -dithiobis(2-nitrobenzoic acid) depressed Ca²⁺-ATPase activity; the effects of the SH group modifying agents used were potentiated in the presence of Fenton''s reagent. It is suggested that ·OH radical-induced oxidant injury may be caused primarily by modification of the key SHgroup(s) on the ATPase molecule of masseter muscle SR vesicles.