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Hydroxyl radical-mediated reduction of Ca(2+)-ATPase activity of masseter muscle sarcoplasmic reticulum
Abstract:
To understand the effect of oxygen free radicals on Ca(2+)-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 (H2O2/FeSO4). H2O2 (10 mM) alone had no effect on Ca(2+)-ATPase activity; in the presence of FeSO4 (0.2 mM), H2O2 inhibited the enzyme activity. Oxygen free radical species generated from H2O2/FeSO4 under the conditions employed in the Ca(2+)-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 (AN = A beta H = 1.49 mT), characteristic of the DMPO-OH spin adduct, was observed. The Ca(2+)-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 Ca(2+)-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 SH group(s) on the ATPase molecule of masseter muscle SR vesicles.
Insights
Oxygen free radicals, generated by Fenton's reagent, inhibit Ca(2+)-ATPase activity in canine masseter muscle. This damage is primarily caused by hydroxyl radical modification of key sulfhydryl groups on the ATPase.
Area of Science:
- Biochemistry
- Membrane Biology
- Oxidative Stress
Background:
- Sarcoplasmic reticulum (SR) Ca(2+)-ATPase is crucial for muscle function.
- Oxidative stress can impair enzyme activity and membrane integrity.
- Understanding radical damage mechanisms is vital for cellular health.
Purpose of the Study:
- To investigate the impact of hydroxyl radicals on Ca(2+)-ATPase activity.
- To elucidate the role of sulfhydryl groups in radical-induced enzyme inhibition.
- To utilize canine masseter muscle SR microsomes as a model system.
Main Methods:
- Generation of hydroxyl radicals using Fenton's reagent (H2O2/FeSO4).
- Assay of Ca(2+)-ATPase activity in SR microsomes.
- Electron spin resonance (ESR) spectroscopy with DMPO spin-trap to detect hydroxyl radicals.
- Assessment of inhibition by antioxidants and sulfhydryl-reducing agents.
Main Results:
- Fenton's reagent significantly inhibited Ca(2+)-ATPase activity.
- Hydroxyl radical generation was confirmed by ESR spectroscopy.
- Enzyme inhibition correlated inversely with hydroxyl radical levels.
- Catalase, DMSO, and sulfhydryl reducing agents protected against inhibition.
- SH group modifying agents potentiated Fenton's reagent effects.
Conclusions:
- Hydroxyl radicals induce oxidant injury to Ca(2+)-ATPase.
- Damage likely results from modification of critical sulfhydryl groups on the enzyme.
- SR Ca(2+)-ATPase is susceptible to oxidative damage in muscle tissue.