Hydroxyl radical-mediated reduction of Ca(2+)-ATPase activity of masseter muscle sarcoplasmic reticulum.
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.
Lee
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Department of Pharmacology, Kanagawa Dental College, Japan.
Okabe
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E
D000251:Adenosine Triphosphatases; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003545:Cysteine / Q000494:pharmacology; D004285:Dogs; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D008406:Masseter Muscle; D010100:Oxygen; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D005609:Free Radicals; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; D003545:Cysteine; D010100:Oxygen