HYDROXYL RADICAL-MEDIATED REDUCTION OF CA2-ATPASE ACTIVITY OF MASSETER MUSCLE SARCOPLASMIC-RETICULUM()

To understand the effect of oxygen free radicals on Ca2+-ATPase, we us ed sarcoplasmic reticulum (SR) microsomes of canine masseter muscle as a model system in which to explore the effects of oxidation on a biol ogical 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 Ca2+-ATPase activity; in the presence of FeSO4 (0.2 m M), H2O2 inhibited the enzyme activity. Oxygen free radical species ge nerated from H2O2/FeSO4 under the conditions employed in the Ca2+-ATPa se assay were verified by highly sensitive electron spin resonance spe ctroscopy 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)(beta)=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 als o inhibited by sulfhydryl (SH) group reducing agents, cysteine and dit hiothreitol. 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 i n the presence of Fenton's reagent. It is suggested that OH radical-in duced oxidant injury may be caused primarily by modification of the ke y SH group(s) on the ATPase molecule of masseter muscle SR vesicles.