Interaction of an Aromatic Dibromoisothiouronium Derivative with the Ca2+-ATPase of Skeletal Muscle Sarcoplasmic Reticulum

Isothiouronium compounds [Hoving, S., Bar-Shimon, M., Tijmes, J. J., Goldshleger, R., Tal, D. M., and Karlish, S. J. (1995) J. Biol. Chem. 270, 29788−29793] act as high-affinity competitive antagonists for Na+ and K+ (Rb+) on the renal Na+/K+-ATPase where they favor the E1 conformation. We have now characterized the effects of 1,3-dibromo-2,4,6-tris(methylisothiouronium)benzene (Br2-TITU) on the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum. Br2-TITU inhibited the Ca2+-ATPase, both transport and catalytic activity, with a K 0.5 of 5−15 μM. Maximum inhibition was at 10 min with t 0.5 of 3−5 min. Br2-TITU, 100 μM, quenched Trp autofluorescence by 80%, but the residual signal still responded to Ca2+ binding. Maximum quenching of fluorescence was at pH 9.0. Total E-P levels, during the steady state of turnover of the Ca2+-ATPase, were increased from 0.5 to 5.8 nmol·mg-1 by Br2-TITU at pH 6.8. Trinitrophenyl-ATP (TNP-ATP) superfluorescence, which monitors hydrophobicity of the ATP site, was increased 3−4-fold, suggesting that Br2-TITU favors an “E2”-like state. Fluorescence was also increased 3−5-fold when E-P was induced with Pi plus EGTA. Br2-TITU increased the rate constants of induction of superfluorescence with ATP plus Ca2+ from 0.32 to 0.69 s-1 and with Pi plus EGTA from 0.84 to 7.45 s-1. Br2-TITU also decreased rate constants for “off” reactions from 2.9 to 0.66 s-1 and from 10.9 to 0.73 s-1 for the ATP and Pi reactions, respectively. Br2-TITU, which competitively inhibits the Na+/K+-ATPase, has a novel effect on the Ca2+-ATPase. It promotes accumulation of E2-P species due to increased rate of formation and decreased rate of hydrolysis and quenches tryptophan autofluorescence. Br2-TITU could be a useful inhibitor to probe intermediate reactions of the Ca2+-ATPase that link catalysis with Ca2+ translocation.