Interaction of an aromatic dibromoisothiouronium derivative with the Ca(2+)-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 (Br(2)-TITU) on the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum. Br(2)-TITU inhibited the Ca(2+)-ATPase, both transport and catalytic activity, with a K(0.5) of 5-15 microM. Maximum inhibition was at 10 min with t(0.5) of 3-5 min. Br(2)-TITU, 100 microM, quenched Trp autofluorescence by 80%, but the residual signal still responded to Ca(2+) binding. Maximum quenching of fluorescence was at pH 9.0. Total E-P levels, during the steady state of turnover of the Ca(2+)-ATPase, were increased from 0.5 to 5.8 nmol x mg(-1) by Br(2)-TITU at pH 6.8. Trinitrophenyl-ATP (TNP-ATP) superfluorescence, which monitors hydrophobicity of the ATP site, was increased 3-4-fold, suggesting that Br(2)-TITU favors an "E2"-like state. Fluorescence was also increased 3-5-fold when E-P was induced with P(i) plus EGTA. Br(2)-TITU increased the rate constants of induction of superfluorescence with ATP plus Ca(2+) from 0.32 to 0.69 s(-1) and with P(i) plus EGTA from 0.84 to 7.45 s(-1). Br(2)-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 P(i) reactions, respectively. Br(2)-TITU, which competitively inhibits the Na(+)/K(+)-ATPase, has a novel effect on the Ca(2+)-ATPase. It promotes accumulation of E2-P species due to increased rate of formation and decreased rate of hydrolysis and quenches tryptophan autofluorescence. Br(2)-TITU could be a useful inhibitor to probe intermediate reactions of the Ca(2+)-ATPase that link catalysis with Ca(2+) translocation.
Berman
M C
MC
Division of Chemical Pathology, Health Science Faculty, University of Cape Town, Observatory 7925, Cape Town, South Africa. mervyn@chempath.uct.ac.za
Karlish
S J
SJ
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D005456:Fluorescent Dyes; D006868:Hydrolysis; D066298:In Vitro Techniques; D007550:Isothiuronium / Q000031:analogs & derivatives* / Q000494:pharmacology*; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D014364:Tryptophan / Q000737:chemistry
C108416:1,3-dibromo-2,4,6-tris-(methylisothiouronium)benzene; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007550:Isothiuronium; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases