title: High affinity binding of 9-[3H]methyl-7-bromoeudistomin D to the caffeine-binding site of skeletal muscle sarcoplasmic reticulum.
abstract: 3H-Labeled 9-methyl-7-bromoeudistomin D ([3H] MBED), the most powerful inducer of Ca2+ release from sarcoplasmic reticulum (SR), was successfully prepared with a high specific activity of 10.2 Ci/mmol. [3H]MBED bound to terminal cisternae (TC) of skeletal muscle SR in a replacable and saturable manner, indicating the existence of its specific binding site. Caffeine inhibited the [3H]MBED binding to the TC-SR membranes from skeletal muscle with an IC50 value of 0.8 mM, in close agreement with a concentration that causes Ca2+ release from SR. Scatchard analysis gave values of KD = 40 nM and Bmax = 10 pmol/mg protein. The KD value was increased by caffeine, while that of Bmax was not changed, indicating a competitive mode of inhibition. Adenosine 5'-(beta, gamma-methylene)triphosphate enhanced [3H]MBED binding, but ryanodine and Ca2+ did not affect it. [3H]MBED binding to TC-SR membranes was inhibited by procaine, a representative blocker of Ca(2+)-induced Ca2+ release channels, whereas that was not changed by Mg2+, suggesting that procaine but not Mg2+ may exert its inhibitory effect on Ca(2+)-induced Ca2+ release by affecting the caffeine-binding sites. These results suggest that MBED shares the same binding site as that of caffeine in TC-SR. The [3H]MBED is the first radiolabeled ligand for caffeine-binding sites in Ca2+ release channels and thus may provide an essential biochemical tool for elucidating this site.
authors.affiliation: Department of Pharmaceutical Molecular Biology, Tohoku University, Sendai, Japan.
authors.forename: Y I
authors.initials: YI
authors.lastname: Fang
authors.forename: M
authors.initials: M
authors.lastname: Adachi
authors.forename: J
authors.initials: J
authors.lastname: Kobayashi
authors.forename: Y
authors.initials: Y
authors.lastname: Ohizumi
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002110:Caffeine / Q000378:metabolism* / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002243:Carbolines / Q000378:metabolism* / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000187:drug effects / Q000378:metabolism*; D011343:Procaine / Q000494:pharmacology; D011817:Rabbits; D012430:Ruthenium Red / Q000494:pharmacology; D012433:Ryanodine / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*
chemical_list: D002243:Carbolines; D012430:Ruthenium Red; C060741:9-methyl-7-bromoeudistomin D; D012433:Ryanodine; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D002110:Caffeine; D011343:Procaine; D000255:Adenosine Triphosphate; D008274:Magnesium; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D002118:Calcium