THE SPECIFIC BINDING-SITE OF 9-[H-3]METHYL-7-BROMOEUDISTOMIN-D, A CAFFEINE-LIKE CA2-MICROSOMES IS DISTINCT FROM THAT IN SKELETAL SARCOPLASMIC-RETICULUM( RELEASER, IN LIVER)

H-3-labeled 9-methyl-7-bromoeudistomin D([H-3]MBED), a powerful caffei ne-like Ca2+ releaser, binds to the caffeine binding site of terminal cisternae (TC) of skeletal muscle sarcoplasmic reticulum (SR) (Fang, Y -I., Adachi, M., Kobayashi, J., and Ohizumi, Y. (1993). J. Biol. Chem. 268, 18622-18625.) and activates Ca2+-induced Ca2+ release (CICR). [H -3]MBED, however, bound to rabbit hepatic microsomes with a comparable affinity (K(d) = 50 nM) and with a more than 30-fold greater receptor density (B(max) = 350 pmol/mg of protein), compared with those in SR. Caffeine (0.1-10 mM) caused a concentration dependent inhibition of [ H-3]MBED binding to hepatic microsomes with the IC50 value of 0.3 mM. The mode of inhibition by caffeine was allosteric, indicating that the binding site of the ligand is distinct from but related to that of ca ffeine. Procaine (1-10 mM), a representative inhibitor of CICR, which supresses [H-3]MBED binding to TC-SR, inhibited ligand binding to hepa tic microsomes only slightly. Moreover, ligand binding to the hepatic binding site was not affected by adenosine 5'-(beta,gamma-methylene) t riphosphate (AMP-PCP) (10-100 muM), which is an activator of CICR and potentiates [H-3]MBED binding to TC-SR. Inhibitors of [H-3]MBED bindin g to liver microsomes other than caffeine were nucleotides such as ADP , ATP, GTP, UTP (1 muM), while CTP, cAMP, AMP, adenosine (1 mM), ryano dine (0.1-100 mM) and inositol 1,4,5-trisphosphate (1 muM) were not ef fective. These features of the hepatic microsomal [H-3]MBED binding si te distinguish it from that of skeletal muscle SR. [H-3]MBED, which bi nds to the different sites which are both sensitive to caffeine, is us eful as a probe to investigate the actions of caffeine at the molecula r level.