N-methyl-bromoeudistomin D, a potent Ca++ releaser, binds to the caffeine binding sites in the microsomal membranes from bovine aorta

• Published in: Japanese Journal of Pharmacology Vol. 52; no. suppl-1.1; p. 120
• Main Authors: Fang, Yan-Il, Honda, Hiromi, Kobayashi, Jun’ichi, Ohizumi, Yasushi
• Format: Journal Article
• Language: English
• Published: The Japanese Pharmacological Society 1990
• ISSN: 0021-5198; 1347-3506
• DOI: 10.1016/S0021-5198(19)55187-3

We have previously reported that N-methyl-bromoeudistomin D(MBED) specificially binds to the caffeine binding sites on Ca^++ release channels in sarcoplasmic reticulum(SR) of skeletal muscle. In the present study, we have characterized the properties of MBED binding to the microsomal membranes prepared from bovine aorta. [^^3 H]MBED(10.2 Ci/mmol) binding was inhibited by unlabeled MBED with an IC_50 value of 0.8 μM. The specific binding was saturable and of high affinity (dissociation constant=50 nM, binding capacity=20 pmol/mg). Caffeine inhibited [^^3 H]MBED binding to the microsomal membranes with an IC_50 value of 0.12 mM. Scatchard analysis of [^^3 H]MBED binding revealed a competitive mode of inhibition of MBED binding by caffeine. These results suggest that MBED shares the same binding sites as that of caffeine in the microsomal membranes prepared from aortic smooth muscle. Thus, MBED may provide an essential biochemical tool for the identification or the characterization of the caffeine binding sites.