Identification of 30 kDa protein for Ca(2+) releasing action of myotoxin a with a mechanism common to DIDS in skeletal muscle sarcoplasmic reticulum.
The molecular mechanism of Ca(2+) release by myotoxin a (MTYX), a polypeptide toxin isolated from the venom of prairie rattlesnakes (Crotalus viridis viridis), was investigated in the heavy fraction of sarcoplasmic reticulum (HSR) of rabbit skeletal muscles. [(125)I]MYTX bound to four HSR proteins (106, 74, 53 and 30 kDa) on polyvinylidene difluoride (PVDF) membrane. DIDS, 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, bound predominantly to 30 kDa protein on the PVDF membrane, the molecular weight of which was similar to one of the MYTX binding proteins. The maximum (45)Ca(2+) release induced by caffeine (30 mM) was further increased in the presence of MYTX (10 microM) or DIDS (30 microM), whereas that induced by DIDS (30 microM) was not affected by MYTX (10 microM). MYTX inhibited [(3)H]DIDS binding to HSR in a concentration-dependent manner. Furthermore, [(125)I]MYTX binding to 30 kDa protein was inhibited by DIDS in a concentration-dependent manner. These results suggest that MYTX and DIDS release Ca(2+) from HSR in a common mechanism. The 30 kDa protein may be a target protein for the Ca(2+) releasing action of MYTX and DIDS.
Hirata
Y
Y
Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-Ku, Sendai 980-8578, Japan.
Nakahata
N
N
Ohkura
M
M
Ohizumi
Y
Y
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D003435:Crotalid Venoms / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D009124:Muscle Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects
D002121:Calcium Channel Blockers; D003435:Crotalid Venoms; D009124:Muscle Proteins; D002110:Caffeine; C018932:myotoxin A; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium