Identification of a 97-kDa mastoparan-binding protein involving in Ca(2+) release from skeletal muscle sarcoplasmic reticulum.
Mastoparan (MP) and radiolabeled [Tyr(3)]MP caused a transient Ca(2+) release from the heavy fraction of sarcoplasmic reticulum, which was inhibited by ryanodine. MP enhanced [(3)H]ryanodine binding in a concentration-dependent manner with an EC(50) value of approximately 0.3 microM. The (45)Ca(2+) release was accelerated by MP, [Tyr(3)]MP, or caffeine in a concentration-dependent manner. The EC(50) values for MP, [Tyr(3)]MP, and caffeine were approximately 2. 0 microM, 7.7 microM, and 1.8 mM, respectively. MP, like caffeine, shifted the stimulatory limb of a bell-shaped curve of Ca(2+) dependence to the left. (45)Ca(2+) release induced by caffeine was completely inhibited by typical blockers of Ca(2+)-induced Ca(2+) release, such as Mg(2+), ruthenium red, or procaine. However, (45)Ca(2+) release induced by MP was completely inhibited by Mg(2+), but it was only partially inhibited by ruthenium red or procaine. The rate of (45)Ca(2+) release induced by MP was further increased in the presence of caffeine, showing that the MP binding site is different from that of caffeine on Ca(2+) release channels. We succeeded in the synthesis of (125)I-[Tyr(3)]MP with a high specific activity. (125)I-[Tyr(3)]MP bound specifically to heavy fraction of sarcoplasmic reticulum with a K(d) value of 4.0 microM and a B(max) value of 3.0 nmol/mg. Furthermore, (125)I-[Tyr(3)]MP specifically cross-linked to the 97-kDa protein without direct binding to ryanodine receptor. The protein was not triadin or Ca(2+)-pump, because antitriadin antibody and anti-Ca(2+)-pump antibody did not immunoprecipitate the protein. These results suggest that the 97-kDa MP-binding protein may have an important role in the excitation-contraction coupling of skeletal muscle.
Hirata
Y
Y
Department of Pharmaceutical Molecular Biology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-Ku, Sendai, Japan.
Nakahata
N
N
Ohizumi
Y
Y
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D066298:In Vitro Techniques; D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D008970:Molecular Weight; D009124:Muscle Proteins / Q000032:analysis / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism; D010455:Peptides; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D014862:Wasp Venoms / Q000138:chemical synthesis / Q000378:metabolism*
D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D009124:Muscle Proteins; D010455:Peptides; D019837:Ryanodine Receptor Calcium Release Channel; D014862:Wasp Venoms; C037500:mastoparan; D002118:Calcium