Abstract
Mastoparan (MP) and radiolabeled [Tyr3]MP caused a transient Ca2+ release from the heavy fraction of sarcoplasmic reticulum, which was inhibited by ryanodine. MP enhanced [3H]ryanodine binding in a concentration-dependent manner with an EC50 value of approximately 0.3 μM. The45Ca2+ release was accelerated by MP, [Tyr3]MP, or caffeine in a concentration-dependent manner. The EC50 values for MP, [Tyr3]MP, and caffeine were approximately 2.0 μM, 7.7 μM, and 1.8 mM, respectively. MP, like caffeine, shifted the stimulatory limb of a bell-shaped curve of Ca2+ dependence to the left.45Ca2+ release induced by caffeine was completely inhibited by typical blockers of Ca2+-induced Ca2+ release, such as Mg2+, ruthenium red, or procaine. However, 45Ca2+ release induced by MP was completely inhibited by Mg2+, but it was only partially inhibited by ruthenium red or procaine. The rate of45Ca2+ 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 Ca2+ release channels. We succeeded in the synthesis of125I-[Tyr3]MP with a high specific activity.125I-[Tyr3]MP bound specifically to heavy fraction of sarcoplasmic reticulum with a Kdvalue of 4.0 μM and a Bmax value of 3.0 nmol/mg. Furthermore, 125I-[Tyr3]MP specifically cross-linked to the 97-kDa protein without direct binding to ryanodine receptor. The protein was not triadin or Ca2+-pump, because antitriadin antibody and anti-Ca2+-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.
Footnotes
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Send reprint requests to: Professor Norimichi Nakahata, Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-Ku, Sendai 980-8578, Japan. E-mail: nakahata{at}mail.pharm.tohoku.ac.jp
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This work was partially supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (Y.H.) and Grant-in-Aid for Scientific Research from the Ministry of Education, Sciences and Culture of Japan (N.N. and Y.O.).
- Abbreviations:
- RyR
- ryanodine receptor
- SR
- sarcoplasmic reticulum
- MP
- mastoparan
- HSR
- heavy fraction of sarcoplasmic reticulum
- SR Ca2+-pump
- sarco/endoplasmic reticulum Ca2+-ATPase
- Sulfo-SANPAH
- sulfosuccinimidyl-6-[4′-azido-2′-nitrophenylamino] hexanoate
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- Fluo-3
- 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid
- PAGE
- polyacrylamide gel electrophoresis
- MOPS
- 3-(N-morpholino)propanesulfonic acid
- Ck
- creatinine kinase
- Received August 30, 1999.
- Accepted February 15, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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