The properties of specific binding site of 125I-radioiodinated myotoxin a, a novel Ca++ releasing agent, in skeletal muscle sarcoplasmic reticulum.
It was found that myotoxin a (MYTX), which is isolated from prairie rattlesnakes (Crotalus viridis viridis), is a powerful Ca++ releaser in the heavy fraction of sarcoplasmic reticulum (HSR). 125I-labeled MYTX (125I-MYTX), which has high Ca(++)-releasing ability, was successfully prepared. It specifically bound to a single class of binding sites in HSR with a KD of 0.4 microM and Bmax of 6 nmol/mg of protein. 125I-MYTX binding was markedly inhibited by Na+ and K+, whereas it was little affected by Ca++ and Mg++. The binding activity was markedly decreased by spermine, a blocker of Ca++ releasing channels, and was not affected by the other modulators of Ca++ release such as caffeine, procaine or ruthenium red. Spermine decreased the binding in a concentration-dependent manner with the IC50 value of 20 microM. Scatchard analysis of 125I-MYTX binding indicated that the Bmax value was decreased by spermine, although the KD value was not changed, which indicates a noncompetitive mode of inhibition. 125I-MYTX did not bind to the purified ryanodine receptor. Ca++ electrode experiments indicated that MYTX induced Ca++ release from HSR at doses of 0.1 microM or more; this was abolished by spermine. The maximal response to MYTX (10 microM) was further increased by caffeine (10 mM) in 45Ca++ release, which probably indicates that the effects of MYTX and caffeine are synergistic in Ca(++)-releasing action. These results suggest that MYTX binds to an important regulatory protein of Ca++ release, which is not the ryanodine receptor.
Ohkura
M
M
Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Furukawa
K
K
Oikawa
K
K
Ohizumi
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D003435:Crotalid Venoms / Q000378:metabolism*; D004563:Electrochemistry; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D011817:Rabbits; D011869:Radioligand Assay; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D003435:Crotalid Venoms; D007457:Iodine Radioisotopes; C018932:myotoxin A; D002118:Calcium