Properties of specific binding site of myotoxin a, a powerful convulsant, in brain microsomes

Myotoxin a, a small basic polypeptide from prairie rattlesnakes (Crotalus viridis viridis), induces myonecrosis and binds to a single class of binding sites in skeletal muscle sarcoplasmic reticulum. In the present study, [125I]myotoxin a with a high specific activity was prepared and it was shown to bind mainly to microsomes in rat whole brain. [125I]Myotoxin a was further shown to bind to microsomes prepared from all regions tested in brain. Its specific binding to whole brain microsomes was of approximately 1.9 times lower affinity (KD = 0.76 µM; Bmax = 13.1 nmol/mg) than that to skeletal muscle sarcoplasmic reticulum. [125I]Myotoxin a binding to brain microsomes was displaced by unlabeled myotoxin a with an IC50 value of 4.5 µM. [125I]Myotoxin a binding was markedly reduced by treatment of microsomes with trypsin, suggesting that the binding site of [125I]myotoxin a is partially proteins. The binding was significantly inhibited by Mg2+ at concentrations above 1 mM. Having looked at several drugs, we noted that [125I]myotoxin a binding was noncompetitively inhibited by spermine, whereas it was enhanced by heparin. On the other hand, the i.c.v. injection of myotoxin a in mice induced potent convulsive effects at 0.05 nmol/mouse or more. This paper is the first to show that the specific binding site of myotoxin a is present in mouse brain and that myotoxin a is a novel peptidic convulsant in mice.Key words: myotoxin a, specific binding site, brain microsomes, powerful convulsion, central nervous system.

Mechanism of inhibition of Ca2+-ATPase by myotoxin a

The peptide DCRQKWKCCKKGSG [myotoxin-(29-42)], corresponding to residues 29-42 of myotoxin a, inhibits the activity of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum, with a Kd value of 19.4 microM at pH 7.5, in 100 mM KCl. The peptide YKQCHKKGGHCFPKEK, corresponding to residues 1-16 of myotoxin a, is a less potent inhibitor. Inhibition by myotoxin-(29-42) is reduced at low pH and at high ionic strength, suggesting that charge interactions are important in binding to the ATPase. Inhibition of the ATPase has been shown to follow from a decrease in the rate of dephosphorylation, with no effect on the rate of phosphorylation of the ATPase or on the rate of the Ca2+ transport step (E1PCa2-->E2P). Binding of myotoxin-(29-42) decreased the affinity of the ATPase for Ca2+ and Mg2+, and increased the rate of dissociation of the outer Ca2+ ion from the ATPase. Unlike the amphipathic peptide melittin, it is suggested that myotoxin-(29-42) does not bind significantly to the lipid bilayer portion of the sarcoplasmic reticulum. Fluorescence quenching studies suggest that it could bind to the ATPase in the vicinity of Cys-344 in the phosphorylation domain and Lys-515 in the nucleotide binding domain. Inhibition of the ATPase is observed when the ATPase is reconstituted in monomeric form in sealed vesicles, suggesting that aggregation of the ATPase is not involved in inhibition.