MECHANISM OF INHIBITION OF CA2-ATPASE BY MYOTOXIN A()

The peptide DCRQKWKCCKKGSG [myotoxin-(29-42)], corresponding to residu es 29-42 of myotoxin a, inhibits the activity of the Ca2+-ATPase of sk eletal muscle sarcoplasmic reticulum, with a K-d value of 19.4 mu M 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 b y myotoxin-(29-42) is reduced at low pH and at high ionic strength, su ggesting that charge interactions are important in binding to the ATPa se. Inhibition of the ATPase has been shown to follow from a decrease in the rate of dephosphorylation, with no effect on the rate of phosph orylation of the ATPase or on the rate of the Ca2+ transport step (E1P Ca(2) --> E2P). Binding of myotoxin-(29-42) decreased the affinity of the ATPase for Ca2+ and Mg2+, and increased the rate of dissociation o f the outer Ca2+ ion from the ATPase. Unlike the amphipathic peptide m elittin, it is suggested that myotoxin-(29-42) does not bind significa ntly to the lipid bilayer portion of the sarcoplasmic reticulum. Fluor escence quenching studies suggest that it could bind to the ATPase in the vicinity of Cys-344 in the phosphorylation domain and Lys-515 in t he nucleotide binding domain. Inhibition of the ATPase is observed whe n the ATPase is reconstituted in monomeric form in sealed vesicles, su ggesting that aggregation of the ATPase is not involved in inhibition.