Mechanism of inhibition of Ca2+-ATPase by myotoxin a

Abstract

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.

An investigation of the mechanism of inhibition of the Ca2+-ATPase by phospholamban

“…We have shown that Cys-containing peptides can quench the fluorescence of the ATPase labelled with a variety of fluorescence probes [38]. Figure 2(B) shows the effect of PLB(25-52) on the intensity of the tryptophan fluorescence of the ATPase, as a function of the molar ratio of peptide to ATPase at a fixed molar ratio of DOPC to ATPase of 2000 : 1.…”

“…We have shown that melittin and myotoxin inhibit the Ca# + -ATPase by decreasing the rate of dephosphorylation of the phosphorylated ATPase [38,39]. The rate of dephosphorylation can be determined either by first phosphorylating the ATPase with [$#P]P i at pH 6.0 in the absence of Ca# + and the presence of DMSO followed by mixing with an excess of a pH 7.5 buffer containing KCl and ATP to induce dephosphorylation, or by first phosphorylating the ATPase with [γ-$#P]ATP in the presence of Ca# + and then mixing with an excess of unlabelled ATP.…”

The Mechanism of Inhibition of the Ca2+-ATPase by Mastoparan

“…Tryptophan fluorescence was monitored (at 25°C) by exciting the sample at 280 nm and measuring the emission above 320 nm using a cut off filter. 4 , 100 M CaCl 2 , and 0.075 mg/ml ATPase. Two stocks of labeled ATP were made up to cover the range of ATP concentrations up to 100 M, with specific activities of 10 and 100 Ci/mol.…”

Regulation of Mg2+‐independent Ca2+‐ATPase by a low molecular mass protein purified from bovine brain

“…A recent report describes the role of PDC-109, the major secretory protein from bull seminal vesicles as a stimulator of bovine sperm membrane Ca 2+ -ATPase [19]. Apart from these, certain exogenous peptides like mellitin [1], myotoxin a [2], mastoparan [9] etc. are potent inhibitors of sarcoplasmic reticulum Ca 2+ -ATPase (SERCA).…”