Crystallization of the Ca²⁺‐ATPase of skeletal muscle sarcoplasmic reticulum Inhibition by myotoxin a

Abstract

Decavanadate produces extensive ordered arrays of Ca 2+-ATPase molecules on sarcoplasmic reticulum (SR) vesicle surfaces [(1984) J. Bioenerg. Biomembranes 16, 491-505] and the basic unit of these crystalline structures seems to be a dimer of Ca 2 +-ATPase [(1983) J. Ultrastruct. Res. 24, 454-464; (1984) J. Mol. Biol. 174, 193-204]. Myotoxin a, isolated from the venom of the prairie rattlesnake Crotalus viridis viridis, is a muscle-degenerating polypeptide and its primary site of interaction is the SR membrane, where it uncouples CaZ+-translocation from CaZ+-dependent ATP hydrolysis [(1986) Arch. Biochem. Biophys. 246, 90 97]. The effect of myotoxin a on decavanadate-induced two-dimensional Ca 2 +-ATPase crystals of SR membranes has been investigated. The toxin inhibits the formation of two-dimensional SR-membrane crystals and disrupts previously formed crystals in a time-and concentration-dependent manner, which parallels the uncoupling of ATP hydrolysis from Ca 2 + translocation. Two-dimensional crystalline arrays of the SR membrane have a typical diffraction pattern which, after myotoxin a treatment, displays a progressive loss of order. Decavanadate is an uncompetitive inhibitor of the Ca 2 +-ATPase enzyme-myotoxin a complex. The present results suggest that a Ca / +-ATPase dimer is required for coupling Ca 2 + translocation to Ca 2 +-dependent ATP hydrolysis.

Mechanism of inhibition of Ca2+-ATPase by myotoxin a

“…Binding in this region would have to be such as not to affect binding of ATP, since neither the ATPdependence of ATPase activity nor the rate of phosphoenzyme formation are affected (results not shown). It has also been reported that inhibition of the ATPase by myotoxin a and decavanadate are non-competitive [56], suggesting that myotoxin a does not bind close to the phosphate-binding site on the ATPase.…”