Characterization of phenylmaleimide inhibition of the Ca2+-ATPase from skeletal-muscle sarcoplasmic reticulum

The Ca2+-ATPase from sarcoplasmic reticulum reacts with phenylmaleimide, pr oducing the inhibition of the ATPase activity following a pseudo-first-orde r kinetic with a rate constant of 19 M-1 s(-1). Calcium and ATP binding are not altered upon phenylnaleimide inhibition. However, the presence of mill imolar calcium, and to a lesser extent magnesium, in the inhibition medium enhances the effect of phenylmaleimide, causing a higher degree of inhibiti on. Solubilization with C12E8 does not affect the ATPase inhibition, exclud ing any kind of participation of the lipid bilayer, Phosphorylation with AT P in steady-state conditions as well as phosphorylation with inorganic phos phate in equilibrium conditions were strongly inhibited. Conversely, we hav e found that the occupancy of the phosphorylation site by ortovanadate full y protects against the inhibitory effect of phenylmaleimide, indicating a c onformational transition associated with the phosphorylation reaction. (C) 1999 Academic Press.