Characterization of phenylmaleimide inhibition of the Ca(2+)-ATPase from skeletal-muscle sarcoplasmic reticulum.

The Ca(2+)-ATPase from sarcoplasmic reticulum reacts with phenylmaleimide, producing the inhibition of the ATPase activity following a pseudo-first-order kinetic with a rate constant of 19 M(-1) s(-1). Calcium and ATP binding are not altered upon phenylmaleimide inhibition. However, the presence of millimolar calcium, and to a lesser extent magnesium, in the inhibition medium enhances the effect of phenylmaleimide, causing a higher degree of inhibition. Solubilization with C(12)E(8) does not affect the ATPase inhibition, excluding any kind of participation of the lipid bilayer. Phosphorylation with ATP in steady-state conditions as well as phosphorylation with inorganic phosphate in equilibrium conditions were strongly inhibited. Conversely, we have found that the occupancy of the phosphorylation site by ortovanadate fully protects against the inhibitory effect of phenylmaleimide, indicating a conformational transition associated with the phosphorylation reaction.