CYCLOPIAZONIC ACID EFFECT ON CA2-DEPENDENT CONFORMATIONAL STATES OF THE SARCOPLASMIC-RETICULUM ATPASE - IMPLICATION FOR THE ENZYME TURNOVER()

The affinity of sarcoplasmic reticulum Ca2+-ATPase for cyclopiazonic a cid is dependent on the conformational state of the enzyme. It is high in the absence of Ca2+ but low in its presence. When Ca2+ was added t o the enzyme in the presence of equimolar toxin, the apparent rate con stant for Ca2+ binding was 0.6 min(-1) when measured at 37 degrees C. The apparent equilibrium constant for Ca2+ dissociation increased from 0.2 to 0.6 mu M at neutral pH, and from 5.9 to 37 mu M at pH 6.0. The apparent equilibrium constant for Ca2+ dissociation increased progres sively as the amount of toxin increased above an equimolar level, Cycl opiazonic acid decreased phosphorylation by ATP and Ca2+ when the enzy me in the absence of Ca2+ was incubated in the presence of toxin, alth ough no effect was observed after a preliminary incubation with Ca2+ a t 37 degrees C. Cyclopiazonic acid incubated with the enzyme in the pr esence of Ca2+ could be eliminated with a Sephadex column. However, th e toxin could not be removed when it was incubated with the enzyme in the absence of Ca2+. In the latter case, cyclopiazonic acid was elimin ated when the enzyme in the presence of toxin was incubated with Ca2at 37 degrees C. Under turnover conditions and in the presence of 10 m u M ATP, the toxin-enzyme interaction can be characterized by an appar ent Kd of 7 nM. With an ATP concentration of 1 mM, the enzyme was inhi bited completely at a toxin/enzyme molar ratio of similar to 10. Furth ermore, enzyme activity was observed to recover at a toxin/enzyme mola r ratio of 1 when the Ca2+ concentration was raised, which is consiste nt with the competitive character of cyclopiazonic acid and Ca2+. It i s concluded that ATP and Ca2+ can protect against cyclopiazonic acid i nhibition.