ON THE INHIBITION-MECHANISM OF SARCOPLASMIC OR ENDOPLASMIC-RETICULUM CA2-ATPASES BY CYCLOPIAZONIC ACID()

Ca2+-ATPase inhibition by stoichiometric and substoichiometric concent rations of cyclopiazonic acid was studied in sarcoplasmic reticulum pr eparations from rabbit fast-twitch muscle. The apparent affinity of th e nonphosphorylated enzyme for ATP showed a K-d of similar to 3 mu M i n the absence of cyclopiazonic acid and similar to 28 mu M in the pres ence of the drug. Fractional saturation of the enzyme by cyclopiazonic acid was accompanied by the appearance of two ATP-binding populations (enzyme with and without drug) and a progressive increase in the half -maximal concentration for saturating the ATP-binding sites. Enzyme tu rnover in the presence of stoichiometric concentrations of cyclopiazon ic acid dis played lower apparent affinity for ATP and lower maximal h ydrolytic activity than in the absence of the drug. When cyclopiazonic acid is in the substoichiometric range, the observed kinetic paramete rs will correspond to the simultaneous contribution of two different r eaction cycles sustained by the enzyme with and without drug. The inhi bition could be elicited by adding ATP to allow the enzyme turnover wh en cyclopiazonic acid was preincubated with the enzyme in the presence of Ca2+. The onset of inhibition during enzyme cycling was observed o ver a period of seconds, revealing the existence of a low inhibition r ate constant, It is concluded that cyclopiazonic acid decreases enzyme affinity for ATP in non-turnover conditions by approximately one orde r of magnitude. This allows enzyme cycling after drug binding, provide d that a high ATP concentration is used. Cyclopiazonic acid and ATP do not compete for the same binding site.