On the inhibition mechanism of sarcoplasmic or endoplasmic reticulum Ca2+-ATPases by cyclopiazonic acid.
Ca2+-ATPase inhibition by stoichiometric and substoichiometric concentrations of cyclopiazonic acid was studied in sarcoplasmic reticulum preparations from rabbit fast-twitch muscle. The apparent affinity of the nonphosphorylated enzyme for ATP showed a Kd of approximately 3 microM in the absence of cyclopiazonic acid and approximately 28 microM in the presence 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 turnover in the presence of stoichiometric concentrations of cyclopiazonic acid displayed lower apparent affinity for ATP and lower maximal hydrolytic activity than in the absence of the drug. When cyclopiazonic acid is in the substoichiometric range, the observed kinetic parameters will correspond to the simultaneous contribution of two different reaction cycles sustained by the enzyme with and without drug. The inhibition could be elicited by adding ATP to allow the enzyme turnover when cyclopiazonic acid was preincubated with the enzyme in the presence of Ca2+. The onset of inhibition during enzyme cycling was observed over a period of seconds, revealing the existence of a low inhibition rate constant. It is concluded that cyclopiazonic acid decreases enzyme affinity for ATP in non-turnover conditions by approximately one order of magnitude. This allows enzyme cycling after drug binding, provided that a high ATP concentration is used. Cyclopiazonic acid and ATP do not compete for the same binding site.
Plenge-Tellechea
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F
Departamento de Bioquimica y Biologia Molecular A, Edificio de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30071 Murcia, Spain.
Soler
F
F
Fernandez-Belda
F
F
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology*; D007700:Kinetics; D008956:Models, Chemical; D018656:Muscle Fibers, Fast-Twitch / Q000201:enzymology; D018482:Muscle, Skeletal / Q000201:enzymology*; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D004791:Enzyme Inhibitors; D007211:Indoles; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium; C000543:cyclopiazonic acid