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 acid 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 to the enzyme in the presence of equimolar toxin, the apparent rate constant 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 microM at neutral pH, and from 5.9 to 37 microM at pH 6.0. The apparent equilibrium constant for Ca2+ dissociation increased progressively as the amount of toxin increased above an equimolar level. Cyclopiazonic acid decreased phosphorylation by ATP and Ca2+ when the enzyme in the absence of Ca2+ was incubated in the presence of toxin, although no effect was observed after a preliminary incubation with Ca2+ at 37 degrees C. Cyclopiazonic acid incubated with the enzyme in the presence of Ca2+ could be eliminated with a Sephadex column. However, the toxin could not be removed when it was incubated with the enzyme in the absence of Ca2+. In the latter case, cyclopiazonic acid was eliminated when the enzyme in the presence of toxin was incubated with Ca2+ at 37 degrees C. Under turnover conditions and in the presence of 10 microM ATP, the toxin-enzyme interaction can be characterized by an apparent Kd of 7 nM. With an ATP concentration of 1 mM, the enzyme was inhibited completely at a toxin/enzyme molar ratio of approximately 10. Furthermore, enzyme activity was observed to recover at a toxin/enzyme molar ratio of 1 when the Ca2+ concentration was raised, which is consistent with the competitive character of cyclopiazonic acid and Ca2+. It is concluded that ATP and Ca2+ can protect against cyclopiazonic acid inhibition.
Soler
F
F
Departamento de Bioquimica y Biologia Molecular A, Edificio de Veterinaria, Universidad de Murcia, Spain.
Plenge-Tellechea
F
F
Fortea
I
I
Fernandez-Belda
F
F
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D006873:Hydroquinones / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology*; D007700:Kinetics; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D019284:Thapsigargin / Q000494:pharmacology
D006873:Hydroquinones; D007211:Indoles; C019359:2,5-di-tert-butylhydroquinone; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium; C000543:cyclopiazonic acid