Cyclopiazonic Acid Effect on Ca²⁺-Dependent Conformational States of the Sarcoplasmic Reticulum ATPase. Implication for the Enzyme Turnover

Abstract

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.

Pharmacological Modulation of Sarcoplasmic Reticulum Function in Smooth Muscle

“…With an ATP concentration of 1 mM, SERCA is completely inhibited at a CPA/SERCA molar ratio of approximately 10 (Soler et al, 1998). Furthermore, enzyme activity is observed to recover at a CPA/ SERCA molar ratio of 1 when the Ca 2ϩ concentration is raised, which is consistent with the competitive character of CPA and Ca 2ϩ (Soler et al, 1998). These results suggest that ATP and Ca 2ϩ can protect against CPA inhibition.…”

“…Nonetheless, CPA effect on the enzyme is likely not restricted to the ATP-binding process since it causes additional inhibition of the ATPase at concentrations above the stoichiometric levels (Plenge-Tellechea et al, 1997). With an ATP concentration of 1 mM, SERCA is completely inhibited at a CPA/SERCA molar ratio of approximately 10 (Soler et al, 1998). Furthermore, enzyme activity is observed to recover at a CPA/ SERCA molar ratio of 1 when the Ca 2ϩ concentration is raised, which is consistent with the competitive character of CPA and Ca 2ϩ (Soler et al, 1998).…”

Insight into the Uncoupling Mechanism of Sarcoplasmic Reticulum ATPase Using the Phosphorylating Substrate UTP

“…8B). The uncoupled state of the enzyme was protected from CPA, an E 2 -directed inhibitor of the enzyme (33). Such inhibition by CPA was patent when the lumenal Ca 2ϩ load was relieved by the addition of Ca 2ϩ ionophore (Fig.…”

“…The E 1 -E 2 cycle (coupled route) involves dissociation of lumenal Ca 2ϩ from E 1 P⅐Ca 2 and further release of P i from E 2 P. The E 1 cycle (uncoupled route) consists of the release of P i from E 1 P⅐Ca 2 before Ca 2ϩ dissociation to the cytoplasmic medium and the absence of E 2 and E 2 P species. The mechanism of intramolecular uncoupling was confirmed by using the high affinity inhibitor CPA (31)(32)(33) and measuring UTP hydrolysis under the conditions described in Fig. 8.…”

Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase

“…Intriguingly, early kinetic analyses on SERCA (29) showed that the inhibitor cyclopiazonic acid (CPA) modifies SERCA in a way very similar to the CPZ modification of NKA. Transport studies have also shown that CPA uncouples SERCA and reduces the transport stoichiometry to one-half (30).…”