Binding of sesquiterpene lactone inhibitors to the Ca2+-ATPase

Abstract

The mechanism of inhibition of the Ca(2+)-ATPase from sarcoplasmic reticulum by the sesquiterpene lactones thapsigargin, trilobolide and thapsivillosin A (TvA) has been determined. A decrease in the affinity of the ATPase for Ca2+ is observed in the presence of the inhibitors (I), consistent with a shift in the E1/E2 equilibrium for the ATPase towards E2 forms. Amounts of inhibitor beyond a 1:1 molar ratio with ATPase produce no further decrease in affinity for Ca2+, inconsistent with the formation of a dead-end complex. Measurements of the rate of quenching of the tryptophan fluorescence of the ATPase by TvA are consistent with an association step to give E2I followed by an isomerization to a modified state E2AI. The kinetics of the reversal of the effects of TvA by Ca2+ at sub-stoichiometric amounts of TvA are bi-exponential, with a fast component whose rate is independent of TvA concentration and equal to the rate observed in the absence of TvA, and a slow component whose rate decreases with increasing TvA concentration. These observations are also consistent with the formation of a modified state E2AI following the initial binding of I to E2. The equilibrium constant E2AI/E2I increases in the order TvA < trilobolide < thapsigargin. The results suggest that the effects of the inhibitors on the overall ratio of E2 to E1 forms of the ATPase follow largely from the formation of E2AI from E2I, and that binding constants are very similar for E1Ca2, E1 and E2.

The Endoplasmic Reticulum in PC12 Cells

“…Phosphorylation was carried out in the presence of 50 M Ca 2ϩ alone, in the presence of excess EGTA, and in the presence of 1 M thapsigargin. The inhibitor was always preincubated with the samples before addition of Ca 2ϩ , since the presence of Ca 2ϩ greatly slows the rate of binding of thapsigargin by SERCA (30).…”

Effects of Inhibitors on Luminal Opening of Ca2+ Binding Sites in an E2P-like Complex of Sarcoplasmic Reticulum Ca22+-ATPase with Be22+-fluoride

“…TG also did not greatly affect the superfluorescent state of TNP-AMP bound to the ATPase complex with beryllium fluoride (24). On the other hand, it was previously shown that TG altered the affinity of Ca 2ϩ -free ATPase for ATP (16), and it was suggested that the interaction of ATPase with thapsigargin (or BHQ) might result in formation of modified E2 species (25)(26)(27). More specifically, TG was also shown to induce pretty large structural changes in two-dimensional crystals of the E2P-related E2⅐VO 4 species (28), and TG was suspected from Trp fluorescence experiments (but with no direct evidence) to close, in E2P-related states, the postulated Ca 2ϩ release pathway from the ATPase sites toward the SR lumen (24).…”

Effects of pH on phosphorylation of the Ca2+-ATPase of sarcoplasmic reticulum by inorganic phosphate

“…Thapsivillosin A also binds to the ATPase with high affinity, to give a modified E2 state of the ATPase [26]. In the presence of Ca# + and ATP at pH 6.0, the ATPase was maximally phosphorylated (Table 1 ; [29]) ; labelling the ATPase with DMC had no effect on 4 2 − can bind to the ATPase, but only H 2 PO 4 − can phosphorylate it.…”

“…Ammonium vanadate was dissolved in 100 mM KOH to give a 100 mM stock solution. Thapsivillosin A was purified from roots of Thapsia ellosa as described by Wictome et al [26].…”