BINDING OF SESQUITERPENE LACTONE INHIBITORS TO THE CA2-ATPASE()

The mechanism of inhibition of the Ca2+-ATPase from sarcoplasmic retic ulum by the sesquiterpene lactones thapsigargin, trilobolide and thaps ivillosin A (TvA) has been determined. A decrease in the affinity of t he ATPase for Ca2+ is observed in the presence of the inhibitors (I), consistent with a shift in the E1/E2 equilibrium for the ATPase toward s E2 forms. Amounts of inhibitor beyond a 1:1 molar ratio with ATPase produce no further decrease in affinity for Ca2+, inconsistent with th e formation of a dead-end complex. Measurements of the rate of quenchi ng 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 E2(A)I. 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 a nd equal to the rate observed in the absence of TvA, and a slow compon ent whose rate decreases with increasing TvA concentration. These obse rvations are also consistent with the formation of a modified state E2 (A)I following the initial binding of I to E2. The equilibrium constan t E2(A)I/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 formatio n of E2(A)I from E2I, and that binding constants are very similar for E1Ca(2), E1 and E2.