Unravelling the interaction of thapsigargin with the conformational states of Ca2+-ATPase from skeletal sarcoplasmic reticulum

Preincubation of thapsigargin with sarcoplasmic reticulum vesicles in the presence of high Ca2+ or the addition of high Ca2+ to microsomal vesicles preincubated with thapsigargin in the absence of Ca2+ allowed full enzyme phosphorylation by ATP. However, the enzyme activity was not protected by high Ca2+ even when the samples were subjected to gel filtration before ATP addition. Our data indicate that: (i) the enzyme in the Ca2+-bound conformation can be stabilized in the presence of thapsigargin; (ii) the conformational transition from the Ca2+-free to the Ca2+-bound state can be elicited by Ca2+ when thapsigargin is present; (iii) thapsigargin binding occurs whether or not the enzyme is in the presence of Ca2+, and so a ternary complex enzyme-Ca2+-thapsigargin may be formed; (iv) thapsigargin can be dissociated from the enzyme with a slow kinetics after dilution under drastic conditions; (v) the kinetics of Ca2+ binding is clearly slowed down by thapsigargin; and (vi) thapsigargin does not affect the hydrolysis rate of phosphorylating substrates when measured in the absence of Ca2+, indicating that thapsigargin specifically inhibits the Ca2+-dependent activity.