SELF-ASSOCIATION ACCOMPANIES INHIBITION OF CA-ATPASE BY THAPSIGARGIN

Recent studies have demonstrated a relationship between the activity o f the Ca-ATPase of sarcoplasmic reticulum and its state of self-associ ation. In the present study, the effects of thapsigargin (TG), a toxin that specifically inhibits the Ca-ATPase of rabbit skeletal muscle sa rcoplasmic reticulum membrane, were studied by detecting the time-reso lved phosphorescence anisotropy (TPA) decay of the Ca-ATPase that had been labeled with the phosphorescent probe erythosin-isothiocyanate (E rITC). Anisotropy decays were fit to a function that consisted of thre e exponential decays plus a constant background, as well as to a funct ion describing explicitly the uniaxial rotation of proteins in a membr ane. In the absence of TG, the anisotropy was best-fit by a model repr esenting the rotation of three populations, corresponding to different -sized oligomeric species in the membrane. The addition of stoichiomet ric amounts of TG to the Ca-ATPase promptly decreased the overall appa rent rate of decay, indicating decreased rotational mobility. A detail ed analysis showed that the principal change was not in the rates of r otation but rather in the population distribution of the Ca-ATPase mol ecules among the different-sized oligomers. TG decreased the proportio n of small oligomers and increased the proportion of large ones. Prein cubation of the ErITC-SR in 1 mM Ca2+, which stabilizes the E1 conform ation relative to E2, was found to protect partially against the chang es in the TPA associated with the presence of the inhibitor. These res ults are consistent with the hypothesis that TG inhibits the Ca-ATPase by stabilizing it in an E2-like conformation, which promotes the form ation of larger aggregates of the enzyme. When combined with the effec ts of other inhibitors on the Ca-ATPase, these results support a gener al model for the coupling of enzyme conformation and self-association in this system.