Self-association accompanies inhibition of Ca-ATPase by thapsigargin.
Recent studies have demonstrated a relationship between the activity of the Ca-ATPase of sarcoplasmic reticulum and its state of self-association. In the present study, the effects of thapsigargin (TG), a toxin that specifically inhibits the Ca-ATPase of rabbit skeletal muscle sarcoplasmic reticulum membrane, were studied by detecting the time-resolved phosphorescence anisotropy (TPA) decay of the Ca-ATPase that had been labeled with the phosphorescent probe erythrosin-isothiocyanate (ErITC). Anisotropy decays were fit to a function that consisted of three exponential decays plus a constant background, as well as to a function describing explicitly the uniaxial rotation of proteins in a membrane. In the absence of TG, the anisotropy was best-fit by a model representing the rotation of three populations, corresponding to different-sized oligomeric species in the membrane. The addition of stoichiometric amounts of TG to the Ca-ATPase promptly decreased the overall apparent rate of decay, indicating decreased rotational mobility. A detailed analysis showed that the principal change was not in the rates of rotation but rather in the population distribution of the Ca-ATPase molecules among the different-sized oligomers. TG decreased the proportion of small oligomers and increased the proportion of large ones. Preincubation of the ErITC-SR in 1 mM Ca2+, which stabilizes the E1 conformation relative to E2, was found to protect partially against the changes in the TPA associated with the presence of the inhibitor. These results are consistent with the hypothesis that TG inhibits the Ca-ATPase by stabilizing it in an E2-like conformation, which promotes the formation of larger aggregates of the enzyme. When combined with the effects of other inhibitors on the Ca-ATPase, these results support a general model for the coupling of enzyme conformation and self-association in this system.
Mersol
J V
JV
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.
Kutchai
H
H
Mahaney
J E
JE
Thomas
D D
DD
D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D004923:Erythrosine / Q000031:analogs & derivatives; D066298:In Vitro Techniques; D017879:Isothiocyanates; D049449:Luminescence; D046911:Macromolecular Substances; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D013729:Terpenes / Q000378:metabolism / Q000494:pharmacology*; D019284:Thapsigargin
D017879:Isothiocyanates; D046911:Macromolecular Substances; D013729:Terpenes; D019284:Thapsigargin; C022232:erythrosine isothiocyanate; D000252:Calcium-Transporting ATPases; D004923:Erythrosine