Unravelling the interaction of thapsigargin with the conformational states of Ca(2+)-ATPase from skeletal sarcoplasmic reticulum.
Preincubation of thapsigargin with sarcoplasmic reticulum vesicles in the presence of high Ca(2+) or the addition of high Ca(2+) to microsomal vesicles preincubated with thapsigargin in the absence of Ca(2+) allowed full enzyme phosphorylation by ATP. However, the enzyme activity was not protected by high Ca(2+) even when the samples were subjected to gel filtration before ATP addition. Our data indicate that: (i) the enzyme in the Ca(2+)-bound conformation can be stabilized in the presence of thapsigargin; (ii) the conformational transition from the Ca(2+)-free to the Ca(2+)-bound state can be elicited by Ca(2+) when thapsigargin is present; (iii) thapsigargin binding occurs whether or not the enzyme is in the presence of Ca(2+), and so a ternary complex enzyme-Ca(2+)-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 Ca(2+) 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 Ca(2+), indicating that thapsigargin specifically inhibits the Ca(2+)-dependent activity.
Fortea
M I
MI
Departamento de Bioquimica y Biologia Molecular A, Facultad de Veterinaria, Universidad de Murcia, 30071 Espinardo, Murcia, Spain.
Soler
F
F
Fernandez-Belda
F
F
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects* / Q000201:enzymology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D019284:Thapsigargin / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D019284:Thapsigargin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases