title: Mechanism of thermal uncoupling of Ca2+-ATPase of sarcoplasmic reticulum as revealed by thapsigargin stabilization.
abstract: Thermal uncoupling of the Ca2+ pump of skeletal muscle sarcoplasmic reticulum is specifically blocked by binding of Ca2+ to the high affinity sites, having identical characteristics to the Ca2+ transport sites (Berman, M.C., McIntosh, D.B. and Kench, J.E. (1977) J. Biol. Chem. 252, 994-1001). The present study has investigated the nature of the decreased net Ca2+ transport in the uncoupling process. Ca2+ uptake in the presence and absence of oxalate, Ca2+ retention following passive Ca2+ loading and Ca2+-dependent ATPase activity were inactivated at pH 7.0 and 37 degrees C, with rate constants of 0.12, 0.047, 0.053 and 0.001 min-1, respectively. Activation energies were in the range 72-76 kcal/mol, indicating a common irreversible protein conformational transition. A thermodynamic analysis of parallel or consecutive inactivation pathways revealed that loss of Ca2+ transfer and ATPase activity occurred on the same pump unit, making the existence of a predominant uncoupled intermediate unlikely. Decreased passive Ca2+ loading, an index of the number of intact vesicles, correlated with decreased active uptake in the absence of oxalate, indicating increased vesicle permeability. Thapsigargin, at a 1:1 stoichiometry, stabilised the Ca-ATPase against thermal inactivation, while previously inactivated Ca-ATPases appeared not to bind TG. Protection by TG suggests that the origin of inactivation is in the transmembrane and stalk regions of the ATPase. We propose that protein unfolding results in inefficient gating of a small percentage of ATPases with subsequent uncoupling of the entire vesicle.
authors.affiliation: Department of Chemical Pathology, University of Cape Town Medicial School, South Africa.
authors.forename: G A
authors.initials: GA
authors.lastname: Davidson
authors.forename: M C
authors.initials: MC
authors.lastname: Berman
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004795:Enzyme Stability; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013729:Terpenes / Q000378:metabolism / Q000494:pharmacology*; D019284:Thapsigargin; D013816:Thermodynamics
chemical_list: D004791:Enzyme Inhibitors; D013729:Terpenes; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium