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• W2029984583 abstract "Thapsigargin is a potent inhibitor of sarcoplasmic reticulum Ca²⁺-ATPase. It binds the Ca²⁺-free E2 conformation in the picomolar range, supposedly resulting in a largely catalytically inactive species. We now find that thapsigargin has little effect on medium P_i↔ HOH oxygen exchange and that this activity is greatly stimulated (up to 30-fold) in the presence of 30% (v/v) Me₂SO. Assuming a simple two-step mechanism, we have evaluated the effect of thapsigargin and Me₂SO on the four rate constants governing the reaction of P_i with Ca²⁺-ATPase. The principal effect of thapsigargin alone is to stimulate <i>E</i>P hydrolysis (<i>k</i> ₋₂), whereas that of Me₂SO is to greatly retard P_i dissociation (<i>k</i> ₋₁), accounting for its well known effect on increasing the apparent affinity for P_i. These effects persist when the agents are used in combination and substantially account for the activated oxygen exchange (<i>v</i> _exchange =<i>k</i> ₋₂[<i>E</i>P]). Kinetic simulations show that the overall rate constant for the formation of <i>E</i>P is very fast (∼300 s⁻¹) when the exchange is maximal. Thapsigargin greatly stabilizes Ca²⁺-ATPase against denaturation in detergent in the absence of Ca²⁺, as revealed by glutaraldehyde cross-linking, suggesting that the membrane helices lock together. It seems that the reactions at the phosphorylation site, associated with the activated exchange reaction, are occurring without much movement of the transport site helices, and we suggest that they may be associated solely with an occluded H⁺ state." @default.
• W2029984583 created "2016-06-24" @default.
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• W2029984583 date "2001-12-01" @default.
• W2029984583 modified "2023-10-17" @default.
• W2029984583 title "Thapsigargin and Dimethyl Sulfoxide Activate Medium Pi ↔ HOH Oxygen Exchange Catalyzed by Sarcoplasmic Reticulum Ca2+-ATPase" @default.
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• W2029984583 doi "https://doi.org/10.1074/jbc.m106320200" @default.
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