Thapsigargin and dimethyl sulfoxide activate medium P(i)<-->HOH oxygen exchange catalyzed by sarcoplasmic reticulum Ca2+-ATPase.
Thapsigargin is a potent inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase. It binds the Ca(2+)-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) Me(2)SO. Assuming a simple two-step mechanism, we have evaluated the effect of thapsigargin and Me(2)SO on the four rate constants governing the reaction of P(i) with Ca(2+)-ATPase. The principal effect of thapsigargin alone is to stimulate EP hydrolysis (k(-2)), whereas that of Me(2)SO is to greatly retard P(i) dissociation (k(-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 (v(exchange) = k(-2)[EP]). Kinetic simulations show that the overall rate constant for the formation of EP is very fast (approximately 300 s(-1)) when the exchange is maximal. Thapsigargin greatly stabilizes Ca(2+)-ATPase against denaturation in detergent in the absence of Ca(2+), 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.
Seekoe
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Department of Chemical Pathology, University of Cape Town Medical School, Observatory 7925, Cape Town, South Africa.
Peall
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McIntosh
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DB
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D005976:Glutaral / Q000494:pharmacology; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D009132:Muscles / Q000201:enzymology; D010100:Oxygen / Q000378:metabolism*; D010710:Phosphates / Q000737:chemistry*; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D019284:Thapsigargin / Q000494:pharmacology*; D013997:Time Factors
D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D010710:Phosphates; D019284:Thapsigargin; D006859:Hydrogen; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; D010100:Oxygen; D005976:Glutaral; D004121:Dimethyl Sulfoxide