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    Thapsigargin: A potent inhibitor of the calcium ATPase in sarcoplasmic reticulum

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    Author
    Sagara, Yutaka
    Advisor
    Inesi, Giuseppe
    Date
    1992
    Type
    dissertation
    
    Metadata
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    Abstract
    A Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase, a membranous protein in sarcoplasmic reticulum (SR) of skeletal muscle, is an enzyme that catalyzes transport of cytoplasmic Ca{dollar}\sp{lcub}2+{rcub}{dollar} into the SR lumen upon ATP hydrolysis. The endoplasmic reticulum (ER) Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase from rat liver cells, but not SR Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPases from skeletal or cardiac cells, was inhibited by thapsigargin, a sesquiterpene lactone from an umbelliferous plant Thapsia garganica. In this thesis project, thapsigargin (TG) was shown to be a potent inhibitor of all the SR/ER-type Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPases, but not the plasma membrane Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase in erythrocytes or the ryanodine receptor in skeletal muscle. TG inhibition is stoichiometric and potent: the affinity of TG to the Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase is high with the dissociation constant in subnanomolar. TG binding to the ATPase is apparently irreversible, even though the interaction is non-covalent. TG inhibits the steady state activities, Ca{dollar}\sp{lcub}2+{rcub}{dollar} transport and ATP hydrolysis of the SR Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase, as well as partial activities of the ATPase: Ca{dollar}\sp{lcub}2+{rcub}{dollar} and ATP binding at equilibrium and phosphoenzyme formation from inorganic phosphate are equally affected. Kinetic studies revealed that TG tightly binds an ATPase intermediate, which is produced during the ATPase catalytic cycle. The ATPase conformation (intermediate) sensitive to TG interaction can be also produced by the removal of Ca{dollar}\sp{lcub}2+{rcub}{dollar} by EGTA. The complex of TG and the ATPase intermediate does not react with any ATPase ligand and, therefore, is a dead-end complex. Spectroscopic and biochemical studies showed that this dead-end complex is in a different conformational state from the original intermediate that TG is initially binding. The changes in the ATPase conformation upon TG binding may be responsible for the global inhibition of ATPase partial activities. Since these partial activity sites (the nucleotide binding site, Ca{dollar}\sp{lcub}2+{rcub}{dollar} binding site, and the phosphorylation site) affected by TG are distantly located in the ATPase, the potent and stoichiometric inhibition of the ER/SR-type Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase by TG may occur at either a location that is critical in the function of all the partial activities of the ATPase or a location essential for transduction of the signals between the sites. Determination of the TG interaction site showed that TG is not binding to the phosphorylation site, the Ca{dollar}\sp{lcub}2+{rcub}{dollar} binding site, or the nucleotide binding site. (Abstract shortened with permission of author.)
    Description
    University of Maryland, Baltimore. Biochemistry and Molecular Biology. Ph.D. 1992
    Keyword
    Biology, Cell
    Chemistry, Biochemistry
    Chemistry, Pharmaceutical
    Sarcoplasmic Reticulum Calcium-Transporting ATPases--antagonists & inhibitors
    Thapsigargin
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/2528
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    Theses and Dissertations All Schools
    Theses and Dissertations School of Medicine

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