Characterization of the inhibition of intracellular Ca2+ transport ATPases by thapsigargin
The effects of thapsigargin (TG), a specific inhibitor of intracellular Ca(2+)-ATPases, were studied on vesicular fragments of sarcoplasmic reticulum (SR) membranes. Inhibition of Ca2+ transport and ATPase activity was observed following stoichiometric titration of the membrane bound enzyme with TG....
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Veröffentlicht in: | The Journal of biological chemistry 1992-06, Vol.267 (18), p.12606-12613 |
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Sprache: | eng |
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Zusammenfassung: | The effects of thapsigargin (TG), a specific inhibitor of intracellular Ca(2+)-ATPases, were studied on vesicular fragments
of sarcoplasmic reticulum (SR) membranes. Inhibition of Ca2+ transport and ATPase activity was observed following stoichiometric
titration of the membrane bound enzyme with TG. When Ca2+ binding to the enzyme was measured in the absence of ATP, or when
one cycle of Ca(2+)-dependent enzyme phosphorylation by ATP was measured under conditions preventing turnover, protection
against TG by Ca2+ was observed. The protection by Ca2+ disappeared if the phosphoenzyme was allowed to undergo turnover,
indicating that a state reactive to TG is produced during enzyme turnover, whereby a dead end complex with TG is formed. Enzyme
phosphorylation with Pi, ATP synthesis, and Ca2+ efflux by the ATPase in its reverse cycling were also inhibited by TG. However,
under selected conditions (millimolar Ca2+ in the lumen of the vesicles, and 20% dimethyl sulfoxide in the medium) TG permitted
very low rates of enzyme phosphorylation with Pi and ATP synthesis in the presence of ADP. It is concluded that the mechanism
of ATPase inhibition by TG involves mutual exclusion of TG and high affinity binding of external Ca2+, as well as strong (but
not total) inhibition of other partial reactions of the ATPase cycle. TG reacts selectively with the state acquired by the
ATPase in the absence of Ca2+. This state is obtained either by enzyme exposure to EGTA, or by utilization of ATP and consequent
displacement of bound Ca2+ during catalytic turnover. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(18)42320-4 |