Mechanism of inhibition of the plasma membrane Ca(2+)-ATPase by barbiturates.
We have demonstrated that sodium pentobarbital inhibited the activation of the human red blood cell plasma membrane Ca(2+)-ATPase produced by dimerization of enzyme monomers or by calmodulin binding to enzyme monomers. The effects of the barbiturate were dose-dependent. Both Vmax and Ca2+ affinity were reduced. The Ca(2+)-ATPase activity of the dimeric enzyme was distinctly less sensitive with respect to the effective inhibitory concentrations of pentobarbital and to the rate of onset of inhibition than was the calmodulin-dependent activation of enzyme monomers. Temperature dependence of the inhibition was in agreement with direct, nonpolar interactions of pentobarbital with a water-exposed nonpolar patch on the surface of this transmembrane protein. The barbiturate prevented the increase of intrinsic tryptophan fluorescence associated with substrate Ca2+ binding to the enzyme dimer. On the basis of the barbiturate effects we propose a model for the action of detergent-like compounds on the enzyme. They inhibit Ca(2+)-ATPase activity by binding to a nonpolar patch on the water-exposed dimerization surface of the enzyme monomer, part of which is also the binding site for calmodulin. The model assumes that their binding to the nonpolar patch on the monomer interferes with dimerization and weakens but does not prohibit calmodulin binding, whose activation of the enzyme is then submaximal. The model should be applicable to other proteins as the two activation pathways studied have been demonstrated for various enzymes.
Department of Anesthesiology/CCM, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
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D
Kosk-Kosicka
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Fomitcheva
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Lopez
D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002147:Calmodulin / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology*; D004910:Erythrocyte Membrane / Q000201:enzymology; D006801:Humans; D006993:Hypnotics and Sedatives / Q000494:pharmacology*; D010424:Pentobarbital / Q000494:pharmacology*
D002147:Calmodulin; D006993:Hypnotics and Sedatives; D000252:Calcium-Transporting ATPases; D010424:Pentobarbital