Distinct mechanisms of inhibition of purified cardiac sarcolemma Ca(2+)-ATPase by two calmodulin antagonists.
The effects of calmidazolium and compound 48/80 were studied in four different states of activation of the purified Ca(2+)-ATPase from cardiac sarcolemma: "basal" or unactivated, activated by calmodulin, activated by phosphatidylserine, and activated by controlled trypsinization. When assayed in the presence of phosphatidylcholine as the sole phospholipid (basal state), the purified enzyme was resistant to inhibition by calmidazolium (0.1 to 3 microM). In the same range, calmidazolium inhibited the enzyme activated by controlled proteolysis as well as the calmodulin-activated enzyme regardless of the calmodulin concentration. The phosphatidylserine-activated enzyme was inhibited at higher calmidazolium concentrations due to non-specific trapping of the inhibitor by the excess of phospholipid. Addition of calmidazolium did not modify the K0.5 for calcium activation of ATP hydrolysis by the enzyme. The inhibition by calmidazolium was counteracted by Pi. Compound 48/80 also had no effect on the enzyme when only phosphatidylcholine was present and, like calmidazolium, it inhibited the calmodulin-activated enzyme and the phosphatidylserine-activated enzyme. The apparent Ki for inhibition by compound 48/80 was dependent on the calmodulin concentration. However, the enzyme activated by controlled trypsinization was insensitive to compound 48/80. Binding of 48/80 to the enzyme in the presence of phosphatidylserine or calmodulin reversed the increased affinity for Ca2+ caused by these activators.
Pasa
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Departamento de Bioquimica, Universidade Federal do Rio de Janeiro, Brasil.
Otero
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A
Barrabin
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Scofano
H M
HM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D007093:Imidazoles / Q000494:pharmacology*; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology*; D010718:Phosphatidylserines / Q000494:pharmacology; D012508:Sarcolemma / Q000201:enzymology; D014357:Trypsin; D003189:p-Methoxy-N-methylphenethylamine / Q000494:pharmacology*
D002147:Calmodulin; D007093:Imidazoles; D010718:Phosphatidylserines; D003189:p-Methoxy-N-methylphenethylamine; C031938:calmidazolium; D000255:Adenosine Triphosphate; D014357:Trypsin; D000252:Calcium-Transporting ATPases