The effect of Ca2+ and calmodulin on the inhibition of Ca2(+)+Mg2(+)-ATPase in erythrocyte ghost membranes by nonpolar and polar carbodiimides.
N,N'-dicyclohexylcarbodiimide (DCCD) and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide (CMCD) inhibited calmodulin-dependent Ca2(+)+Mg2(+)-ATPase activity in erythrocyte ghost membranes. The extent of the inhibition caused by carbodiimides strongly depended on their hydrophobicity. Hydrophobic DCCD was a more potent inhibitor then hydrophilic CMCD. Calmodulin (CaM) protected the enzyme against the former carbodiimide, whereas Ca2+ did the same against the latter. In contrast to previous observations made by Villalobo et al., on the purified enzyme, neither carbodiimide affected the calmodulin-independent ATPase activity in ghost membranes. Inhibition of the calmodulin-dependent ATPase activity was due to a decrease of the maximum activity, whereas the Km value for Ca2+ remained unchanged. Titration of erythrocyte ghost membranes with CaM revealed a biphasic response of ATPase to this activator. Two affinity constants were found for CaM, 0.64 nM and 14 nM. DCCD affected the interaction with CaM at high- and low-affinity binding sites in a competitive manner. CMCD acted as a noncompetitive inhibitor for CaM low-affinity sites, whereas it behaved in a competitive way against CaM interaction with high-affinity sites. In E2 form (stabilized by vanadate and EGTA) ATPase was more sensitive to carbodiimides than in E1 form (induced by La3+).
Famulski
K S
KS
Department of Cellular Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Pikula
S
S
Wrzosek
A
A
Wojtczak
A B
AB
D000818:Animals; D001665:Binding Sites; D003028:CME-Carbodiimide / Q000031:analogs & derivatives*; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors*; D002118:Calcium / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002147:Calmodulin / Q000494:pharmacology*; D002234:Carbodiimides / Q000494:pharmacology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000201:enzymology*; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D013552:Swine; D014638:Vanadates / Q000494:pharmacology
D002147:Calmodulin; D002234:Carbodiimides; C060331:1-cyclohexyl-3-(2-(4-morpholinyl)ethyl)carbodiimide; D003028:CME-Carbodiimide; D014638:Vanadates; D004024:Dicyclohexylcarbodiimide; D007811:Lanthanum; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium