Effect of carbonylcyanide m-chlorophenylhydrazone on the calcium-stimulated ATPase activity of erythrocyteghosts.
Incubation of etythrocyte ghosts with carbonylcyanide m-chlorophenyl-hydrazone (CCCP) plus Ca-2+ resulted in inactivation of the Ca-2+ -stimulated ATPase activity. Omission of Ca-2+ or lowering of the temperature below 25 degrees C eliminated the inhibitory effect, as also did the presence of ATP during the incubation. On the other hand, the addition of beta-mercaptoethanol did not influence the Ca-2+ -dependent inhibition by CCCP. Compared with the level of CCCP which uncouples oxidative phosphorylation, a rather high level (0.5 mM) of CCCP was required to inhibit the ATPase activity in ghosts. However, once the inhibition had been accomplished, almost all of the CCCP could be removed from the ghost membrane by washing with a Ca-2+ -containing solution, without affecting the inhibition of ATPase. If ethylene-glycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid was included in the washing medium, the inhibition of ATPase was nearly completely reversed by washing. The results indicate that only the Ca-2+ -stimulated, Mg-2+ -ATPase was inhibited by 0.5 mM CCCP, while the remaining components of the ATPase activity were slightly inhibited by higher levels of the uncoupler. Low levels of CCCP (0.1 mM) stimulated the Mg-2+ -ATPase slightly. CCCP was much more specific for the Ca-2+ -stimulated ATPases than N-(1-naphthyl)maleimide, an unusually effective sulfhydryl reagent, and the requirement of Ca-2+ for inactivation was also quite specific.
D000251:Adenosine Triphosphatases / Q000097:blood*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D004912:Erythrocytes / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009284:Naphthols / Q000494:pharmacology; D009570:Nitriles / Q000494:pharmacology*; D011188:Potassium / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology; D013552:Swine; D013997:Time Factors
D008301:Maleimides; D009284:Naphthols; D009570:Nitriles; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D012964:Sodium; D000251:Adenosine Triphosphatases; D008274:Magnesium; D011188:Potassium; D002118:Calcium