Progressive inactivation of plasma membrane (Ca2++Mg2+)ATPase by Cd2+ in the absence of ATP and reversible inhibition during catalysis.
The influence of Cd2+ ions on plasma membrane (Ca2++Mg2+)ATPase activity from red cells was investigated. When the membranes were preincubated with Cd2+ in the absence of ATP, basal (Ca2++Mg2+)ATPase activity (no calmodulin) was slowly and irreversibly inhibited (inactivated) following first-order kinetics (k/Ki = 0.0057 microM-1 min-1 at [Cd2+] = 0.25-1 microM). However, preincubation with Cd2+ did not affect the degree of stimulation by calmodulin added to the assay medium together with ATP. Inactivation was not released by prolonged exposure of membranes to EGTA prior to catalysis, but it was strongly attenuated when the pH in the preincubation medium was lowered from 7.2 to 6.4. When the reaction was started by supplying membranes simultaneously with Cd2+ and ATP (no preincubation), (Ca2++Mg2+)ATPase was inhibited by increasing concentrations of the CdATP complex ([CdATP]50 = 7.2 microM). In this condition, however, even total inhibition of the pump was almost completely released after addition of enough EGTA to decrease CdATP concentrations to the nanomolar range. These results, taken together, indicate that inactivation of the unphosphorylated enzyme by Cd2+ is influenced by dissociation of amino acid residues exhibiting pK between 6.0 and 7.0, and that recognition by the pump of the physiological modulator calmodulin is preserved in the preincubated pump molecules which did not undergo inactivation. In addition, they show that the catalytic site is a target for reversible inhibition of the pump by CdATP and that occupancy of the nucleotide binding site prevents inactivation.
Toledo-Maciel
A
A
Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brasil.
Gonçalves-Gomes
S
S
de Gouveia Castex
M
M
Vieyra
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors* / Q000097:blood*; D002104:Cadmium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013379:Substrate Specificity / Q000187:drug effects; D013997:Time Factors
D002147:Calmodulin; D002104:Cadmium; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase