Pseudosubstrate hydrolysis by the erythrocyte plasma membrane Ca(2+)-ATPase: kinetic evidence for a modified E1 conformation in dimethylsulfoxide.
The purified Ca(2+)-ATPase of pig red cells displays a phosphatase activity towards p-nitrophenylphosphate which is inhibited by Ca2+ in the absence of solvents, and activated by calmodulin. This activity has been attributed to the E2 conformation of the enzyme. Here we show that the pNPPase activity in the absence of Ca2+ is stimulated 10-25-fold by the presence of the organic solvent dimethylsulfoxide (Me2SO). This is an activation that surpasses by severalfold that induced by calmodulin in the absence of the solvent. At 30% Me2SO, activation by calmodulin disappears. In the absence of calmodulin and at pH 7.2, the Ca2+ concentration needed for half-maximal inhibition of the pNPPase activity (K1) increases from 130 microM in the absence of Me2SO to 860 microM at 30% Me2SO. This effect of Me2SO is enhanced at pH 8.0: the K for Ca2+ increases from 2.7 microM in the absence of the solvent to 2.0 mM in its presence. However, the K0.5 for Ca2+ activation of the ATPase activity decreases from 8.3 to 2.6 microM following addition of the same Me2SO concentration. This indicates that, even in the presence of Me2SO, microM Ca2+ concentrations shift the equilibrium towards E1 but the decrease in activity that would be expected if pNPP hydrolysis were catalysed exclusively by the E2 conformation is not observed. The affinity for pNPP as a substrate increases from 2.6 mM in the absence of Me2SO to 1.6 mM in the presence of 20% Me2SO. These results suggest that Me2SO induces multiple effects in the Ca(2+)-ATPase that (i) increase the reactivity of E2 towards substrate: (ii) surpass the activation by calmodulin and, (iii) allow the enzyme to hydrolyze pNPP even when Ca2+ is bound to the high-affinity sites of the enzyme. The change in reactivity is attributed to an increase on substrate catalysis rather than on pNPP binding.
Freire
M M
MM
Departamento de Bioquímica Médica, ICB, Universidade Federal do Rio de Janeiro, Brazil.
Carvalho-Alves
P C
PC
Barrabin
H
H
Scofano
H M
HM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D011487:Protein Conformation; D013552:Swine
D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium; D004121:Dimethyl Sulfoxide