The dephosphorylation reaction of the Ca(2+)-ATPase from plasma membranes

Abstract

The breakdown of phosphoenzyme (EP) of the Ca(2+)-ATPase from pig red blood cell membranes was studied at 37 degrees C by means of a rapid chemical quenching technique. When the enzyme was phosphorylated with [gamma-32P]ATP in media without added MgCl2, all the EP formed disappeared along two single exponential curves, a rapid one with k(app) = 90 +/- 10 s-1 and a slow one with k(app) = 0.7 +/- 0.3 s-1. The amount of EP involved in each reaction was close to 50% of the EP present at the beginning. Only EP of rapid breakdown could account for the steady-state hydrolysis of ATP observed under the same experimental conditions. ADP accelerated the slow reaction 45-fold (k(app) = 31 +/- 9 s-1) with K0.5 = 740 +/- 120 microM as if this reaction represented the decay of CaE1P, which donated its phosphate to water slowly in the forward direction and rapidly to ADP in the reverse direction of the cycle. Combination of Mg2+ with K0.5 = 26.3 +/- 5.0 microM at a single class of site in E1 before phosphorylation increased EP of rapid breakdown at the expense of ADP-sensitive EP so that, at nonlimiting concentrations of Mg2+ in the phosphorylation media, all EP decomposed at high rate. Rapid decomposition was observed even with enough CDTA to chelate most of the Mg2+ remaining from phosphorylation, suggesting that the role of Mg2+ during dephosphorylation was to accelerate the transition CaE1P-->CaE2P, preparing EP for hydrolysis. The combination of ATP at a single class of site with Km = 845 +/- 231 microM accelerated the hydrolysis of CaE2P. Calmodulin alone had no effects on dephosphorylation but enhanced acceleration of hydrolysis of CaE2P by ATP making the decay of EP under these conditions the fastest among those measured. Comparison of the rates of dephosphorylation of EP made in the presence of Mg2+ with those of steady-state Ca(2+)-ATPase activity with and without calmodulin showed that the transition CaE1P-->CaE2P and decomposition of CaE2P by hydrolysis are compatible with their role as obligatory intermediate reactions in the cycle of hydrolysis of ATP by the Ca(2+)-ATPase.