THE DEPHOSPHORYLATION REACTION OF THE CA2-ATPASE FROM PLASMA-MEMBRANES()

The breakdown of phosphoenzyme (EP) of the Ca2+-ATPase from pig red bl ood cell membranes was studied at 37 degrees C by means of a rapid che mical quenching technique. When the enzyme was phosphorylated with [ga mma-P-32]ATP in media without added MgCl2, all the EP formed disappear ed 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 stead y-state hydrolysis of ATP observed under the same experimental conditi ons. ADP accelerated the slow reaction 45-fold (k(app) = 31 +/- 9 s(-1 )) with K-0.5 = 740 +/- 120 mu M as if this reaction represented the d ecay of CaE(1)P, which donated its phosphate to water slowly in the fo rward direction and rapidly to ADP in the reverse direction of the cyc le. Combination of Mg2+ with K-0.5 = 26.3 +/- 5.0 mu M at a single cla ss of site in E(1) before phosphorylation increased EP of rapid breakd own at the expense of ADP-sensitive EP so that, at nonlimiting concent rations of Mg2+ in the phosphorylation media, all EP decomposed at hig h rate. Rapid decomposition was observed even with enough CDTA to chel ate most of the Mg2+ remaining from phosphorylation, suggesting that t he role of Mg2+ during dephosphorylation was to accelerate the transit ion CaE(1)P --> CaE(2)P, preparing EP for hydrolysis. The combination of ATP at a single class of site with K-m = 845 +/- 231 mu M accelerat ed the hydrolysis of CaE(2)P. Calmodulin alone had no effects on depho sphorylation but enhanced acceleration of hydrolysis of CaE(2)P 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 Ca2+-ATPase activity w ith and without calmodulin showed that the transition CaE(1)P --> CaE( 2)P and decomposition of CaE(2)P by hydrolysis are compatible with the ir role as obligatory intermediate reactions in the cycle of hydrolysi s of ATP by the Ca2+-ATPase.