Pre-steady-state kinetic study of the effects of K+ on the partial reactions of the catalytic cycle of the plasma membrane Ca2+-ATPase

Abstract

The effects of 100 mM K+ on the partial reactions that take place during ATP hydrolysis by the calcium ion-dependent ATPase from plasma membrane (PM-Ca2+-ATPase) were studied at 37 °C on fragmented intact membranes from pig red cells by means of a rapid chemical quenching technique. At 10 μM [γ-32P]ATP plus non-limiting concentrations of Ca2+ and Mg2+, K+ increased the kapp of formation by 140% to 84±11 s-1 and the steady-state level of phosphoenzyme (EP) by 25% to 3.4±0.17 pmol/mg of protein. If added together with [γ-32P]ATP at the beginning of phosphorylation, K+ was much less effective than if added earlier, indicating that it did not act on the phosphorylation reaction. Measurements of the E2 → E1 transition by phosphorylation showed that in medium with Ca2+ and Mg2+, K+ increased the kapp of the transition by 55% to 14±3 s-1 and the apparent concentration of E1 by 45%, suggesting that this may be the cause of the increased rate of phosphorylation observed in enzyme preincubated with K+. The presence of K+ did not change the slow decay of EP without Mg2+ but activated the decay of EP made with Mg2+, increasing its kapp by 60% to 91±12 s-1. In contrast with observations made during phosphorylation, if added at the beginning of dephosphorylation K+ was fully effective in favouring decomposition of EP made in medium containing no K+. In the presence of either 3 mM ATP or 3 mM ATP plus calmodulin, which activate hydrolysis of CaE2P, the effect of K+ on dephosphorylation was conserved. Because the sites for K+ are intracellular and the concentration of K+ in normal red cells is above 100 mM, the effects described here must be taken into account to describe the catalytic cycle of the PM-Ca2+-ATPase under physiological conditions.