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

摘要

pThe effects of 100 mM Ksup+/sup on the partial reactions that take place during ATP hydrolysis by the calcium ion-dependent ATPase from plasma membrane (PM-Casup2+/sup-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-sup32/supP]ATP plus non-limiting concentrations of Casup2+/sup and Mgsup2+/sup, Ksup+/sup increased the ik/isubapp/sub of formation by 140% to 84±11 ssup-1/sup and the steady-state level of phosphoenzyme (EP) by 25% to 3.4±0.17 pmol/mg of protein. If added together with [γ-sup32/supP]ATP at the beginning of phosphorylation, Ksup+/sup was much less effective than if added earlier, indicating that it did not act on the phosphorylation reaction. Measurements of the Esub2/sub → Esub1/sub transition by phosphorylation showed that in medium with Casup2+/sup and Mgsup2+/sup, Ksup+/sup increased the ik/isubapp/sub of the transition by 55% to 14±3 ssup-1/sup and the apparent concentration of Esub1/sub by 45%, suggesting that this may be the cause of the increased rate of phosphorylation observed in enzyme preincubated with Ksup+/sup. The presence of Ksup+/sup did not change the slow decay of EP without Mgsup2+/sup but activated the decay of EP made with Mgsup2+/sup, increasing its ik/isubapp/sub by 60% to 91±12 ssup-1/sup. In contrast with observations made during phosphorylation, if added at the beginning of dephosphorylation Ksup+/sup was fully effective in favouring decomposition of EP made in medium containing no Ksup+/sup. In the presence of either 3 mM ATP or 3 mM ATP plus calmodulin, which activate hydrolysis of CaEsub2/subP, the effect of Ksup+/sup on dephosphorylation was conserved. Because the sites for Ksup+/sup are intracellular and the concentration of Ksup+/sup 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-Casup2+/sup-ATPase under physiological conditions./p