CA2-RETICULUM ATPASE REVISITED .1. MECHANISM OF AFFINITY AND COOPERATIVITY MODULATION BY H+ AND MG2+( BINDING TO SARCOPLASMIC)

H+ and Mg2+ are known to inhibit Ca2+ binding to the transport sites o f sarcoplasmic reticulum-ATPase. Evaluation of the affinity for the Ca 2+ binding sites requires measurement of the amount of Ca2+ bound to A TPase as a function of the free Ca2+ concentration imposed by a Ca2+ c helator. The choice of the chelator is crucial as it determines the ac curacy of the free Ca2+ concentration. At pH > 7, the EGTA affinity fo r Ca2+ is higher than that of ATPase, inducing artifacts that alter th e shape of the binding curves. Thus, we have used ,2-bis(2-aminophenox y)ethane-N,N,N',N'-tetraacetic acid (BAPTA), whose affinity is unchang ed at pH greater-than-or-equal-to 7. Ca2+ binding was studied at equil ibrium, from pH 6 to pH 8 and from 0 to 10 mM Mg2+, using EGTA and/or BAPTA and [Ca-45]Ca2+. Under all conditions, the stoichiometry was 2 C a2+/ATPase. At variance with previous studies, the Hill coefficient wa s 1.1-2 and higher at pH 6 than at pH 8. In addition, it decreased in the presence of Mg2+. The Ca2+ binding curves were analyzed according to a model in which they result from a sequential binding of two Ca2+, each binding step being modified by H+ and Mg2+. The effect of H+ is described by two steps involving two H+ and one H+, with pK 7 and 7.9, respectively. At pH 6, ATPase must lose two H+ for the first Ca2+ to bind and a third H+ for the second Ca2+ to bind. At pH 9, both Ca2+ bi nd without any H+ exchange. Mg2+ can bind to all species, except to th at saturated with Ca2+. The species having lost two H+ has a higher af finity for Mg2+ (less-than-or-equal-to mM) than the species having bou nd three H+ (4 mM). The above model allows us to analyze the effects o f H+ and Mg2+ at each Ca2+ binding step and to explain the changes in the apparent affinity and cooperativity.