The pH dependence of the Ca2+, Mg2+-ATPase of sarcoplasmic reticulum: evidence that the Ca2+ translocator bears a doubly negative charge.

Abstract

The pH dependence of the Ca2+, Mg2+-ATPase pump of rabbit skeletal sarcoplasmic reticulum has been analyzed. Active uptake progress curves of the free luminal Ca2+ concentrationvs. time were obtained by fluorometric readout. The average rates (evaluated att =2 sec) and steady-state maximal uptakes ([Ca2+] i were determined at variable external [Ca2+] o , set by a Ca2+/EGTA buffer. The average rates (t=2 sec) and the [Ca2+] i values showed the same dependence on [Ca2+] o and pH. At pH 7.0, a second-order dependence on [Ca2+] o was observed with aKm (equal to [Ca2+] i for half-maximal rate) of 7.3×10−8m. Both the average rate and the maximal internal Ca2+ level achieved had identicalKm values. The apparent affinity of the pump for Ca2+ (Kapp=1/Km) shows little pH dependence between pH values of 7.0 and 8.0. The apparent affinity drops markedly with decreasing pH below pH 7.0, showing a slope of 1.63 on a log (Kapp)vs. pH plot. This is interpreted as competition between 2H+ and Ca2+ for occupation of each of the two outwardly oriented translocators. The maximal values of average rate (t=2 sec) and [Ca2+] i , were analyzed at saturating [Ca2+] o values to giveVm and [Ca2+]i, max values as a function of pH. These two parameters showed identical pH dependence, with a pH optimum between 6.0 and 6.5. These results are explained both qualitatively and quantitatively by a preliminary model of the steady-state behavior of the pump. The model assumes that enzyme dephosphorylation and countertranslocation represent the rate-limiting step of the cycle and that all other steps are at equilibrium. According to the model, the enzyme has two translocator sites, each bearing a doubly negative charge at pH 7.0 and above. Occupation of both sites by Ca2+ is necessary for transport. Partial or full protonation of the negative charges on the outwardly oriented translocators destroys their capacity for Ca2+ transport. This process is responsible for the decrease in apparent translocator affinity with decreasing pH. A pK a of 7.2±0.3 was determined for the outwardly oriented translocator. Transport of 2 Ca2+ is followed by their release to the lumen. Return of the carrier requires the loading of a charge-stoichiometric amount of alkali cation and H+. The pH dependence ofVm and [Ca2+]i, max is explained by the dual effect of protonation to lower the apparent affinity of the inwardly oriented translocator for internal Ca2+ and to produce single and doubly protonated forms of the translocator capable of high rates of enzyme dephosphorylation and return. A pK a of 6.4±0.3 was determined for the inwardly oriented form. Computer analysis shows that the model is capable of predicting the pH dependence of theKm,Vm and [Ca2+]i, max values. The liminations of the model are evaluated. The structural and physiological significance of the findings is discussed.