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

Autor: Haynes, Duncan, Mandveno, Alan, Haynes, D H, Mandveno, A
Zdroj: Journal of Membrane Biology; 1983, Vol. 74 Issue 1, p25-40, 16p
Abstrakt: The pH dependence of the Ca, Mg-ATPase pump of rabbit skeletal sarcoplasmic reticulum has been analyzed. Active uptake progress curves of the free luminal Ca concentration vs. time were obtained by fluorometric readout. The average rates (evaluated at t =2 sec) and steady-state maximal uptakes ([Ca] were determined at variable external [Ca], set by a Ca/EGTA buffer. The average rates ( t=2 sec) and the [Ca] values showed the same dependence on [Ca] and pH. At pH 7.0, a second-order dependence on [Ca] was observed with a K (equal to [Ca] for half-maximal rate) of 7.3×10 m. Both the average rate and the maximal internal Ca level achieved had identical K values. The apparent affinity of the pump for Ca ( K=1/ K) 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 ( K) vs. pH plot. This is interpreted as competition between 2H and Ca for occupation of each of the two outwardly oriented translocators. The maximal values of average rate ( t=2 sec) and [Ca], were analyzed at saturating [Ca] values to give V and [Ca] 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 Ca is necessary for transport. Partial or full protonation of the negative charges on the outwardly oriented translocators destroys their capacity for Ca transport. This process is responsible for the decrease in apparent translocator affinity with decreasing pH. A pK of 7.2±0.3 was determined for the outwardly oriented translocator. Transport of 2 Ca 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 of V and [Ca] is explained by the dual effect of protonation to lower the apparent affinity of the inwardly oriented translocator for internal Ca and to produce single and doubly protonated forms of the translocator capable of high rates of enzyme dephosphorylation and return. A pK 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 the K, V and [Ca] values. The liminations of the model are evaluated. The structural and physiological significance of the findings is discussed. [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index
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