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Abstract:
The interdependence of the competition between Ca2+ and hydrogen ions for the internally located low‐affinity Ca2+ binding sites of sarcoplasmic reticulum vesicles and the pH‐dependent splitting rate of phosphoenzyme was investigated. Sarcoplasmic reticulum vesicles were preincubated at a selected pH and passive Ca2+ loading, active Ca2+ uptake at the same pH as well as active Ca2+ uptake at a distinct pH (pH‐jump method) were observed. In addition, Cai‐Cao exchange in the absence and presence of ADP and ATP‐ADP exchange were measured. The overall ATP splitting rate was assayed with leaky vesicles in the presence of varied Ca2+ concentration and four different pH. All experiments were carried out at Ca2+ concentrations sufficient to saturate the externally located activating high‐affinity binding sites at all pH and in the absence of affecting concentrations of monovalent cations.
Active Ca2+ transport (particularly evident applying the pH‐jump method) is facilitated at low intravesicular pH, reflecting the favoured Ca2+ release to the intravesicular space, in contrast to the reverse pH‐dependence of passive Ca2+ accumulation and the initial rate of Cai‐Cao exchange, both favoured by elevated internal Ca2+ binding capacity. The rates of ATP splitting, the continuing slow rate of Cai‐Cao exchange, and the ATP‐ADP exchange are optimal at an intermediate proton concentration, reflecting the influence of protons on partial reaction steps occurring later in the reaction cycle and the accelerated exchange of Ca2+ at the internal low‐affinity sites as well as the establishment of a new pseudo equilibrium between the possible reaction intermediates. The pool of rapidly exchangeable Ca2+ is enlarged whereas the rate of slow exchange is unaltered or diminished (pH 7.8) by ADP.
