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Time-Resolved Charge Movements in the Sarcoplasmatic Reticulum Ca-ATPase

Department of Biology, University of Konstanz, 78457 Konstanz, Germany

Correspondence: Address reprint requests to Hans-Jürgen Apell, Dept. of Biology, University of Konstanz, Fach M635, 78457 Konstanz, Germany. Tel.: +49-7531-88-2253; Fax: +49-7531-88-3183; E-mail: h-j.apell{at}uni-konstanz.de.

The time-resolved kinetics of the Ca²⁺-translocating partial reaction of the sarcoplasmatic reticulum Ca-ATPase was investigated by ATP-concentration jump experiments. ATP was released by an ultraviolet light flash from its inactive precursor and charge movements in the membrane domain of the ion pumps were detected by the fluorescent styryl dye 2BITC. Two oppositely directed cation movements were found, which were assigned to Ca²⁺ release and H⁺ binding. The faster process with a typical time constant of 30 ms reports the rate-limiting process before Ca²⁺ release, probably the conformation transition E₁ E₂. The following, slow uptake of positive charge had a pH-dependent time constant, which was 1 s at low pH and 3 s at pH > 8. This process is assigned to an electrically silent conformational relaxation of the state P-E₂ preceding H⁺ binding. This interpretation is in agreement with the observation that the fast process was independent of the substrate concentrations (i.e., when [Ca²⁺] > 200 nM, and [ATP] > 20 µM). The slow process was independent of the Ca²⁺ concentration. The activation energy of the resolved processes was between 80 kJ/mol and 90 kJ/mol, which is comparable to the activation energy of the enzymatic activity (92 kJ/mol) and these high values point to conformational changes underlying rate-limiting steps of the pump cycle.

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