Time-resolved charge movements in the sarcoplasmatic reticulum Ca-ATPase.

Peinelt, Christine; Apell, Hans-Jürgen (2004). Time-resolved charge movements in the sarcoplasmatic reticulum Ca-ATPase. Biophysical journal, 86(2), pp. 815-824. Biophysical Society 10.1016/S0006-3495(04)74157-0

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The time-resolved kinetics of the Ca(2+)-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 Ca(2+) release and H(+) binding. The faster process with a typical time constant of 30 ms reports the rate-limiting process before Ca(2+) release, probably the conformation transition E(1) --> E(2). The following, slow uptake of positive charge had a pH-dependent time constant, which was 1 s at low pH and approximately 3 s at pH > 8. This process is assigned to an electrically silent conformational relaxation of the state P-E(2) preceding H(+) binding. This interpretation is in agreement with the observation that the fast process was independent of the substrate concentrations (i.e., when [Ca(2+)] > 200 nM, and [ATP] > 20 micro M). The slow process was independent of the Ca(2+) 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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