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Electrogenic partial reactions of the SR-Ca-ATPase investigated by a fluorescence method

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1999

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Journal of Membrane Biology. 1999, 168(2), pp. 169-181. ISSN 0022-2631. eISSN 1432-1424. Available under: doi: 10.1007/s002329900507

Zusammenfassung

A fluorescence method was adapted to investigate active ion transport in membrane preparations of the SR-Ca-ATPase. The styryl dye RH421 previously used to investigate the Na,K-ATPase was replaced by an analogue, 2BITC, to obtain optimized fluorescence changes upon substrate-induced partial reactions. Assuming changes of the local electric field to be the source of fluorescence changes that are produced by uptake/release or by movement of ions inside the protein, 2BITC allowed the determination of electrogenic partial reactions in the pump cycle. It was found that Ca2+ binding on the cytoplasmic and on the lumenal side of the pump is electrogenic while phosphorylation and conformational transition showed only minor electrogenicity. Ca2+ equilibrium titration experiments at pH 7.2 in the two major conformations of the protein indicated cooperative binding of two Ca2+ ions in state E1 with an apparent half-saturation concentration, KM of 600 nm. In state P-E2 two KM values, 5 μm and 2.2 mM, were determined and are in fair agreement with published data. From Ca2+ titrations in buffers with various pH and from pH titrations in P-E2, it could be demonstrated that H+ binding is electrogenic and that Ca2+ and H+ compete for the same binding site(s). Tharpsigargin-induced inhibition of the Ca-ATPase led to a state with a specific fluorescence level comparable to that of state E1 with unoccupied ion sites, independent of the buffer composition.

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570 Biowissenschaften, Biologie

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Calcium binding, Ion transport, Binding site, Electrogenicity, Styryl dyes, pH dependence

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ISO 690BUTSCHER, Christine, Milena ROUDNA, Hans-Jürgen APELL, 1999. Electrogenic partial reactions of the SR-Ca-ATPase investigated by a fluorescence method. In: Journal of Membrane Biology. 1999, 168(2), pp. 169-181. ISSN 0022-2631. eISSN 1432-1424. Available under: doi: 10.1007/s002329900507
BibTex
@article{Butscher1999Elect-6520,
  year={1999},
  doi={10.1007/s002329900507},
  title={Electrogenic partial reactions of the SR-Ca-ATPase investigated by a fluorescence method},
  number={2},
  volume={168},
  issn={0022-2631},
  journal={Journal of Membrane Biology},
  pages={169--181},
  author={Butscher, Christine and Roudna, Milena and Apell, Hans-Jürgen}
}
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