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

Butscher, Christine; Roudna, Milena; Apell, Hans-Jürgen

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

Dateien

Electrogenic_partial_reactions_of_the.pdfGröße: 934.94 KBDownloads: 504

Datum

1999

Autor:innen

Butscher, Christine

Roudna, Milena

Apell, Hans-Jürgen

Open Access-Veröffentlichung

Open Access Green

Sammlungen

Biologie

Publikationstyp

Zeitschriftenartikel

Publikationsstatus

Published

Erschienen in

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.

Fachgebiet (DDC)

570 Biowissenschaften, Biologie

Schlagwörter

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

Zitieren

ISO 690

BUTSCHER, 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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