Electrogenic Partial Reactions of the SR-Ca-ATPase Investigated by a Fluorescence Method

Electrogenic Partial Reactions of the SR-Ca-ATPase Investigated by a Fluorescence Method 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, K M of 600 nm. In state P-E2 two K M 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

Electrogenic Partial Reactions of the SR-Ca-ATPase Investigated by a Fluorescence Method

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Publisher
Springer-Verlag
Copyright
Copyright © Inc. by 1999 Springer-Verlag New York
Subject
Life Sciences; Biochemistry, general; Human Physiology
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s002329900507
Publisher site
See Article on Publisher Site

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