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

A fluorescence method was adapted to investigate active ion transport in me mbrane preparations of the SR-Ca-ATPase. The styryl dye RH421 previously us ed to investigate the Na,K-ATPase was replaced by an analogue, 2BITC, to ob tain optimized fluorescence changes upon substrate-induced partial reaction s. Assuming changes of the local electric field to be the source of fluores cence changes that are produced by uptake/release or by movement of ions in side the protein, 2BITC allowed the determination of electrogenic partial r eactions in the pump cycle. It was found that Ca2+ binding on the cytoplasm ic and on the lumenal side of the pump is electrogenic while phosphorylatio n and conformational transition showed only minor electrogenicity. Ca2+ equ ilibrium titration experiments at pH 7.2 in the two major conformations of the protein indicated cooperative binding of two Ca2+ ions in state E-1 wit h an apparent half-saturation concentration, K-M of 600 nM. In state P-E-2 two K-M values, 5 mu M and 2.2 mM, were determined and are in fair agreemen t with published data. From Ca2+ titrations in buffers with various pH and from pH titrations in P-E-2, it could be demonstrated that H+ binding is el ectrogenic and that Ca2+ and H+ compete for the same binding site(s). Tharp sigargin-induced inhibition of the Ca-ATPase led to a state with a specific fluorescence level comparable to that of state E-1 with unoccupied ion sit es, independent of the buffer composition.