DISSOCIATION OF CA2-RETICULUM CA2+-ATPASE AND CHANGES IN FLUORESCENCEOF OPTICALLY SELECTED TRP RESIDUES, EFFECTS OF KCL AND NACL AND IMPLICATIONS FOR SUBSTEPS IN CA2+ DISSOCIATION( FROM SARCOPLASMIC)

Sequential dissociation of the two Ca2+ ions bound to non-phosphorylat ed sarcoplasmic reticulum Ca2+-ATPase was triggered by addition, in a stopped-flow experiment, of quin2, which acted both as a high-affinity chelator and as a Ca2+-sensitive fluorescent probe, The kinetics of C a2+ dissociation were deduced from the observed changes' in quin2 fluo rescence in the visible region (with lambda(ex) = 313 nm), while fluor escence detection in the UV region (with lambda(ex) = 290 nm) made it possible to monitor the tryptophan fluorescence changes accompanying t his dissociation under the same ionic conditions. In the absence of KC l or NaCl, at pH 6 or 7, the observed changes in quin2 fluorescence we re monoexponential, with rate constants very close to those of the cha nges in ATPase tryptophan fluorescence, which also appeared monophasic , In the presence of 100 mM KCl, quin2 fluorescence changes, although still monoexponential, were faster than in the absence of the monovale nt ions but distinctly slower than the changes in tryptophan fluoresce nce, which were accelerated to a larger extent. In addition, the appar ent kinetics of the Trp fluorescence changes depended on the excitatio n wavelength. Using an excitation wavelength of 296 nm, the Trp fluore scence drop was still faster than with an excitation wavelength of 290 nm, and in the presence of NaCl it even displayed a clear undershoot. We conclude that in the presence of KCl or NaCl and with an excitatio n wavelength of 290 nm, the rapid drop in tryptophan fluorescence main ly monitors the dissociation of the first of the two Ca2+ ions to be r eleased from Ca2+ ATPase, while excitation at 296 nm optically selects a subpopulation of Trp residues whose fluorescence level is lower in the ATPase species with one Ca2+ ion bound than in the Ca2+-deprived A TPase species. The latter conditions result in an initial drop in Trp fluorescence whose apparent rate constant (in single-exponential analy sis) is faster than the true rate of dissociation of the first Ca2+ io n and in a subsequent slower rise related to dissociation of the secon d Ca2+ ion. The difference between results obtained in the absence and in the presence of K+ or Na+ is due to an antagonizing effect of thes e cations on proton-induced conformational rearrangement of Ca2+-free ATPase, a conformational rearrangement which changes the ATPase Trp fl uorescence level and significantly affects the cooperativity of Ca2+ b inding at equilibrium.