| Abstract: | Absorbance and fluorescence changes of oxacarbocyanine dyes during ATP-induced Ca2+ transport in rabbit sarcoplasmic reticulum were analyzed. The response of the probes is complex and contains contributions from the binding of Ca2+ and ATP to the membrane. In a medium of 0.12 M KCl and 5 mM MgCl2, the fluorescence of Di-O-C5(3) is decreased by Ca2+ or ATP with apparent dissociation constants of 0.2 and 5 micron, respectively. This suggests that oxacarbocyanines respond to binding of Ca2+ and ATP at the active site of Ca2+ transport ATPase. The effect of ATP is observed in the absence of divalent cations. Further changes in the fluorescence or absorbance of cyanine dyes occur at millimolar concentrations of Ca2+ or during ATP-induced Ca2+ uptake, which can be related to Ca2+ binding to low affinity, relatively nonspecific binding sites on the membrane, that can also bind K+ and Mg2+. The optical changes due to Ca2+ accumulation are most pronounced in media of 0.25 M sucrose and much reduced in 0.12 M KCl and 5 mM MgCl2, in accord with competition by K+ and Mg2+ for the low affinity Ca2+ binding sites. These effects must be taken into account in the evaluation of the magnitude and direction of membrane potential in sarcoplasmic reticulum vesicles during Ca2+ uptake and release. |
