Terbium as a fluorescent probe for analysis of the nature of Ca2+-binding sites of rat intestinal mucosal membranes.

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1. The addition of Tb3+ to rat intestinal mucosal membranes resulted in a marked enhance-ment of Tb3+ fluorescence at 550 nm and some decrease in the native fluorescence of the membranes at 330nm. 2. The pH dependence profile of Tb3+ fluorescence indicates that the development of Tb3+ fluorescence upon its addition to the membranes is related to changes in the states of tryptophan and/or tyrosine residues of the membrane proteins. 3. More than one type of Tb3+-binding site appears to be present in the membranes; the apparent dissociation constant of one type of Tb3+-membrane complex is 10.2 μM at pH 7.4. 4. Tb3+ fluorescence increased in a sigmoidal fashion with increasing concentration of Tb3+ in the presence of Ca2+, but the Hill coefficient (n_??_2) was almost the same regardless of the presence of Ca2+. 5. Addition of Ca2+ or Mg2+ to the medium induced marked decreases in both Tb3+ fluorescence and native fluorescence of the membranes. The apparent dissociation constants of Ca2+- and Mg2+-membrane complexes were estimated from the changes in Tb3+ fluorescence to be 2.89 and 5.35mM, respectively. Mg2+ markedly reduced the binding affinity of Ca2+ for the membranes. 6. The Ca2+-induced reduction of the fluorescence of Tb3+-membrane complex was attenuated at high ionic strength.On the basis of these results, the nature of the Ca2+-binding sites of rat intestinal mucosal membranes is discussed.
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