Ca2+-induced conformation changes of intestinal brush border membranes. Studies with fluorescence probes and sulfhydryl reagent.
We have applied the fluorescent probes 1-anilino-8-naphthalene sulfonate (ANS) and pyrene, and an SH-directed reagent, 5,5'-dithiobis(2-nitrobenzoate) (DTNB) in order to analyze Ca2+-induced conformational changes in porcine intestinal brush border membranes. Addition of Ca2+ to the ANS-membrane complex resulted in enhancement of the fluorescence intensity accompanied by an increase of the affinity of the dye for the membranes, shift of the transition temperature of the ANS fluorescence toward a higher temperature range, and disappearance of the slope in the thermal Perrin plot of the fluorescence polarization. These results suggest that the membrane structure becomes more rigid upon binding of Ca2+ to the membranes. This hypothesis was further confirmed by a marked decrease of the excimer formation efficiency of pyrene in the membranes with increasing Ca2+ concentration. Reaction of the membranes with DTNB suggested that there are at least two classes of SH groups with different pseudo-first-order that constants, 1.48 x 10(-3)s-1 (SHf) and 0.25 x 10(-3)s-1 (SHs) at pH 7.4 in the absence of Ca2+. Of the estimated 52.2 nmol of SH groups per mg protein in the presence of sodium dodecyl sulfate, about 23.8 nmol are reactive with DTNB under the same conditions. The reactivity of SH groups with DTNB is responsive to Ca2+ concentration and the maximum number of reactive SH groups was increased by the addition of Ca2+. Based on these results, the conformational changes of the membrane structure induced by Ca2+ are discussed.
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects*; D004228:Dithionitrobenzoic Acid; D005456:Fluorescent Dyes; D007413:Intestinal Mucosa / Q000187:drug effects / Q000648:ultrastructure; D008565:Membrane Proteins / Q000032:analysis; D008871:Microvilli / Q000187:drug effects* / Q000648:ultrastructure; D011485:Protein Binding; D011487:Protein Conformation; D011721:Pyrenes; D013050:Spectrometry, Fluorescence; D013552:Swine
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D011721:Pyrenes; C027132:1-anilino-8-naphthalenesulfonate; D004228:Dithionitrobenzoic Acid; C030984:pyrene; D002118:Calcium