Fluorescence probe studies on the interaction of phlorizin with rabbit intestinal brush border membranes.
Spectrofluorometric studies on the interaction of phlorizin with rabbit intestinal brush border membranes under various conditions were carried out using 1-anilino-8-naphthalene sulfonate (ANS) and N-(1-anilinonaphthyl-4)maleimide (ANM). The fluorescence intensity of membrane-bound ANS was markedly decreased upon addition of phlorizin. The apparent dissociation constant of phlorizin and the membranes at pH 7.4 was determined to be approximately 24 microM from the slope of a plot of the fluorescence change of membrane-bound ANS against phlorizin concentration. Addition of D-glucose diminished the quenching effect of phlorizin on the ANS fluorescence by lowering the binding affinity of phlorizin for the membranes. The interaction between phlorizin and the membranes was influenced by temperature, ionic strength and divalent cations. The binding affinity of phlorizin for the membranes is markedly reduced at higher temperature, showing a transition between 25 and 30 degrees C. Imposition of an ionic strength gradient across the membrane vesicles (out greater than in) increased the binding affinity. On the other hand, divalent cations decreased the affinity; Mg2+ was more effective than Ca2+. The binding affinity of ANS for the membranes was evidently decreased, and the reactivity of SH groups of the membrane proteins with ANM was apparently reduced, upon binding of phlorizin to the membranes. Based on these results, the relationships among modification of membrane structures, the extent of phlorizin binding and phlorizin-induced conformational changes in the membrane proteins are discussed.
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D005456:Fluorescent Dyes; D005947:Glucose / Q000494:pharmacology; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D008301:Maleimides; D008871:Microvilli / Q000378:metabolism*; D010695:Phlorhizin / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D008301:Maleimides; C027132:1-anilino-8-naphthalenesulfonate; C010854:N-(1-anilinonaphthalyl)-4-maleimide; D010695:Phlorhizin; D005947:Glucose