Peerce B E
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550.
J Biol Chem. 1990 Jan 25;265(3):1737-41.
Conformations of the Na+/glucose cotransporter were examined using tryptophan fluorescence and substrates to induce cotransporter conformational changes. Addition of Na+ but not K+ or TMA+ resulted in a saturable quenching of tryptophan fluorescence with a K0.5 for Na+ of 28 mM. In the presence of saturating Na+ concentrations, d-glucose but not l-glucose, fructose, or phlorizin resulted in a partial return of tryptophan fluorescence to approximately 70% of the substrate-free levels. This return of tryptophan fluorescence was a saturable function of d-glucose concentration with a K0.5 of 43 microM. The three conformations were compared with respect to their sensitivity to tryptophan quench reagents. Acrylamide quenching was unaffected by substrates. In contrast, I- quenching decreased 40% in the presence of Na+, while Cs+ quenching increased 64%. Addition of saturating d-glucose concentrations resulted in the return of I- quenching to 90% of the substrate-free values and reduced Cs+ quenching to substrate-free levels. In contrast, phlorizin did not mimic the effect of d-glucose on tryptophan fluorescence. These results are interpreted in terms of a second substrate-induced cotransporter conformational change which based on similar substrate specificities appears directly related to cotransporter-mediated Na+ and d-glucose transport.
利用色氨酸荧光和底物诱导共转运体构象变化，对钠/葡萄糖共转运体的构象进行了研究。添加Na⁺而非K⁺或TMA⁺会导致色氨酸荧光的饱和猝灭，Na⁺的K0.5为28 mM。在饱和Na⁺浓度存在的情况下，d-葡萄糖而非l-葡萄糖、果糖或根皮苷会使色氨酸荧光部分恢复至无底物水平的约70%。色氨酸荧光的这种恢复是d-葡萄糖浓度的饱和函数，K0.5为43 microM。比较了三种构象对色氨酸猝灭试剂的敏感性。丙烯酰胺猝灭不受底物影响。相比之下，在存在Na⁺的情况下，I⁻猝灭降低了40%，而Cs⁺猝灭增加了64%。添加饱和d-葡萄糖浓度会使I⁻猝灭恢复至无底物值的90%，并将Cs⁺猝灭降低至无底物水平。相比之下，根皮苷并未模拟d-葡萄糖对色氨酸荧光的影响。这些结果根据第二种底物诱导的共转运体构象变化进行解释，基于相似的底物特异性，该变化似乎与共转运体介导的Na⁺和d-葡萄糖转运直接相关。
