Peerce B E
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550.
Biochemistry. 1991 Apr 30;30(17):4186-92. doi: 10.1021/bi00231a012.
The glucose derivative, 2,2,6,6-tetramethylpiperidine-1-oxylglucose (TEMPO-glucose) was synthesized and examined for its ability to substitute for glucose as a substrate for the intestinal brush border membrane Na+/glucose cotransporter. TEMPO-glucose inhibited Na(+)-dependent phlorizin binding with an apparent KI of 18 microM and Na(+)-dependent glucose uptake with an apparent KI of 70 microM. The transport competence of TEMPO-glucose was examined by using two measures of transport. The first involved comparing the reversal of trans Na+ inhibition by D-glucose and TEMPO-glucose. The second directly examined Na(+)-dependent TEMPO-glucose uptake by using TEMPO-glucose quenching of intravesicular fluorescein sulfonate fluorescence. Tryptophan fluorescence was sensitive to TEMPO-glucose in a Na(+)-dependent, glucose-inhibitable manner. The bulk of these tryptophans appeared to be located in hydrophobic environments based on Cs(+)-insensitivity. With the reconstituted cotransporter, TEMPO-glucose, and tryptophan quench reagents, the cotransporter was compared in three transport modes: zero trans uptake, zero trans uptake in the presence of a shunt of membrane potential, and substrate exchange. The results suggest that the cotransporter conformation varies depending on its mode of operation and that TEMPO-glucose may be a useful probe for localizing amino acid residues involved in glucose transport.
合成了葡萄糖衍生物2,2,6,6-四甲基哌啶-1-氧基葡萄糖（TEMPO-葡萄糖），并检测了其替代葡萄糖作为肠刷状缘膜Na⁺/葡萄糖共转运体底物的能力。TEMPO-葡萄糖抑制Na⁺依赖性根皮苷结合，其表观抑制常数（KI）为18μM，抑制Na⁺依赖性葡萄糖摄取，表观KI为70μM。通过两种转运测量方法检测了TEMPO-葡萄糖的转运能力。第一种方法是比较D-葡萄糖和TEMPO-葡萄糖对反式Na⁺抑制的逆转情况。第二种方法是通过利用囊泡内荧光素磺酸盐荧光的TEMPO-葡萄糖淬灭直接检测Na⁺依赖性TEMPO-葡萄糖摄取。色氨酸荧光以Na⁺依赖性、葡萄糖可抑制的方式对TEMPO-葡萄糖敏感。基于Cs⁺不敏感性，这些色氨酸中的大部分似乎位于疏水环境中。利用重组的共转运体、TEMPO-葡萄糖和色氨酸淬灭试剂，在三种转运模式下比较了共转运体：零转摄取、存在膜电位分流时的零转摄取和底物交换。结果表明，共转运体的构象因其操作模式而异，并且TEMPO-葡萄糖可能是定位参与葡萄糖转运的氨基酸残基的有用探针。
