Peerce B E, Cedilote M, Clarke R D
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550.
Am J Physiol. 1993 Feb;264(2 Pt 1):G294-9. doi: 10.1152/ajpgi.1993.264.2.G294.
The role of sulfhydryl (SH) and carboxylic acid residues in Na(+)-dependent glucose uptake, Na(+)-dependent phlorizin binding, and substrate exchange by the rabbit small intestinal brush-border membrane (BBM) Na(+)-glucose cotransporter was examined in sodium dodecyl sulfate-BBM vesicles. The sulfhydryl reagent p-chloromercuribenzoate (PCMB) inhibited all three measures of cotransporter function in a dithiothreitol-sensitive manner with similar K0.5 values (concn of PCMB resulting in 50% inhibition). PCMB sulfonate had no effect on Na(+)-glucose cotransporter function < 250 microM. The carboxylic acid reagent 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide no effect on Na(+)-glucose cotransporter function. N,N'-dicyclohexylcarbodiimide (DCCD) inhibited all three measures of cotransporter function with similar K0.5 values for inhibition. Inhibition by DCCD did not require addition of a nucleophile. In contrast, PCMB-pretreated cotransporter was insensitive to DCCD in the absence of added nucleophile with respect to substrate transport (Na(+)-dependent glucose uptake) but not Na(+)-dependent phlorizin binding. These results indicate an intravesicular or lipophilic environment for both the PCMB-reactive SH residue and the DCCD-reactive carboxylic acid residues, suggesting that a SH residue may act as an endogenous nucleophile for interaction of DCCD with the Na(+)-glucose cotransporter and suggesting that different carboxylic acid residues may be involved in Na(+)-dependent glucose uptake and Na(+)-dependent phlorizin binding.
在十二烷基硫酸钠 - 小肠刷状缘膜（BBM）囊泡中，研究了巯基（SH）和羧酸残基在兔小肠刷状缘膜Na⁺依赖性葡萄糖摄取、Na⁺依赖性根皮苷结合以及底物交换过程中，对Na⁺ - 葡萄糖共转运蛋白的作用。巯基试剂对氯汞苯甲酸（PCMB）以二硫苏糖醇敏感的方式抑制共转运蛋白功能的所有三项指标，其半数抑制浓度（K0.5值）相似（PCMB浓度导致50%抑制）。在浓度低于250微摩尔时，PCMB磺酸盐对Na⁺ - 葡萄糖共转运蛋白功能无影响。羧酸试剂1 - 乙基 - 3 -（4 - 氮杂 - 4,4 - 二甲基戊基）碳二亚胺对Na⁺ - 葡萄糖共转运蛋白功能无影响。N,N' - 二环己基碳二亚胺（DCCD）以相似的K0.5抑制值抑制共转运蛋白功能的所有三项指标。DCCD的抑制作用不需要添加亲核试剂。相反，在没有添加亲核试剂的情况下，对于底物转运（Na⁺依赖性葡萄糖摄取），经PCMB预处理的共转运蛋白对DCCD不敏感，但对Na⁺依赖性根皮苷结合仍敏感。这些结果表明，PCMB反应性SH残基和DCCD反应性羧酸残基都处于囊泡内或亲脂性环境中，这表明一个SH残基可能作为内源性亲核试剂，参与DCCD与Na⁺ - 葡萄糖共转运蛋白的相互作用，也表明不同的羧酸残基可能参与Na⁺依赖性葡萄糖摄取和Na⁺依赖性根皮苷结合。
