ROLE OF CARBOXYL AND SULFHYDRYL RESIDUES ON RABBIT SMALL INTESTINAL BRUSH-BORDER MEMBRANE NA+-GLUCOSE COTRANSPORTER

The role of sulfydryl (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 sulfydryl 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 muM. The carboxylic acid reagent 1-ethyl-3-(4-azonia-4,4-dimethyl-pentyl)carbodiimide had no effect on Na+-glucose cotransporter function. NN'-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.