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Abstract

D-Glucose-binding polypeptides in the Na⁺-D-glucose cotransporter from pig renal cortex were identified by affinity labeling with two D-glucose analogs, 10-N-(N-[4-azido-2-nitrophenyl]-beta-alanyl)amino-1-decyl-beta-D- glucopyranoside (NapADG) and 10-N-(bromoacetyl)amino-1-decyl-beta-D-glucopyranoside (BADG). During short-term incubation in the dark, NapADG and BADG are reversible inhibitors of Na⁺ gradient-dependent D-glucose uptake and Na⁺-dependent phlorizin binding with K_i values of about 40 and 400 microM, respectively. Irreversible inhibition of Na⁺-dependent phlorizin binding, which was prevented by D-glucose or phlorizin, was measured after a 1-h incubation with BADG. Both NapADG and BADG selectively labeled polypeptides with apparent molecular weights of 82,000, 75,000, 64,000, and 47,000. Since labeling of the Mr 82,000 and 75,000 polypeptides by both analogs was partially dependent on the presence of Na⁺ and was partially protected by D-glucose or phlorizin but not by L-glucose or D-mannose, these polypeptides are thought to be components of the renal Na⁺-D-glucose cotransporter which contain D-glucose-binding sites. For the Mr 64,000 and 47,000 polypeptides, Na⁺ dependence and D-glucose protection were not constantly observed. However, also, these polypeptides are thought to be components or proteolytic splitting products of the Na⁺-D-glucose cotransporter since we observed that three monoclonal antibodies showed cross-reaction with the BADG-labeled Mr 82,000, 64,000, and 47,000 polypeptides (K. Korn, A. Raszeja-Specht, S. Bernotat-Danielowski, and H. Koepsell, manuscript in preparation). When the BADG-labeled Mr 82,000 and 75,000 polypeptides were analyzed after two-dimensional separation by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, three-labeled, D-glucose-protectable polypeptides with the respective molecular weights and isoelectric points of 82,000 and 5.6, 75,000 and 5.4, and 75,000 and 6.9 were distinguished. The data indicate that renal brush-border membranes contain several polypeptides which are components of the Na⁺-D-glucose cotransporter and contain D-glucose-binding sites.