Interaction of the sugar carrier of intestinal brush-border membranes with HgCl 2

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HgCl 2 was used as an inhibitor and potential label for the glucose carrier of intestinal brush-border membranes. Half-maximal inhibition of Na +-dependent d-glucose uptake was reached with micromolar concentrations of HgCl 2 when the protein concentration was 1.2 mg/ml. Similar concentrations were found to inhibit the binding of [ 3H]phlorizin, a reversible competitive inhibitor of sugar transport. Inhibition was reversed by dithioerythritol but only marginally by EDTA. The data support the involvement of a sulfhydryl group in the inhibitory process. Deoxycholate-extracted membranes, which are enriched in specific phlorizin binding activity, were used for labeling studies using 203HgCl 2. The polypeptides were separated by gel electrophoresis and analyzed by protein staining and autoradiography. Non-specific 203HgCl 2 labeling was minimized by pre-treatment with sulfhydryl reagents which do not inhibit phlorizin binding. Several bands, which are lost from the autoradiographic pattern during a negative purification of the phlorizin binding sites, could be ruled out as essential components of the sugar carrier. The polypeptide profile was also analyzed following proteolysis, which abolished phlorizin binding. Those radioactive bands of which apparent M r values were altered by the treatment were considered as possible candidates. Finally, samples in which inhibition was reversed by thiols were also studied. The possible identity of the polypeptide(s) involved in glucose translocation is discussed in the light of these observations.