Characterization of an essential disulfide bond associated with the active site of the renal brush-border membrane D-glucose transporter.
In a previous report (J. Biol. Chem. 258 (1983) 3565-3570) we have demonstrated that the disulfide-reducing agent dithiothreitol has two effects on the sodium-dependent outer cortical brush border membrane D-glucose transporter; the first results in a reversible increase in the affinity of the transporter for the non-transported competitive inhibitor phlorizin, while the second results in a partially reversible loss of phlorizin binding and glucose-transport activity. Evidence was presented that both of these effects are the result of the reduction of disulfide bonds on the transport molecule. In the present paper we extend our observations on the inactivation of the transporter by dithiothreitol. We provide evidence here (i) that the inactivation of the transporter by dithiothreitol is independent of the effect of the reducing agent on the affinity of the transporter, (ii) that this inactivation process is first-order in dithiothreitol and thus presumably due to the reduction of a single disulfide bond essential to the functioning of the transporter. (iii) that it is the reduction of this disulfide bond and not some subsequent conformational or other change in the transporter which results in its inactivation, (iv) that phlorizin and substrates of the transporter provide protection against inactivation by dithiothreitol and that the degree of protection provided correlates well with the known specificity and phlorizin-binding properties of the transporter, and (iv) that the reactivity of the transporter with dithiothreitol is pH-dependent, decreasing with increasing pH over the pH range 6.5-8.5. We conclude that this site of action of dithiothreitol is a single essential disulfide bond intimately associated with the glucose-binding site on the transport molecule.
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000037:antagonists & inhibitors; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007687:Kidney Tubules, Proximal / Q000378:metabolism; D007700:Kinetics; D009004:Monosaccharide Transport Proteins; D010695:Phlorhizin / Q000378:metabolism; D011817:Rabbits; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D004220:Disulfides; D009004:Monosaccharide Transport Proteins; D010695:Phlorhizin; D004229:Dithiothreitol