Abstract

The effects of noncompetitive inhibitors of Na(+)-dependent glucose uptake, p-chloromercuribenzoate N,N'-dicyclohexylcarbodiimide (DCCD), on substrate-induced cotransporter conformational changes were examined using fluorescein isothiocyanate (FITC) and tryptophan fluorescence. p-chloromercuribenzoate (PCMB) inhibited both substrate-induced conformational changes with similar concentration required for 50% quenching/enhancement of tryptophan or FITC fluorescence. In contrast, DCCD inhibited the Na(+)-induced conformational change with an apparent concentration resulting in 50% inhibition (K0.5) of 18 microM and the glucose-induced conformational change with an apparent K0.5 of 5 microM. DCCD slightly increased the apparent K0.5 for the Na+ concentration required for Na(+)-induced conformational change. DCCD and PCMB altered tryptophan accessibility to quench reagents in all three conformations. Tryptophan residues on the PCMB-treated cotransporter were more Cs+ than I- sensitive in contrast to the unlabeled cotransporter. The PCMB-treated cotransporter had a reduced response to Na+, suggesting that the mode of PCMB inactivation of cotransporter activity resulted from conformational changes in the substrate-free cotransporter. DCCD had a smaller effect on cotransporter tryptophan quench reagent susceptibility. However, DCCD-labeled cotransporter was equally accessible to I- and Cs+, and the DCCD-labeled cotransporter did not respond to substrates. Loss of charge distribution around cotransporter tryptophans correlated with loss of substrate-induced conformational changes.

Full Text
Paper version not known

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call