TRYPTIC DIGESTION OF THE HUMAN ERYTHROCYTE GLUCOSE-TRANSPORTER - EFFECTS ON LIGAND-BINDING AND TRYPTOPHAN FLUORESCENCE

The conformation of the human erythrocyte glucose transport protein ha s been shown to determine its susceptibility to enzymatic cleavage on a large cytoplasmic loop. We took the converse approach and investigat ed the effects of tryptic digestion on the conformational structure of this protein. Exhaustive tryptic digestion of protein-depleted erythr ocyte ghosts decreased the affinity of the residual transporter for cy tochalasin B by 3-fold but did not affect the total number of binding sites. Tryptic digestion also increased the affinity of the residual t ransporter for D-glucose and inward-binding sugar phenyl beta-D-glucop yranoside but decreased that for the outward-binding 4,6-O-ethylidene glucose. These results suggest that tryptic cleavage stabilized the re maining transporter in an inward-facing conformation, but one with dec reased affinity for cytochalasin B. The steady-state fluorescence emis sion scan of the purified reconstituted glucose transport protein was unaffected by tryptic,digestion. Addition of increasing concentrations of potassium iodide resulted in linear Stern-Volmer plots, which were also unaffected by prior tryptic digestion. The tryptophan oxidant N- bromosuccinimide was investigated to provide a more sensitive measure of tryptophan environment. This agent irreversibly inhibited 3-O-methy lglucose transport in intact erythrocytes and cytochalasin B binding i n protein-depleted ghosts, with a half-maximal effect observed for eac h activity at about 0.3-0.4 mM. Treatment of purified glucose transpor t protein with N-bromosuccinimide resulted in a time-dependent quench of tryptophan fluorescence, which was resolved into two components by nonlinear regression using global analysis. Tryptic digestion retarded the rate of oxidation of the more slowly reacting class of tryptophan s. Thus, tryptic digestion caused the residual transporter to assume a n inward-facing conformation different from that induced by transporte r ligands.