The Na+-independent D-glucose transporter in the enterocyte basolateral membrane: orientation and cytochalasin B binding characteristics.
Phloridzin-insensitive, Na+-independent D-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed a Kt of 48 mM and a Vmax of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lower Kt of 23 mM and a Vmax of 0.20 nmol glucose/mg protein/sec. D-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. The Ki for cytochalasin B as an inhibitor of glucose transport was 0.11 microM and the KD for binding to the carrier was 0.08 microM. D-glucose-sensitive sensitive binding of cytochalasin B to the membrane preparation was maximized with L- and D-glucose concentrations of 1.25 M. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na+-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and the Kt for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002918:Chymotrypsin / Q000494:pharmacology; D003571:Cytochalasin B / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism*; D007421:Intestine, Small / Q000378:metabolism*; D007700:Kinetics; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D012964:Sodium / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D009004:Monosaccharide Transport Proteins; D003571:Cytochalasin B; D012964:Sodium; D002918:Chymotrypsin; D014357:Trypsin; D005947:Glucose