The Na+-independentd-glucose transporter in the enterocyte basolateral membrane: Orientation and cytochalasin B binding characteristics |
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Authors: | D D Maenz C I Cheeseman |
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Institution: | (1) Department of Physiology, University of Alberta, T6G 2H7 Edmonton, Alberta, Canada |
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Abstract: | Summary Phloridzin-insensitive, Na+-independentd-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 aK
t of 48mm and aV
max of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lowerK
t of 23mm and aV
max 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. TheK
t for cytochalasin B as an inhibitor of glucose transport was 0.11 m and theK
D for binding to the carrier was 0.08 m.d-glucose-sensitive binding of cytochalasin B to the membrane preparation was maximized withl- andd-glucose concentrations of 1.25m. 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 theK
t 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. |
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Keywords: | Small intestine glucose transport cytochalasin B binding membrane vesicles |
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