Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:The Na+-independent D-glucose transporter in the enterocyte basolateral membrane: orientation and cytochalasin B binding characteristics. 362 59

Phlorizin is a reversible inhibitor of the renal and small intestinal Na+/D-glucose cotransporter. In an attempt to purify the Na+/D-glucose cotransporter from a pig kidney brush border membrane fraction, we used an Affi-Gel affinity chromatography column to which 3-aminophlorizin had been coupled. A protein, composed according to crosslinking experiments of at least 3 subunits of molecular weight 60 kDa, was found to bind specifically to the phlorizin column. This protein was subsequently identified as catalase by sequence homology of three of its tryptic fragments to the sequence of several mammalian catalases as well as by its enzymatic activity. Although bovine liver catalase was bound tightly to the affinity matrix, phlorizin had no effect on the ability of the enzyme to degrade H2O2. In contrast, the Aspergillus niger and Neurospora crassa catalases did not bind to the phlorizin column. This difference may be related to the fact that mammalian catalases, but not the fungal catalases, contain an NADPH binding site with a yet unknown function. Interestingly, bovine liver catalase could be eluted with 50 microM NADPH from phlorizin columns. Irradiation in the presence of [3H]4-azidophlorizin allowed photolabeling of bovine liver catalase, which was prevented by the presence of 10 microM NADPH. After digestion of photolabeled catalase with chymotrypsin, a radioactive peptide was detected that was absent in catalase protected with NADPH. Docking simulations suggested that phlorizin can bind to the NADPH binding site with high affinity.
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PMID:Interaction of phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, with the NADPH-binding site of mammalian catalases. 800 87