Partial purification of the sugar carrier of intestinal brush border membranes. Enrichment of the phlorizin-binding component by selective extractions.
The [3H] phlorizin-binding component of brush border vesicles was enriched in situ by negative purification. Several procedures, known to effect selective solubilization of membrane components, were used separately or in combination to remove proteins unrelated to the binding. Deoxycholate ruptured the vesicles and released 67% of their protein, thereby increasing the specific [3H] phlorizin-binding activity of the pellet three-to fourfold. Extracting the deoxycholate-pellets with either NaI or alkaline solutions released up to 38% of the deoxycholate-insoluble protein without significantly affecting phlorizin binding. The polypeptide composition of the membranes at the different stages was analyzed by NaDodSO4-polyacrylamide gel electrophoresis. A number of polypeptides present in the original vesicles could be ruled out as essential components of the [3H] phlorizin binding entity. Intact and deoxycholate-treated vesicles were subjected to proteolytic attack. Papain liberated sucrase and isomaltase from intact vesicles, but affected neither other Coomassie-stained bands nor phlorizin binding. Neither the protein composition nor the binding properties of sealed vesicles were influenced by trypsin or chymotrypsin. However, all the proteolytic enzymes tested on deoxycholate-treated membranes substantially reduced [3H] phlorizin binding and produced concomitantly the disappearance of several bands from the electrophoretic profile. Pretreatment of vesicles with papain, followed by deoxycholate extraction and incubation in alkaline media, increased the specific binding activity of the membranes up to ninefold by removing close to 90% of the protein. A limited number of polypeptides are suggested as possible candidates for the glycoside-binding site of intestinal brush borders.
D000818:Animals; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000302:isolation & purification*; D002462:Cell Membrane / Q000378:metabolism*; D003840:Deoxycholic Acid; D005947:Glucose / Q000378:metabolism*; D007421:Intestine, Small / Q000378:metabolism*; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism*; D008970:Molecular Weight; D010447:Peptide Hydrolases; D010695:Phlorhizin / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits
D002352:Carrier Proteins; D003840:Deoxycholic Acid; D010695:Phlorhizin; D010447:Peptide Hydrolases; D005947:Glucose