Distribution of sulfhydryl groups in intestinal brush border membranes. Localization of side-chains essential for glucose transport and phlorizin binding.
1. Brush border membrane vesicles from rabbit small intestine were found to contain 46 nmol SH groups/mg protein, 52% of which could react with 4,4'-dithiodipyridine, a membrane permeating probe. Only 18% of the total SH-groups reacted with the impermeant probe 5,5'-dithiobis(2-nitrobenzoic acid), indicating that only this fraction is externally located. 2. Brush border membrane vesicles could be disrupted by a gentle treatment with deoxycholate, releasing most of their electron-dense core material. In deoxycholate-treated vesicles most of the SH groups that reacted with 4,4'-dithiodipyridine react with 5,5'-dibiobis(2-nitrobenzoic acid), suggesting that both membrane surfaces became exposed to the extravesicular medium. 3. In intact vesicles (1.2 mg protein/ml), the binding of phlorizin (a competitive inhibitor of the monosaccharide transport system) was 50% inhibited by 67 microM of the penetrating organomercurial p-chloromercuribenzoate, but was about ten times less sensitive to the poorly permeating p-chloromercuriphenylsulfonate. In contrast, binding of phlorizin to leaky (deoxycholate-treated) membranes was equally susceptible to either reagent. 4. Mercurial inhibition of phlorizin binding could be reversed by dithioerythritol in both sealed and leaky membranes, whereas the less permeant thiol L-glutathione (reduced form) could only revert the inhibition in leaky membranes.
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002462:Cell Membrane / Q000032:analysis*; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003840:Deoxycholic Acid / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000648:ultrastructure; D008871:Microvilli / Q000032:analysis*; D010695:Phlorhizin / Q000378:metabolism*; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000032:analysis*
D002729:Chloromercuribenzoates; D013438:Sulfhydryl Compounds; D003840:Deoxycholic Acid; D004228:Dithionitrobenzoic Acid; D010695:Phlorhizin; D005947:Glucose; D004229:Dithiothreitol