Distribution of sulfhydryl groups in intestinal brush border membranes: Localization of side-chains essential for glucose transport and phlorizin binding

Abstract

1. 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. 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. 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 μM 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. 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.