Reconstitution of a partially purified Na+-independent D-glucose transport system from rat jejunal basolateral membranes.
1. Basolateral membranes of rat small intestine were first solubilized in a 0.6% cholate buffer and then the insoluble fraction was reextracted with a 1.2 or 1.6% cholate buffer. 2. Proteoliposomes reconstituted from the 1.2 or 1.6% cholate-extracted membrane fraction demonstrated characteristic Na+-independent D-glucose transport of the native basolateral membrane vesicles: inhibitable by mercuric chloride and D-galactose. 3. To further purify this D-glucose transport system, the 1.6% cholate-extracted membrane fraction was chromatographed on either hydroxylapatite, concanavalin A, wheat-germ lectin or castor bean lectin-120 affinity gels. 4. Proteoliposomes reconstituted from the membrane proteins adsorbed on hydroxylapatite and subsequently passed through agarose-castor bean lectin-120 showed a 12-fold enrichment of Na+-independent D-glucose transport activity over that of the native membrane vesicles. 5. SDS-electrophoretic analysis showed that the protein composition of the hydroxylapatite-castor bean lectin-120 treated fraction was much simpler than that of both 1.6% cholate-extracted fraction and the native membrane vesicles.
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002845:Chromatography / Q000379:methods; D005947:Glucose / Q000378:metabolism*; D066298:In Vitro Techniques; D007408:Intestinal Absorption / Q000187:drug effects; D007413:Intestinal Mucosa / Q000378:metabolism*; D007583:Jejunum / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008627:Mercuric Chloride; D008628:Mercury / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000494:pharmacology*; D012995:Solubility
D008081:Liposomes; D008627:Mercuric Chloride; D012964:Sodium; D008628:Mercury; D005947:Glucose