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.