Reconstitution of the glucose transporter from rat skeletal muscle.
As a step in the purification and characterization of the glucose transporter from rat skeletal muscle, we have reconstituted glucose transport activity in liposomes. Plasma membranes were prepared from skeletal muscle which display D-glucose reversible binding of cytochalasin B (10 pmol sites/mg protein; KD = 0.3 microM). Older rats gave a slightly lower specific activity and much lower yield of sites per g muscle than young rats. Glucose transport activity was reconstituted into liposomes by the freeze-thaw procedure using either plasma membranes directly or cholate-extracted membrane proteins; the latter gave a 50% higher specific activity. The reconstituted transport activity was stereospecific, saturable, and inhibited by cytochalasin B, phloretin, and mercuric chloride. The optimum cholate concentration for extraction and reconstitution of transport activity was about 1.5%, and the highest specific activity of reconstituted transport was seen only at low ratios of protein to lipid in the reconstitution. Chromatography on agarose lentil lectin and agarose ethanethiol doubled both the specific activity of reconstituted transport and the fraction of glucose uptake which was stereospecific. In all of these respects the results were similar to our results with the bovine heart transporter (T. J. Wheeler and M. A. Hauck, Biochim. Biophys. Acta 818, 171-182 (1985)). Our findings suggest that further purification procedures developed for the heart transporter may be applicable to the skeletal muscle transporter as well.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002847:Chromatography, Agarose; D003571:Cytochalasin B / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008297:Male; D009004:Monosaccharide Transport Proteins / Q000302:isolation & purification* / Q000378:metabolism; D009132:Muscles / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D008081:Liposomes; D009004:Monosaccharide Transport Proteins; D003571:Cytochalasin B