Reconstitution of the D-glucose transporter of bovine thymocyte plasma membrane: partial purification of transport activity by chromatography on agarose lentil lectin and agarose ethanethiol.
The D-glucose transporter of bovine-thymocyte plasma membrane was partially purified using several procedures in sequence. Dimethylmaleic anhydride extraction removed extrinsic membrane proteins (approximately 50% of the total membrane protein) after which sodium cholate solubilized 40% of the residual protein. Reconstitution of solubilized proteins into phospholipid liposomes indicated a 2.5-fold increase in sugar transport specific activity relative to membrane solubilized without dimethylmaleic anhydride extraction. Detergent removal by gel filtration on G-50 Sephadex resulted in reaggregation of intrinsic membrane proteins. Ultracentrifugation of the reaggregated proteins generated a particulate fraction (pellet 1) which contained about 50% of the total D-glucose transport activity of the preparation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of pellet 1 demonstrated removal of a major band at 68,000 daltons and two minor bands not removed by dimethylmaleic anhydride. The 68,000-dalton protein was not removed by any other method tested. Chromatography of resolubilized pellet 1 on a tandem-bed column of agarose ethanethiol and agarose lentil lectin resulted in a 6-fold increase in transport specific activity of nonabsorbed proteins relative to pellet 1. Approximately 15% of the protein (80-90% of the transport activity) applied to the tandem-bed column was recovered in the nonabsorbed fraction. Sodium dodecyl sulfate-gel electrophoresis of proteins in the nonabsorbed fraction showed apparent enrichment of a diffuse zone at 52,000-45,000 daltons. The overall increase in specific activity of the partially purified preparation was about 12-fold relative to unpurified solubilized proteins.
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002847:Chromatography, Agarose / Q000379:methods; D037102:Lectins; D008299:Maleic Anhydrides; D009004:Monosaccharide Transport Proteins; D037121:Plant Lectins; D012685:Sepharose / Q000031:analogs & derivatives; D012995:Solubility; D013438:Sulfhydryl Compounds; D013950:Thymus Gland / Q000378:metabolism*
D002352:Carrier Proteins; D037102:Lectins; D008299:Maleic Anhydrides; D009004:Monosaccharide Transport Proteins; D037121:Plant Lectins; D013438:Sulfhydryl Compounds; C037187:ethanethiol-sepharose; C033619:lentil lectin; C007474:2,3-dimethylmaleic anhydride; D012685:Sepharose