Purification of a putative Na+/D-glucose cotransporter from pig kidney brush border membranes on a phlorizin affinity column.
Phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, was derivatised to 3-aminophlorizin and subsequently coupled to Affi-Gel 15. Affinity chromatography of pig kidney brush border membranes solubilised in Triton X-100 allowed the purification of a 60 kDa protein on this resin. We consider this protein to be the Na+/D-glucose cotransporter, or part of it, for the following reasons: (i) binding of this protein to Affi-Gel 15 specifically requires phlorizin covalently attached to the resin and is lowered when phlorizin is replaced by phloretin; (ii) binding of the 60 kDa protein to a phlorizin affinity column requires the presence of Na+; (iii) polyclonal as well as monoclonal antibodies against the 60 kDa protein inhibit binding of phlorizin to brush border membranes from rabbit and pig kidney.
Kitlar
T
T
Max-Planck-Institut für Systemphysiologie, Dortmund, FRG.
Morrison
A I
AI
Kinne
R
R
Deutscher
J
J
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004121:Dimethyl Sulfoxide; D004591:Electrophoresis, Polyacrylamide Gel; D007124:Immunoenzyme Techniques; D007158:Immunologic Techniques; D007668:Kidney / Q000032:analysis*; D046911:Macromolecular Substances; D008871:Microvilli / Q000032:analysis; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000276:immunology / Q000302:isolation & purification*; D010695:Phlorhizin; D012964:Sodium / Q000494:pharmacology; D013552:Swine
D000906:Antibodies; D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D009004:Monosaccharide Transport Proteins; D012964:Sodium; D010695:Phlorhizin; D004121:Dimethyl Sulfoxide