Isolation and partial purification of a Na+-dependent phlorizin receptor from dog kidney proximal tubule.
This paper describes a new method for solubilization and partial purification of a Na+-dependent phlorizin receptor from dog kidney proximal convoluted tubule. Selective solubilization is carried out with 0.1% Na+-deoxycholate followed by complete solubilization with 0.5% deoxycholate. The 100,000 X g supernatant of the deoxycholate extract is then subjected to a combination of chromatofocusing and gel exclusion chromatography. Purification is monitored by a new column assay which permits detection of the Na+-dependent high affinity phlorizin receptor in solubilized preparations. Na+-dependent phlorizin binding exhibits the same characteristics on the column assay as in intact brush border vesicles. Binding is temperature-dependent, inhibited by proteolytic agents, Na+-dependent, and inhibited by excess cold phlorizin and D-glucose but not L-glucose. Quantitation of specific binding at different stages of the isolation procedure indicates a final purification of approximately 80-140-fold compared to intact brush border membrane fragments. Enrichment of specific phlorizin binding is paralleled by enrichment of a 61-66-kDa polypeptide on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. It is postulated that this polypeptide contains both the Na and the sugar specific binding site and represents a subunit of the intact Na+-dependent glucose transporter from dog kidney proximal tubule brush border membrane.
D000818:Animals; D001665:Binding Sites; D003840:Deoxycholic Acid; D004285:Dogs; D007672:Kidney Cortex / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010206:Papain / Q000494:pharmacology; D010695:Phlorhizin / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology*; D012995:Solubility; D014357:Trypsin / Q000494:pharmacology
D009004:Monosaccharide Transport Proteins; D003840:Deoxycholic Acid; D012964:Sodium; D010695:Phlorhizin; D014357:Trypsin; D010206:Papain