PMID: 3759992Oct 15, 1986Paper

Isolation and partial purification of a Na+-dependent phlorizin receptor from dog kidney proximal tubule.

The Journal of Biological Chemistry
M Silverman, P Speight

Abstract

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 polype...Continue Reading

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