A method for measuring apical glucose transporter site density in intact intestinal mucosa by means of phlorizin binding

Ronaldo P. Ferraris, Jared M. Diamond

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

Phlorizin binding has been widely used to estimate the site density of glucose transporters on intestinal and renal brush-border vesicles. Glucose transport measurements in the intact intestinal mucosa show that changes in transport rate postulated to arise from changes in site density occur under many physiological and pathological conditions. Exploring the basis of these regulatory phenomena would be facilitated by comparing changes in transport rate and site density measured in the same preparation. Hence we developed methods for measuring phlorizin binding in everted sleeves of intact mouse intestine. Specific binding of phlorizin to glucose carriers reached an asymptotic value within 120 sec, while nonspecific binding continued to rise thereafter. Hence we used 120-sec incubations. The rate of dissociation of specifically bound phlorizin was accelerated by Na+-free solutions and even more by 50 mm glucose, while the rate of dissociation of nonspecifically bound phlorizin was independent of these solution changes. Hence we chose a 20-sec rinse in Ringer+50 mm mannitol, because it washes out 30-40% of the nonspecifically bound phlorizin but virtually none of the specifically bound phlorizin. Ligand-binding analysis of specific binding against phlorizin concentration suggested two classes of binding sites, of which the one with stronger affinity for phlorizin probably has the higher capacity for glucose transport in mouse jejunum. The calculated affinity and capacity of this component are independent of whether one estimates the specific component of total binding by adding glucose or by removing Na+.

Original languageEnglish (US)
Pages (from-to)65-75
Number of pages11
JournalThe Journal of Membrane Biology
Volume94
Issue number1
DOIs
StatePublished - Feb 1986

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Physiology
  • Cell Biology

Keywords

  • dietary regulation
  • glucose transport
  • induction
  • phlorizin binding
  • small intestine

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