High affinity phlorizin receptor sites and their relation to the glucose transport mechanism in the proximal tubule of dog kidney

Abstract

A set of high affinity phlorizin receptors in a brush border membrane preparation from dog kidney cortex is described. The dissociation constant, K d is ≈ 0.3 μM (20 mM Tris · HCl, 140 mM Na +, 5 mM EDTA pH 7.45, 37 °C). The number of receptor sites is ≈ 12 · 10 −12 mol/mg membrane protein. Preincubation with sugar substrates shows that the high affinity phlorizin binding is completely abolished by d-glucose (100 mM), 3-deoxy-3-fluoro- d-glucose (125 mM) and α-methyl- d-gluco-pyranoside (125 mM), while 40–50% inhibition is observed with glucose concentration as low as 5 mM, d-Galactose and β-methyl- d-galactopyranoside inhibition 20–40% at 125 mM while 2-deoxy- d-glucose and 2-deoxy- d-galactose inhibit minimally (≈ 25%) at the same concentration. l-Glucose, d-mannose, d-xylose, myoinositol, d-fructose and 3-O- methyl- d-glucose do not inhibit significantly in concentrations up to 600 mM. Unlabelled phlorizin (1 μM) and d-glucose (125 mM) completely wash off bound [ 3H]phlorizin from the high affinity site. In contrast, phloretin (100 μM) is only about 50% as effective in displacing bound [ 3H]phlorizin. Binding decreases with decreasing sodium concentration and is abolished by N-ethylmaleimide (7 mM). No inhibition is observed with ouabain (0.125 mM), cytochalasin B (0.1–42 μM) and concanavalin A (10–10 000 μg/ml). The specificity of inhibition of phlorizin binding in vivo to the luminal membrane of the proximal tubule in dog kidney has also been investigated. α-Methyl- d-glucopyranoside completely washes off bound [ 3H]phlorizin. d-Galactose is only about 10% as effective at equivalent doses. There is no observable wash off of bound [ 3H]phlorizin with d-fructose, myoinositol, d-mannose or 2-deoxy- d-glucose. The relative affinity of monosaccharides for the glucose transport receptor at the brush border was investigated in vivo using the multiple indicator dilution technique to determine their fractional reabsorption under identical conditions of phlorizin blockade. The relative affinities are in the order d-glucose ≈ α-methyl- d-glucopyranoside > d-galactose > 2-deoxy- d-glucose > d-fructose ≈ myoinositol. It is concluded (i) that phlorizin receptors on the brush border of the proximal tubule in vivo are identical to the high affinity phlorizin binding sites in the brush border membrane fraction in vitro and (ii) that these phlorizin receptor sites are either in close proximity, or identical, to the glucose transport receptor.