Phlorizin Receptor Research Articles

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.

The mechanism of maleic acid nephropathy: investigations using brush border membrane vesicles.

Brush border membrane vesicles from dog kidney proximal tubule have been used to investigate the pathogenesis of glucosuria resulting from maleic acid nephropathy. Direct exposure of brush border vesicles to 9 mM maleic acid for up to 36 minutes has no effect of the Na+-dependent D-glucose uptake mechanism. Brush border membrane vesicles were then prepared from dogs in which glucosuria had first been induced by prior administration of maleic acid (250 mg/Kg body weight). The timed uptake of D- and L-glucose was measured in the presence in the presence of both Na+ chemical and electropotential driving forces and compared to uptake in vesicles prepared from matched control kidneys from the same animal. Maleic acid treated and control vesicles were also assayed for the presence of the Na+-dependent high affinity phlorizin receptor. Our results indicate that there is no alteration in behavior of the Na+-dependent transport system for glucose at the brush border membrane in the model Fanconi state produced by maleic acid. Moreover, there is no change in the coupling of sugar transport either to the Na+ chemical or the electrical potential gradient across the brush border membrane. These studies imply that further efforts to define the pathogenesis of maleic acid nephropathy are probably best carried out using intact tissue or cells because the molecular defect is at the level of the Na+ pump and/or at some intracellular site affecting production of ATP.

Sugar uptake into brush border vesicles from dog kidney. I. Specificity

This study describes the specificity of uptake of sugars into osmotically active vesicles derived from a purified membrane fraction from dog kidney cortex. [ 3H]Phlorizin binding experiments have also been carried out in vesiculated and non-vesiculated membrane preparations. 1. 1. We demonstrate the existence of a sodium-dependent, phlorizin-sensitive d-glucose transport system in the vesiculated membrane preparation. 2. 2. This transport system has similar specificity characteristics to those observed in vivo for the glucose transporter in the brush border membrane of the proximal tubule. 3. 3. We also observe a sodium-dependent, glucose-sensitive phlorizin receptor in the same preparation with a K d for phlorizin ⋍0.3 μ M and K I for glucose ⋍3 mM at 37°C, pH 7.4, 100 mM NaCl. 4. 4. Detailed results relating to the specificity of inhibition of high affinity phlorizin binding are obtained using non-vesiculated brush border membrane fragments in the presence of d-glucose, α-methyl- d-glucoside, d-galactose, β-methyl- d-galactoside, 2-deoxy- d-glucose and d-mannose. 5. 5. Uptake studies using vesiculated membrane fragments from newborn dog kidney indicate that the brush border d-glucose transporter is already at an advanced stage of development at birth. Our results demonstrate that the d-glucose transport system from the dog proximal tubule brush border membrane together with its phlorizin receptor moiety is preserved intact in our membrane vesicle preparation.

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

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.

The in vivo localization of high-affinity phlorizin receptors to the brush border surface of the proximal tubule in dog kidney

The interaction of [ 3H]phlorizin (spec. act. 5 Ci/mmole) with canine renal tubular surfaces has been investigated in vivo, using the multiple indicator dilution technique. 1. 1. Over short observation periods there is no observable interaction between tracer phlorizin and the antiluminal (peritubular) surface. Instead, phlorizin appears to have a limited volume of distribution relative to the extracellular reference (creatinine), from the postglomerular circulation. 2. 2. Phlorizin and creatinine are filtered to the same degree at the glomerulus however the urine recovery of [ 3H]phlorizin is 5–10% of the glomerular marker (plasma glucose concentration 60–124 mg/100 ml). 3. 3. The remaining 90–95% can be “washed off” and recovered in the urine within minutes after the systemic administration of a pulse of unlabelled phlorizin or glucose. Preloading of animals with phlorizin or glucose also increases the urine recovery of [ 3H]phlorizin relative to control conditions. In contrast the urine recovery of [ 3H]phlorizin is unaffected by phloretin infusion. 4. 4. It is concluded that only one set of high-affinity phlorizin receptors is present in dog kidney, and that these are located along the brush border of the proximal tubule, in close association with the glucose-transport mechanism.

The chemical and steric determinants governing sugar interactions with renal tubular membranes

Abstract 1. 1. The multiple indicator dilution technique in vivo has been used to study the specificity of interaction of pyranoside derivatives with renal tubular surfaces in dog kidney. The substances used include [ 14 C]-methyl-α- d -glucose, [ 3 H] methyl-β- d -glucoside, d -[ 14 C]mannosamine, d -[ 14 C]fructose, l -[6- 14 C]iodose, d -[ 3 H]-allose, [2- 3 H] myo -inositol, [ 14 C]glucosamine-HCl and d -[6- 3 H]mannosamine hydrochloride. 2. 2. For the luminal (brush border) membrane the specificity characteristics consist of: (i) a pyranose ring in a chair conformation (ii) OH groups on carbons 3 and 6 oriented as in configuration of d -glucose. 3. 3. For the antiluminal membrane the specificity characteristics are (i) a pyranose ring in a chair conformation (ii) hydroxyl groups on carbons 1 and 2 (iii) OH groups if present on carbons 3 and 6 should be oriented equatorially as in the configurations of d -glucose. 4. 4. These data are compatible with the specificity of inhibition of high affinity phlorizin receptors in vitro, and suggest that these receptors are identical to the sites associated with the glucose-transport mechanism. 5. 5. The results also suggest that the dominant feature controlling the dynamics of the interaction between sugars and membrane-bound transport receptors is the position of certain functional hydroxyl groups relative to the plane of the pyranose chair (Cl or 1C), defined by carbons 2, 3, 5, and the ring oxygen. In this regard the axial orientation of a single OH group on the pyranoside may be of special importance to the sugar-receptor interaction.

Phlorizin receptors in isolated kidney brush border membranes Differential enzymatic modification of high-affinity receptors and unspecific binding sites

High-affinity phlorizin receptors in isolated kidney brush border membranes are destroyed by the proteolytic enzymes trypsin and papain. The digested membranes show increased unspecific phlorizin binding. It is proposed, that both enzymes expose a deeper, more hydrophobic layer in the brush border membrane to explain the latter finding.

Formula omitted] labeling of a phlorizin-sensitive d-glucose binding site of brush border membrane from the rat kidney

A glcuose-sensitive high-affinity receptor for phlorizin is prseent in isolated brush border membrane of rat kidney and is thought to be the first station in a chain of processes comprising “transmembrane transport of glucose”. This receptor was labeled with N-[ 14C] ethylmaleimide for identification and future isolation according to the substrate protection effect. Three molcules of N-[ 14C] ethylmaleimide are inhibited from the reaction with the bursh border membrane if one high-affinity binding site is protected by phlorizin. d-Glucose also protects the high-affinity phlorizin receptor from the reaction with N-[ 14C] ethylmaleimide , suggesting that the site labeled is not only part of a phlorizin receptor but also of a glucose binding protein in the brush border membrane.

Inhibition of [ 3H]phlorizin binding to isolated kidney brush border membranes by phlorizin-like compounds

1. 1. Earlier work has demonstrated that (a) isolated brush border membranes from rat kidney cortex possess a high-affinity receptor for phlorizin; binding is dependent upon the presence of sodium and is competitively blocked by d-glucose, (b) phlorizin competitively inhibits active transport of glucose across rat kidney proximal tubules and (c) a series of phlorizin-like compounds possess a characteristic, wide range of inhibitory potency as glucose transport poisons in kidney and intestinal systems. 2. 2. 3H-labeled phlorizin binding to the brush border membrane site has now been shown to be inhibited by this series of phlorizin analogs, and the critical finding is that the previosuly established relative potencies of these compounds as transport inhibitors parallel their ability to block phlorizin binding to the specific membrane receptor. 3. 3. These results increase the weight of evidence for the view that the glucose-transport system is retained as a structurally unaltered component of these isolated brush border fragments. 4. 4. A view of the conformation, which the phlorizin receptor could assume in the matrix of the outer membrane, is presented in the discussion.

Phlorizin Receptors in Isolated Kidney Brush Border Membranes

Abstract Isolated brush border membranes from rat kidney bind [3H]phlorizin specifically. A binding site (Ka (the association constant) = 5.0 x 106 1 x mole-1 at 150 mm sodium, pH 7.4, 37°) is enriched in the brush border membrane and is not present in kidney mitochondria or red blood cell ghosts of the rat. The concentration of the high affinity receptors in the membrane fraction is 40 pmoles per mg of protein, about 17 times higher than in a kidney homogenate. Inhibition studies with monosaccharides demonstrate that the binding curve is heterogeneous. d-Glucose, d-galactose, and 2-deoxy-d-glucose inhibited phlorizin binding at the high affinity site, whereas d-fructose, d-mannose, and l-arabinose interact with another low receptor in the brush border membrane. The inhibition studies show that its affinity for phlorizin is one to two orders of magnitude lower than that of the high affinity receptor. The high affinity receptors are destroyed by treatment with trypsin and sulfhydryl reagents like p-hydroxymercuriphenylsulfonic acid and N-ethylmaleimide. Phlorizin binding is sodium-dependent; the affinity of the high affinity receptor decreases 30-fold when the sodium concentration is lowered from 150 to 10 mm. Kidney brush border membranes contain no detectable phlorizin hydrolase, but the receptors are rapidly degraded by a temperature-dependent process that can be inhibited by ethylenediaminetetraacetate. The stereospecificity of the phlorizin receptors in the isolated luminal membrane of the rat kidney proximal tubule has remarkable similarity to the stereospecificity of the monosaccharide carriers as shown by in vivo experiments (Silverman, M., Aganon, M. A., and Chinard, P. F. (1970) Amer. J. Physiol. 218, 735; 743).

Specificity of monosaccharide transport in dog kidney.

Specificity of monosaccharide transport in dog kidney.

Is phloretin the sugar transport inhibitor in intestine?

Isolated brush border fragments from hamster small intestine epithelium were incubated with tritiated phlorizin and direct measurements of the interaction were made. The adsorption kinetics were found to be unexpectedly complex involving at least two reactions: An instantaneous adsorption, representing the interaction of phlorizin with a readily accessible receptor site, is followed by a much slower secondary uptake of radioactivity by the tissue fragments. The first reaction can be dissected from the other process; it exhibits first order kinetics and fits a Langmuir adsorption isotherm. The dissociation constant for this interaction has a maximum value of 75 μ m. An estimate of the receptor density on the epithelial cell was also obtained from these data. The brush border membrane of each cell possesses from 2 to 4 × 10 8 phlorizin receptor sites. The relationship of this estimate to the number of sugar carrier molecules per cell is discussed. The secondary uptake of radioactivity by the brush border was attributable to the adsorption of [ 3H]phloretin which was enzymatically generated by a β-glucosidase present in the brush border membrane. The implications of this conversion of phlorizin to the aglycone, under common incubation conditions are discussed and the question is raised whether phloretin, rather than the parent glucoside, is the actual inhibitor of the carrier transport mechanism in the intestine. The preparation of tritiated phlorizin is described and the methods which can be used to radiochemically purify the compound are presented.