Phlorizin Binding Sites Research Articles

Phloretin inhibition of glucose transport by the tapeworm Hymenolepis diminuta: A kinetic analysis

1. 1. Phloretin non-competitively inhibits glucose and methionine absorption by Hymenolepis diminuta ( K i = 0.24 and 1.48 mM, respectively). 2. 2. Inhibition of glucose transport by phloretin is reversible. 3. 3. Phloretin and phlorizin binding sites on the surface of H. diminuta are distinct. 4. 4. Phloretin does not inhibit Na + transport independent of glucose transport in H. diminuta. 5. 5. The data suggest that phloretin inhibits absorptive functions across the surface of H. diminuta via non-specific binding sites associated with the lipid portion of the worm outer membrane.

Regulation of sodium-coupled glucose transport by glucose in a cultured epithelium.

Cultured porcine kidney cells (LLC-PK1) form polarized epithelia that transport glucose from apical to basal surface as in the renal proximal tube. The ability of these cells to transport glucose is known to increase as the epithelium forms and matures in culture. We find that epithelia grown in medium containing 25 mM glucose have reduced hexose transport compared to epithelia grown in 5 mM glucose. This difference is not the result of differences in seeding efficiency and can be reversed by changing the concentration of glucose in the growth medium. Increased transport in epithelia grown in 5 mM glucose is the result of increased influx on the sodium-coupled apical membrane transporter rather than changes in efflux. This difference is apparently the result of more apical membrane transporters in epithelia grown in 5 mM glucose. The number of high affinity phlorizin-binding sites is greater in epithelia grown in 5 mM glucose (about 0.8 pmol/10(6) cells) than in 25 mM glucose (about 0.25 pmol/10(6) cells). The increase in the number of glucose transporters induced by the low glucose medium is specific in that there is not a comparable change in activity of marker enzymes (alkaline phosphatase, acid phosphatase, or glucose 6-phosphatase). The nature of the intracellular signal elicited by extracellular glucose remains to be determined.

Evidence for two disulfide bonds important to the functioning of the renal outer cortical brush-border membrane D-glucose transporter.

The effects of the disulfide reducing agent dithiothreitol on the renal outer cortical brushborder membrane D-glucose transporter are studied. Two apparently independent results of dithiothreitol treatment are observed. The first is a completely reversible increase in the affinity of the carrier for the nontransported competitive inhibitor phlorizin. The second is a partially reversible loss of phlorizin binding sites. Similar results are observed with the monothiol disulfide reducing agent beta-mercaptoethanol. Reversal of both effects of dithiothreitol could be prevented by exposing the membranes to an excess of N-ethylmaleimide following treatment with the reducing agent. When transported sugars are included with dithiothreitol treatment, the magnitude of both of its effects on the carrier are influenced; more specifically, the affinity for phlorizin is further increased and the loss of phlorizin binding sites is reduced. Nontransported sugars are without effect. These observations provide strong evidence for the existence of (at least) two disulfide bonds important to the functioning of this transporter.

Synthesis of [ 3H]phlorizin and its binding behavior to renal brush-border membranes

Tetra- and tribromophlorizin have been synthesized under mild brominating conditions. With catalytic debromination in the presence of hydrogen or tritium gas, bromine atoms in the derivatives were completely substituted by hydrogen or tritium. The product was identical to the native phlorizin and was chemically pure. Tritiated phlorizin with extremely high specific radioactivity (45 Ci/mmol) was obtained when hydrogen gas was replaced by tritium gas. While the brominated compounds showed little inhibition of sodium D-glucose co-transport by isolated renal brush-border membranes. [3H]phlorizin had the same binding affinity to the brush-border membranes as native phlorizin and a Ki value of 1.2 microM for the sodium-dependent D-glucose transport. Binding studies performed using a flow-dialysis method resulted in 150 pmol of phlorizin-binding sites per milligram of membrane protein. This radioactive phlorizin can be a useful tool for determining D-glucose-(phlorizin) binding sites at a low phlorizin concentration in membranes, in nonvesicle forms such as collapsed membrane vesicles, and in purified protein fractions.

Partial purification and reconstitution of the Na+-D-glucose cotransport protein from pig renal proximal tubules.

Brush border membranes from renal proximal tubules were solubilized with deoxycholate, and the proteins were incorporated into liposomes formed from cholesterol and phosphatidylserine by a freeze-thaw procedure. In the proteoliposomes Na+-D-glucose cotransport was demonstrated by showing that the D-glucose concentration in the liposomes increased far above the equilibrium value if a Na+ gradient was applied. The initial D-glucose uptake rate, stimulated by an inside directed gradient of 89 mM Na+, was 4 pmol/mg of protein-1 s-1. High affinity phlorizin binding could not be measured. After two precipitation steps with the solubilized membrane proteins, a protein fraction was obtained in which significantly high affinity phlorizin binding was detected. After reconstitution, proteoliposomes were formed in which more than 70% of the protein was represented by two polypeptides with molecular weights of 94,000 and 52,000. An initial Na+ gradient-dependent D-glucose uptake rate of 118 pmol/mg of protein-1 s-1 was obtained. In these liposomes, the D-glucose uptake rate could be inhibited by phlorizin (Ki = 0.3 microM), and 55-pmol phlorizin-binding sites per mg of protein (KD = 0.5 microM) were measured. In different liposomal preparations a correlation between Na+ gradient-dependent D-glucose uptake rate and the amount of 52,000 molecular weight polypeptide was observed.

Radiation inactivation studies of the renal brush-border membrane phlorizin-binding protein.

Renal brush-border membrane vesicles were irradiated in the frozen state with a high energy electron beam. The integral membrane proteins, alkaline phosphatase and 5'-nucleotidase, each showed a single exponential loss of activity with radiation dose, indicating target sizes of 67,000 and 58,000 daltons, respectively. Inactivation of sodium-dependent phlorizin binding to the brush-border membrane D-glucose transporter was more complex. One-half of the phlorizin binding sites were lost after even the smallest doses of radiation suggestive of large functional units (greater than 4 X 10(6) daltons) for a subpopulation of phlorizin binding proteins. The remaining sites behaved as a single radiation target of 110,000 +/- 8,000 daltons and retained the kinetic characteristics commonly associated with phlorizin binding to the glucose transporter. Thus, the data are consistent with the assignment of a molecular weight of 110,000 to the phlorizin binding moiety of the brush-border membrane D-glucose transport protein.

Localization of the Na +-sugar cotransport system in a kidney epithelial cell line (LLC PK 1)

Studies of the localization of the Na +-dependent sugar transport in monolayers of LLC PK 1 cells show that the uptake of a methyl α- d-glucoside, a nonmetabolizable sugar which shares the glucose-galactose transport system, occurs mainly from the apical side of the monolayer. Kinetics of [ 3H]phlorizin binding to monolayers of LLC PK 1 cells were also measured. These studies demonstrate the presence of two distinct classes of receptor sites. The class comprising high affinity binding sites had a dissociation constant ( K d ) of 1.2 μM and a concentration of high affinity receptors of 0.30 μmol binding sites per g DNA. The other class involving low affinity sites had a K d of 240 μM with the number of binding sites equal to 12 μmol/g DNA. Phlorizin binding at high affinity binding sites is a Na +-dependent process. Binding at the low affinity sites on the contrary is Na +-independent. The mode of action of Na + on the high affinity binding sites was to increase the dissociation constant without modifying the number of binding sites. The Na + dependence and the matching of K d for high affinity binding sites with the K i of phlorizin for the inhibition of methyl α- d-glucoside strongly suggest that the high affinity phlorizin binding site is, or is part of the methyl α- d-glucoside transport system. Binding studies from either side of the monolayer also show that the binding of phlorizin at the Na + dependent high affinity binding sites occurs mainly from the apical rather than the basolateral side. The specific location of the Na +-dependent sugar transport system in the apical membrane of LLC PK 1 cells is, therefore, another expression of the functional polarization of epithelial cells that is retained under tissue culture condition. In addition, since this sugar transport almost disappears after the cells are brought into suspension, it can be used as a marker to study the development of the apical membrane in this cell line.

Interaction of the sugar carrier of intestinal brush-border membranes with HgCl 2

HgCl 2 was used as an inhibitor and potential label for the glucose carrier of intestinal brush-border membranes. Half-maximal inhibition of Na +-dependent d-glucose uptake was reached with micromolar concentrations of HgCl 2 when the protein concentration was 1.2 mg/ml. Similar concentrations were found to inhibit the binding of [ 3H]phlorizin, a reversible competitive inhibitor of sugar transport. Inhibition was reversed by dithioerythritol but only marginally by EDTA. The data support the involvement of a sulfhydryl group in the inhibitory process. Deoxycholate-extracted membranes, which are enriched in specific phlorizin binding activity, were used for labeling studies using 203HgCl 2. The polypeptides were separated by gel electrophoresis and analyzed by protein staining and autoradiography. Non-specific 203HgCl 2 labeling was minimized by pre-treatment with sulfhydryl reagents which do not inhibit phlorizin binding. Several bands, which are lost from the autoradiographic pattern during a negative purification of the phlorizin binding sites, could be ruled out as essential components of the sugar carrier. The polypeptide profile was also analyzed following proteolysis, which abolished phlorizin binding. Those radioactive bands of which apparent M r values were altered by the treatment were considered as possible candidates. Finally, samples in which inhibition was reversed by thiols were also studied. The possible identity of the polypeptide(s) involved in glucose translocation is discussed in the light of these observations.

High‐affinity phlorizin binding to brush border membranes from small intestine: Identity with (a part of) the glucose transport system, dependence on the Na+‐gradient, partial purification

In the presence of an NaSCN gradient phlorizin binds with a high affinity (Kd similar or equal to 4.7 micron) to vesicles derived from brush border membranes of intestinal cells of rabbits. The value for Kd corresponds closely to that of Ki determined from phlorizin inhibition of sugar transport. The apparent affinity for phlorizin is decreased if NaCl is substituted for NaSCN and decreased substantially if the gradient of NaSCN is allowed to dissipate prior to the phlorizin binding. The number of high affinity binding sites is about 11 pmol/mg protein. Additional binding to low affinity sites can amount to as much as 600 pmol/mg protein after prolonged exposure to phlorizin (5 min.). The high affinity sites are related to glucose transport based on the similarity of the Kd and Ki values under a variety of conditions and on the inhibition of the binding by D-glucose but not by D-fructose. The transport system and the high affinity phlorizin binding sites can be enriched by a factor of 2-3 by treatment of vesicles with papain, which does not affect the transport system, but considerably hydrolyzes nonrelevant protein.

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.

Demonstration of glucose inhibitable phlorizin binding to renal brushborder proteins separated by gel electrophoresis

The brushborder membrane of the proximal convolution contains a phlorizin binding site, which due to its high affinity for phlorizin and other properties like competitive inhibition by D-glucose and sodium dependence has been suggested to be involved in the transport of D-glucose through the brushborder membrane [l-3] In order to study the chemical and physical interactions between this receptor and the transported substrate an extraction and purification of the molecule is important. In this paper a procedure is described by which the glucose sensitive phlorizin binding site can be extracted from the membranes in a conformation which still shows the glucose sensitive phlorizin binding The binding itself is measured by a technique in which after the separation of the proteins by electrophoresis the gel is incubated with [3H]phIorizin and [ “C]mannose and after washing.the ratio 3H/14C is determined in the gel slices.

Studies on the arrangement of a glucose sensitive phlorizin binding site in the microvilli of isolated rat kidney brushborder

FEBS LettersVolume 25, Issue 2 p. 242-244 ArticleFree Access Studies on the arrangement of a glucose sensitive phlorizin binding site in the microvilli of isolated rat kidney brushborder L. Thomas, L. Thomas Max-Planck-Institut für Biophysik, 6 Frankfurt/M., GermanySearch for more papers by this authorR. Kinne, R. Kinne Max-Planck-Institut für Biophysik, 6 Frankfurt/M., GermanySearch for more papers by this author L. Thomas, L. Thomas Max-Planck-Institut für Biophysik, 6 Frankfurt/M., GermanySearch for more papers by this authorR. Kinne, R. Kinne Max-Planck-Institut für Biophysik, 6 Frankfurt/M., GermanySearch for more papers by this author First published: September 15, 1972 https://doi.org/10.1016/0014-5793(72)80494-0Citations: 11AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.

The Inhibition by Phlorizin of Kidney Microsomal Inorganic Pyrophosphate-Glucose Phosphotransferase and Glucose 6-Phosphatase

Abstract Glucose 6-phosphatase from rat liver and kidney microsomes previously has been shown to catalyze an inorganic pyrophosphate-glucose phosphotransferase reaction. Nordlie and Soodsma recently have suggested that formation and then degradation of glucose 6-phosphate by the combined action of phosphotransferase and phosphohydrolase activities of this enzyme may under certain conditions constitute part of a mechanism for facilitated transport of glucose across the kidney tubular cell. To attempt to gain additional insight regarding the possible significance of such a physiological role for the enzyme, studies of the effect of phlorizin, a potent inhibitor of tubular sugar transport, on both phosphohydrolase and phosphotransferase activities of this enzyme were undertaken. Inhibition, in vitro, of both glucose 6-phosphate phosphohydrolase and pyrophosphate-glucose phosphotransferase activities of kidney homogenates and microsomal suspensions prepared in isotonic sucrose solution was noted over the entire range of phlorizin concentrations studied, 6.7 x 10-5 m to 6.7 mm. Sensitivity of activities to inhibition by this compound was either partially or completely abolished by supplementation of the homogenates or microsomal suspensions, to 0.2% (w/v), with sodium deoxycholate, sodium cholate, digitonin, Triton X-100, or Tween 20. Phlorizin was without discernible effect on activity of partially purified enzyme preparations obtained by fractional ammonium sulfate precipitation of deoxycholate-dispersed microsomal suspensions. Inhibition of phosphotransferase activity in homogenates or microsomes was markedly potentiated by the cationic detergent trimethylhexadecylammonium bromide. Kinetic analyses indicated that inhibition of both phosphohydrolase and phosphotransferase activities by phlorizin was of the classical noncompetitive type. A value of approximately 0.7 mm for Ki for phlorizin was determined in the absence of detergent; the value was reduced to 0.2 InM when trimethylhexadecylammonium bromide was added (to 0.2%, w/v) to enzyme preparations prior to assay for activity. The effects of the administration in vivo of phlorizin on phosphotransferase activity of the enzyme were also studied. Reductions in activity per mg of kidney protein of as much as 18% were produced by intravenous injection of 0.1-g doses of phlorizin to rats. The data obtained appear to rationalize the initially apparently contradictory reports of earlier workers regarding the effects (or lack of effect) of phlorizin on glucose 6-phosphatase. The experimental observations reported in this paper also are interpreted to constitute added support for, but not direct proof of, the possible involvement of synthetic and hydrolytic activities of this enzyme in kidney glucose transport. It is suggested that the modifying effects of various detergents on phlorizin-sensitivity of the enzyme are manifest through selective action on both active and inactive silent phlorizin-binding sites in the enzyme preparation.