Phlorizin Binding Research Articles

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

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+.

Use of phlorizin binding to demonstrate induction of intestinal glucose transporters.

We used specific binding of phlorizin to the intact intestinal mucosa in order to measure glucose transport site density in intestines of mice fed a high-carbohydrate or no-carbohydrate diet. Nonspecific binding varied with intestinal position but showed only modest dependence on diet. Specific binding to glucose transporters was 1.9 times greater in jejunum of high-carbohydrate mice than of no-carbohydrate mice; this ratio was the same as the ratio for Vmax values of active D-glucose uptake between the two diet groups. The gradient in specific binding of phlorizin along the intestine paralleled the gradient in Vmax of glucose transport. These results directly demonstrate that the increase in intestinal glucose transport caused by a high-carbohydrate diet is due to induction of glucose transporters. They also indicate that the normal positional gradient in glucose transport along the intestine arises from a gradient in transporters, induced by the normal gradient in luminal glucose concentration.

Inactivation of the renal outer cortical brush-border membrane D-glucose transporter by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline.

The inactivation of the renal outer cortical brush-border membrane D-glucose transporter by the covalent carboxyl reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is studied by monitoring its effects on sodium-dependent phlorizin binding to the active site of the carrier. In the presence of EEDQ, this component of phlorizin binding decreases exponentially and irreversibly with time. The order of this inactivation reaction is very close to 1, indicating that EEDQ modifies the transporter at a single essential site. This site can be partially protected by glucose and by other substrates of the transporter and completely protected by phlorizin, a nontransported competitive inhibitor. By contrast, sodium, a co-transported activator, has no protective effect. The concentration dependence of the protection provided by glucose and phlorizin indicates that the site of action of EEDQ is at or closely related to the substrate binding site on the carrier. The effects of EEDQ on the transporter are mimicked by another carboxyl specific reagent, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate. The rate of inactivation of the transporter by EEDQ increases dramatically with decreasing pH, consistent with the hypothesis that the rate-limiting step in the inactivation process is a reaction with an essential carboxyl group. The properties of this group indicate, however, that it is distinct from the carboxyl group proposed by others as forming (a part of) the sodium binding site of sodium-coupled sugar carriers.

Adaptive Changes of Sugar Absorption in Rat Small Intestine during Fasting and Refeeding

The effects of fasting and refeeding on the adaptive changes in intestinal sugar absorption was observed by measuring the transmural potential difference (ΔPD) evoked by glucose by infusion of sugars and compared with those in disaccharidase activities, glucose uptake and phlorizin binding activity to the brush border membranes, and morphological parameters.Rats were fasted for 1, 3, or 5 days. The rats fasted for 5 days were refed for 1 or 3 days further. The ΔPD evoked by glucose, maltose, or sucrose in everted jejunal segments decreased within 1 day of fasting, remained at a same level during fasting, and then increased to the control level after refeeding. Maltase and sucrase activities did not change during the first day of fasting, but did so significantly after 3 days of fasting, in terms of both specific and total activities. On refeeding, disaccharidase activities increased rapidly. However, in spite of significant changes in ΔPDs and disaccharidase activities, glucose uptake by and phlorizin-binding to brush border membrane vesicles from jejunal mucosa did not change in either 5 day-fasted or refed animals compared with the control group. On the other hand, jejunal villus height in rats fasted for 5 days decreased significantly compared with that in control rats and was accompanied by a reduction in epithelial cell height and surface area of villi.These results suggest that the absorption of sugar in small intestine could adapt to fasting and refeeding with changes in disaccharidase activity and surface area, without any change in glucose carrier.

Evidence for tyrosyl residues at the Na+ site on the intestinal Na+/glucose cotransporter.

A tyrosine group has been identified at, or near, the Na+-binding site of the Na+/glucose and Na+/proline cotransporters of rabbit intestinal brush-borders. Three tyrosine group-specific reagents, n-acetylimidazole, tetranitromethane, and p-nitrobenzene sulfonyl fluoride, were used to evaluate the role of tyrosyl groups in Na+-dependent glucose transport, Na+-dependent phlorizin binding, and the Na+-induced fluorescence quenching of fluorescein isothiocyanate bound to the glucose site of the carrier. All three reagents inhibited glucose transport, phlorizin binding, and fluorescein isothiocyanate quenching by 50-85% with Ki values in the range 7-50 microM. The presence of Na+ during the exposure of membranes to the reagents completely protected against inhibition, the Na+ concentration required to produce 50% protection was 14-36 mM. Fluorescent derivatives of n-acetylimidazole were synthesized to identify the tyrosyl residues on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A total of five polypeptide bands were labeled with eosin or fluorescein n-acetylimidazole in a Na+-sensitive manner. Two of these bands, previously identified as the glucose (75,000-dalton) and proline (100,000-dalton) binding sites of the glucose and proline carriers, account for 50% of the Na+-sensitive tyrosyl residues. On the basis of these studies, we believe that the Na+/glucose cotransporter contains both the Na+ and glucose active sites on the same polypeptide or that the cotransporter consists of two similar polypeptides, each containing one substrate binding site.

Identification of the D-glucose binding polypeptide of the renal Na +-D-glucose cotransporter with a covalently binding D-glucose analog

The covalently binding D-glucose analog 10- N-(bromoacetyl)amino-1-decyl-β-D-glucopyranoside (BADG) was synthesised and shown to be a high-affinity inhibitor of the renal Na +-D-glucose contransporter. From renal brush-border membranes a protein fraction was isolated, in which the concentration of Na +-dependent phlorizin binding sites per mg protein was enriched 7-fold. In labeling experiments with this protein fraction a polypeptide of M r ~ 79000 was identified as containing the D-glucose binding site of the renal Na +-D-glucose cotransporter. Affinity labeling D-Glucose analog D-Glucose binding site Na±D-glucose cotransporter Brush-border membrane Kidney

Structural state of the Na + / d-glucose cotransporter in calf kidney brush-border membranes Target size analysis of Na +-dependent phlorizin binding and Na +-dependent d-glucose transport

Target sizes of the renal sodium- d-glucose cotransport system in brush-border membranes of calf kidney cortex were estimated by radiation inactivation. In brush-border vesicles irradiated at −50°C with 1.5 MeV electron beams, sodium-dependent phlorizin binding, and Na +-dependent d-glucose tracer exchange decreased exponentially with increasing doses of radiation (0.4–4.4 Mrad). Inactivation of phlorizin binding was due to a reduction in the number of high-affinity phlorizin binding sites but not in their affinity. The molecular weight of the Na +-dependent phlorizin binding unit was estimated to be 230 000 ± 38 000. From the tracer exchange experiments a molecular weight of 345 000 ± 24 500 was calculated for the d-glucose transport unit. The validity of these target size measurements was established by concomitant measurements of two brush-border enzymes, alkaline phosphatase and γ-glutamyltransferase, whose target sizes were found to be 68 570 ± 2670 and 73 500 ± 2270, respectively. These findings provide further evidence for the assumption that the sodium- d-glucose cotransport system is a multimeric structure, in which distinct complexes are responsible for phlorizin binding and d-glucose translocation.

A two sodium ion/D-glucose symport mechanism: membrane potential effects on phlorizin binding.

Apical membrane vesicles isolated from a continuous renal cell line, LLC-PK1, catalyze electrogenic Na+-stimulated hexose transport and Na+-dependent binding of 3H-labeled 1-[2-(beta-D-glucopyranosyloxy)-4, 6-dihydroxyphenyl]-3-(4-hydroxyphenyl)-1-propanone [( 3H]phlorizin), a competitive ligand of this transport system. Phlorizin was not itself transported across the membrane and thus can serve as a probe of the binding step. The stoichiometry of Na+-dependent phlorizin binding in vesicles was 1:1, whereas Na+/hexose cotransport in vesicles exhibited a 2:1 stoichiometry. Na+ increased the affinity of phlorizin binding without affecting the total number of binding sites. An increased number of Na+-dependent phlorizin binding sites was observed under conditions of interior-negative membrane potential. These results are consistent with a model of the Na+/glucose cotransport cycle in which the unloaded transporter is negatively charged and its orientation influenced by membrane potential. Glucose and one sodium ion interact with the transporter, resulting in an uncharged complex. Binding of a second sodium ion triggers translocation of glucose and both sodium ions via formation of a loaded carrier complex bearing a single positive charge.

Hexose regulation of sodium-hexose transport in LLC-PK1 epithelia: the nature of the signal.

We have shown previously that the concentration of glucose in the growth medium regulates sodium-coupled hexose transport in epithelia formed by the porcine renal cell line LLC-PK1. Assayed in physiological salt solution, the ratio of the concentration of alpha-methyl glucoside (AMG) accumulated inside the cell at steady state to its concentration outside, and the number of glucose transporters, as measured by phlorizin binding, was inversely related to the glucose concentration in the growth medium. In this study, using a cloned line of LLC-PK1 cells, we provide evidence that the difference in AMG concentrating capacity is the result of a regulatory signal and not simply due to a selection process where the growth of cells with enhanced glucose transport is favored by low glucose medium or vice-versa. By adding glucose to conditioned medium (collected after 48 hr incubation with cells and therefore containing less than 0.1 mM glucose), we demonstrate that the signal in the growth medium is indeed the concentration of glucose rather than another factor secreted into or depleted from the medium. Fructose and mannose, two sugars not transported by the sodium-dependent glucose transporter, can substitute for glucose as a carbohydrate source in the growth medium and have a modest glucose-like effect on the transporter. Growth in medium containing AMG does not affect the transporter, indicating that the regulatory signal is not a direct effect of the hexose on its carrier but involves hexose metabolism.

Characterization of an essential disulfide bond associated with the active site of the renal brush-border membrane d-glucose transporter

In a previous report (J. Biol. Chem. 258 (1983) 3565–3570) we have demonstrated that the disulfide-reducing agent dithiothreitol has two effects on the sodium-dependent outer cortical brush border membrane d-glucose transporter; the first results in a reversible increase in the affinity of the transporter for the non-transported competitive inhibitor phlorizin, while the second results in a partially reversible loss of phlorizin binding and glucose-transport activity. Evidence was presented that both of these effects are the result of the reduction of disulfide bonds on the transport molecule. In the present paper we extend our observations on the inactivation of the transporter by dithiothreitol. We provide evidence here (i) that the inactivation of the transporter by dithiothreitol is independent of the effect of the reducing agent on the affinity of the transporter, (ii) that this inactivation process is first-order in dithiothreitol and thus presumably due to the reduction of a single disulfide bond essential to the functioning of the transporter. (iii) that it is the reduction of this disulfide bond and not some subsequent conformational or other change in the transporter which results in its inactivation, (iv) that phlorizin and substrates of the transporter provide protection against inactivation by dithiothreitol and that the degree of protection provided correlates well with the known specificity and phlorizin-binding properties of the transporter, and (iv) that the reactivity of the transporter with dithiothreitol is pH-dependent, decreasing with increasing pH over the pH range 6.5–8.5. We conclude that this site of action of dithiothreitol is a single essential disulfide bond intimately associated with the glucose-binding site on the transport molecule.

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.

Small-intestinal Na +/ d-glucose cotransport. Inactivation of sugar transport and phlorizin binding by thiol-group and amino-group reagents

It has previously been shown that mercurials acting from the cytoplasmic side or from within the hydrophobic part of the membrane inactivate the small intestinal Na +/ d-glucose cotransporter by blocking essential SH-groups (Klip, A., Grinstein, S. and Semenza, G. (1979) Biochim. Biophys. Acta 558, 233–245). Another (set of) sulfhydryl(s) which are critical for phlorizin binding and sugar transport function and which may lie on the luminal side of the brush border membrane, can be blocked by DTNB and 4,4′-dithiopyridine but not by N- ethylmaleimide . In addition, modification of amino groups by fluorescamine, reductive methylation and (under certain conditions) DIDS also lead to inactivation of the carrier's binding and transport functions. No evidence was obtained that any of the above groups is directly involved in the binding of either Na +/ d-glucose or phlorizin, since none of these compounds prevented inactivation of the cotransporter.

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.

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.

Polycations. A new class of inhibitors for in vitro small intestinal transport of sugars and amino acids in the rat

Polycationic compounds like polylysine, protamine or polyethylenimine may interfere with a cation-related membrane transport system depending on superficially accessible binding sites for particular cations. In vitro experiments were performed using either everted segments of rat small intestine to measure tissue accumulation or everted sacs to determine mucosal-to-serosal transport. The effect of polycations was also tested using brush-border membrane vesicles of rat jejunum. Polycations inhibited the tissue accumulation of methyl α- d-glucoside as well as binding of phlorizin. Inhibition of accumulation was increased by raising the polycation concentration and by preincubation of the tissue with the polycations. Kinetic experiments revealed a competitive type of inhibition for the uptake of neutral amino acids and actively transported sugars. Using everted sacs to compare the monomeric cations with their corresponding polymeric forms for their inhibitory effect, it was found that only polymers applied to the mucosal compartment impaired active transport. The passive diffusion of solutes, e.g. 2-deoxy- d-glucose or mannitol, was slightly increased by polycations. With some intermediate oligomers of lysine it could be shown that more than 20 cationic groups are required for approximate complete inhibition. That membrane-related events are responsible for the observed inhibition is suggested by the reduced uptake of d-glucose by brush-border membrane vesicles in the presence of polycations. Therefore an interaction with transport-related cation binding sites, i.e. anionic residues, at the mucosal surface may be assumed.

Fluorescence probe studies on the interaction of phlorizin with rabbit intestinal brush border membranes.

Spectrofluorometric studies on the interaction of phlorizin with rabbit intestinal brush border membranes under various conditions were carried out using 1-anilino-8-naphthalene sulfonate (ANS) and N-(1-anilinonaphthyl-4)maleimide (ANM). The fluorescence intensity of membrane-bound ANS was markedly decreased upon addition of phlorizin. The apparent dissociation constant of phlorizin and the membranes at pH 7.4 was determined to be approximately 24 microM from the slope of a plot of the fluorescence change of membrane-bound ANS against phlorizin concentration. Addition of D-glucose diminished the quenching effect of phlorizin on the ANS fluorescence by lowering the binding affinity of phlorizin for the membranes. The interaction between phlorizin and the membranes was influenced by temperature, ionic strength and divalent cations. The binding affinity of phlorizin for the membranes is markedly reduced at higher temperature, showing a transition between 25 and 30 degrees C. Imposition of an ionic strength gradient across the membrane vesicles (out greater than in) increased the binding affinity. On the other hand, divalent cations decreased the affinity; Mg2+ was more effective than Ca2+. The binding affinity of ANS for the membranes was evidently decreased, and the reactivity of SH groups of the membrane proteins with ANM was apparently reduced, upon binding of phlorizin to the membranes. Based on these results, the relationships among modification of membrane structures, the extent of phlorizin binding and phlorizin-induced conformational changes in the membrane proteins are discussed.

Synthesis of phlorizin derivatives and their inhibitory effect on the renal sodium/ d-glucose cotransport system

To characterize further the Na +/ d-glucose cotransport system in renal brush border membranes, phlorizin - a potent inhibitor of d-glucose transport - has been chemically modified without affecting the d-glucose moiety or changing the side groups that are essential for the binding of phlorizin to the Na +/ d-glucose cotransport system. One series of chemical modifications involved the preparation of 3-nitrophlorizin and the subsequent catalytic reduction of the nitro compound to 3-aminophlorizin. From 3-aminophlorizin, 3-bromoacetamido-, 3-dansyl- and 3-azidophlorizin have been synthesized. In another approach, 3′-mercuryphlorizin was obtained by reaction of phlorizin with Hg(II) acetate. The phlorizin derivatives inhibit sodium-dependent but not sodium-independent d-glucose uptake by hog renal brush border membrane vesicles in the following order of potency: 3′-mercuryphlorizin = phlorizin > 3-aminophlorizin > 3-bromoacetamidophlorizin > 3-azidophlorizin > 3-nitrophlorizin > 3-dansylphlorizin. 3-Bromoacetamidophlorizin - a potential affinity label - also inhibits sodium-dependent but not sodium-independent phlorizin binding to brush border membranes. In addition, sodium-dependent phosphate and sodium-dependent alanine uptake are not affected by 3-bromoacetamidophlorizin. The results described above indicate that specific modifications of the phlorizin molecule at the A-ring or B-ring are possible that yield phlorizin derivatives with a high affinity and high specificity for the renal Na +/ d-glucose cotransport system. Such compounds should be useful in future studies using affinity labeling (3-bromoacetamido- and 3-azidophlorizin) or fluorescent probes (3-dansylphlorizin).

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.

4-Azidophlorizin, a high affinity probe and photoaffinity label for the glucose transporter in brush border membranes

A new phlorizin derivative ( 2′-O-(β- d- glucopyranosyl)-4- azidophloretin , 4-azidophlorizin) has been synthesized and its affinity for the d-glucose, Na + co-transport system in brush border vesicles from intestinal and renal membranes has been compared with that of phlorizin. The extent of the reversible interaction of the ligand with the transporter in dim light has been evaluated from three separate measurements: (1) K′ i, the constant for fully-competitive inhibition of (Na +, Δψ)-dependent d-glucose uptake, (2) K′ d, the dissociation constant of 4-azido[ 3H]phlorizin binding in the presence of an NaSCN inward gradient, and (3) K″ i, the constant for fully-competitive inhibition of the specific ((Na +, Δψ)-dependent, d-glucose protectable) high-affinity [ 3H]phlorizin binding. In experiments with vesicles derived from rat kidney, all three constants ( K′ i, K′ d and K″ i) were essentially equal and ranged between 3.2 and 5.2 μM, that is, the azide derivative has almost the same affinity for this transporter as phlorizin itself. On the other hand, compared to phlorizin, the 4-azidophlorizin has a lower affinity for the transporter in vesicles prepared from rabbit; its K′ i values are some 15–20-times larger than those determined with rat membranes. However, the affinity of the azide for the sugar transporter in membranes from either the intestine or kidney of the same animal species (rabbit or rat) was essentially the same. In spite of the lower affinity for the transporter in either membrane system from the rabbit, results described elsewhere (Hosang, M., Gibbs, E.M., Diedrich, D.F. and Semenza, G. (1981) FEBS Lett., 130, 244–248) indicate that 4-azidophlorizin is an effective photoaffinity label in this species also. Photolysis of the azide yields a reactive intermediate which reacts with a 72 kDa protein in rabbit intestine brush borders. Covalent labeling of this protein occurred under conditions which suggests that it is (a component of) the glucose transporter.

Phlorizin as a probe of the small-intestinal Na +, d-glucose cotransporter. A model

(1)‘Uptake’ of phlorizin by intestinal brush border membrane vesicles is stimulated, much as that of d-glucose, by the simultaneous presence of Na out + and Δψ⪡0. However, phlorizin contrary to d-glucose, fulfills all criteria of a non-translocated ligand (i.e., of a fully competitive inhibitor) of the Na +, d-glucose cotransporter. (2) The stoicheiometry of Na +/phlorizin binding is 1, as shown by a Hill coefficient of approx. 1 in the Na out +-dependence of phlorizin binding. (3) The preferred order of binding at Δψ⪡0 is Na + first, phlorizin second (4) The velocity of association of phlorizin to the cotransporter, but not the velocity of its dissociation therefrom, responds to Δψ. These observations while agreeing with the effect of Δψ⪡0 on the K d of phlorizin binding in the steady-state time range, also confirm that the mobile part of the cotransporter bears a negative charge of 1. (5) A model is proposed describing the Na +,Δψ-dependent interaction of phlorizin with the cotransporter and agreeing with a more general model of Na +, d-glucose cotransport. (6) The k on , k off and K d constants of phlorizin interaction with the Na +, d-glucose cotransporter are smaller in the kidney than in the small-intestinal brush border membrane, which results in a number of quantitative differences in the overall behaviour of the two systems.