Phlorizin Binding Research Articles

Isolation and reconstitution of the intestinal Na+/glucose cotransporter.

The intestinal Na+/glucose cotransporter was isolated from brush border membrane vesicles using a three-step procedure and Na(+)-dependent phlorizin binding as the measure of cotransporter enrichment. The initial step was to treat the Ca2(+)-precipitated brush border membrane vesicles with 0.02% sodium dodecyl sulfate (SDS) followed by sucrose gradient centrifugation which resulted in a 5-fold enrichment of the Na+/glucose cotransporter. The second step was chromatofocusing chromatography over the pH range from pH 7.4 to pH 4.0. This step resulted in an additional 20-fold purification as compared with the SDS-brush border membrane vesicle protein which served as the starting material. The final step was affinity chromatography on con A-Sepharose which resulted in a 5-fold enrichment of the chromatofocused protein. The glycoprotein fraction from the concanavalin A column reconstituted into phosphatidyl choline: cholesterol liposomes demonstrated Na(+)-dependent, phlorizin-sensitive, and osmotic strength-sensitive glucose uptake. This fraction consisted of a single 75-kDa polypeptide on SDS-polyacrylamide gel electrophoresis upon staining with silver. On the basis of these criteria it appears that a protocol for the isolation of the Na+/glucose cotransporter has been developed.

Different pattern of differentiation in two LLC-PK1 clones.

Two clones (LD3 and LC3) were isolated from the established renal cell line LLC-PK1. They differed with respect to the development of the Na+-dependent alpha-methyl-D-glucoside (AMG) uptake. The higher uptake capacity in LD3 as compared to LC3 was owing to the expression of a higher number of carrier molecules as was shown by the specific phlorizin binding. The intracellular cyclic AMP level, the D-glucose concentration in the growth medium and the cell density could be excluded as the causes of the difference between both clones. We found that the faster development of the Na+-dependent hexose carrier in LD3 was parallelled by a faster expression of trehalase in this clone. This suggests that the expression of both apical proteins is correlated. From the growth curves it was concluded that renewal of the undifferentiated population was roughly the same in both clones. The recruitment of cells from the undifferentiated to the growth arrested state seems also very similar as was deduced from the development of tight junctions and from the down-regulation of the alpha-methylaminoisobutyric acid (meAIB) uptake. The start of differentiation was identical as was shown by the similar rate of expression found for gamma-glutamyl transferase. The difference between both clones is most likely situated at the traverse to a fully differentiated cell. This process takes more time in LC3 than in LD3. Also the fully differentiated state seemed to be different in both clones. We conclude that the pattern of differentiated characteristics found in both clones is different and a model incorporating these differences is presented.

Dexamethasone blocks adaptive increase of Na +P i cotransport in renal brush border membrane elicited by thyroid hormone

Dexamethasone administered to rats blocks and/or reverses adapative increases in the rate of Na +P i cotransport, and also in the Na +-dependent binding of [ 14 C]-phosphonoformic acid (PFA) by renal brush border membrane (BBM) vesicles elicited by thyroid hormone (T 3 ). In contrast, dexamethasone had no effect on Na +-independent binding of [ 14 C]-phosphonoformic acid, on Na +-dependent transport of D-glucose or on Na +-dependent binding of phlorizin by BBMV which indicates that its inhibitory effect is specific for Na +P i cotransport system of BBM. These findings suggest that glucocorticoids antagonize T 3 -elicited adaptive enhancement of Na +P i cotransport in renal proximal tubules by blocking the T 3 -stimulated de novo synthesis of Na +P i symporters and/or their insertion into BBM.

Different mechanisms of adaptive increase in Na+-Pi cotransport across renal brush-border membrane

We explored the biochemical mechanism by which thyroid hormone (T3) and low-phosphate diet (LPD) cause an adaptive increase in Na+-Pi cotransport across renal brush-border membrane (BBM). The rate of Na+-Pi cotransport was determined by 32Pi uptake by BBM vesicles (BBMV), and the number of Na+-Pi symporters was assessed by binding of [14C]phosphonoformic acid (PFA) on BBMV. In BBMV of both T3-treated rats and LPD-fed rats, the Na+ gradient-dependent 32Pi uptake increased (Vmax increased; Km Pi was not changed). The Na+-dependent [14C]PFA binding on BBMV increased (higher Vmax, no change in Km PFA) in response to T3, but it remained unchanged in rats fed LPD. Both the increase of Na+-Pi cotransport and of Na+-dependent [14C]PFA binding in response to T3 were blocked by actinomycin D or cycloheximide. Addition of benzyl alcohol to BBMV in vitro increased Na+-Pi cotransport, but [14C]PFA binding did not change; the [3H]phlorizin binding and cotransports of other solutes decreased or did not change. The exposure of BBMV to cholesterol decreased Na+-Pi cotransport without changing [14C]PFA binding. We suggest that the adaptive increase of Na+-Pi cotransport elicited by T3 is due to an increase in number of Na+-Pi cotransporters in BBM. In contrast, in response to LPD the number of Na+-Pi cotransporters is unchanged, and the increased Na+-Pi cotransport is due to faster translocation of Na+ with Pi due to enhanced fluidity of BBM.

Protein kinase C activation has dissimilar effects on sodium-coupled uptakes in renal proximal tubular cells in primary culture

Protein kinase C (Ca2+/phospholipid-dependent enzyme) was shown to be present in renal brush border membranes. To evaluate the influence of protein kinase C activation on three apical transport systems, we studied the effect of phorbol myristate acetate (PMA) and of two diacylglycerol analogs, oleoylacetylglycerol and dioctanoylglycerol, on sodium-dependent uptakes of phosphate (Pi), L-alanine, and alpha-methyl-D-glucopyranoside (MGP), as well as on specific phlorizin binding, in cultured rabbit proximal tubular cells. PMA, at 100 ng/ml, decreased the Vmax of Pi and MGP uptake by 30 and 17%, respectively, but not that of alanine uptake. None of the Km values were affected. PMA also decreased the number of phlorizin binding sites by 40%. PMA-induced inhibition of Pi and MGP uptake was time- and concentration-dependent, was mimicked by oleoylacetylglycerol, dioctanoylglycerol, and the diacylglycerol kinase inhibitor R59022, and was reversed by the protein kinase C antagonist 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7). The effects of PMA persisted in the presence of amiloride and dimethyl amiloride, and were potentiated by Ca2+ ionophore A23187. Opening of tight junctions blunted subsequent PMA-induced decrease of MGP uptake, but not of Pi uptake. It is concluded that: (i) activation of protein kinase C does not affect similarly Na-Pi, Na-hexose, and Na-alanine cotransport; and (ii) different pathways are likely to be involved in the observed effects.

Induction of intestinal glucose carriers in streptozocin-treated chronically diabetic rats

The maximal transport capacity (Vmax) for intestinal glucose absorption is increased in experimentally induced chronic diabetes mellitus. Using [3H]phlorizin radioautography, we examined the relation between this increase in transport Vmax and the number and distribution of sodium-glucose co-transporters on the luminal surface of rat ileum. Male Lewis rats were made diabetic with streptozocin. Ninety days later we measured 3-O-methyl-d-glucopyranose absorption and specific [3H]phlorizin binding to the ileal mucosa of the same rats. Net 3-O-methyl-d-glucopyranose flux was 6.9-fold greater in diabetic rats compared with age-matched controls. Specific binding of [3H]phlorizin to the luminal surface was 7.2-fold greater in the diabetic rats. Radioautography revealed that, in chronic diabetes, specific phlorizin binding extends into the midvillus region of the ileum, whereas in age-matched controls, it is confined to villus tips. We believe that, in untreated diabetes, a larger fraction of intestinal villus epithelial cells participate in glucose absorption.

Monoclonal antibodies against the renal Na+-D-glucose cotransporter. Identification of antigenic polypeptides and demonstration of functional coupling of different Na+-cotransport systems.

Eight monoclonal antibodies are described which are directed against the renal Na+-D-glucose cotransporter. In porcine renal brush-border membranes, the antibodies either bind to one or to three polypeptides which have been identified as components of the Na+-D-glucose cotransporter (Neeb, M., Kunz, U., and Koepsell, H., (1987) J. Biol. Chem. 262, 10718-10727). Their molecular weights and isoelectric points are 75,000 and pH 5.5, 60,000 and pH 5.2, and 47,000 and pH 5.4. Six antibodies were able to influence Na+-dependent D-glucose uptake and/or Na+-dependent high affinity phlorizin binding. In the presence of Na+, the binding of all antibodies to native membrane proteins was altered by D-glucose but not by D-mannose. Since this effect was observed with D-glucose concentrations less than 1 x 10(-8) M, a high affinity D-glucose-binding site on the D-glucose transporter has been implied. Some of the antibodies probably interact also with other Na+-coupled transporters since their binding was altered by micromolar concentrations of L-lactate, L-alanine, or L-glutamate but not by the nontransported control substances D-alanine and D-glutamate. L-lactate increased the binding of one antibody in the absence but not in the presence of D-glucose. Effects of L-lactate and L-alanine on the binding of another antibody were only observed when D-glucose was present. Thus, some epitopes on the Na+-D-glucose cotransporter are altered by D-glucose and also by substrates of other Na+ cotransporters. This finding suggests functional coupling of different Na+-cotransport systems.

Na+-coupled sugar transport: membrane potential-dependent Km and Ki for Na+.

Kinetic analysis of the characteristics of phlorizin binding and of the Na+, sugar, and potential dependence of alpha-methylglucoside (alpha-MG) influx into isolated avian intestinal cells has pointed toward two alternative models for the transport mechanism (D. Restrepo and G. A. Kimmich, J. Membr. Biol. 89: 269-280, 1986). One of these models envisions a potential-dependent Na+ binding event (Na+ well concept) as a part of the molecular mechanism. The data reported here show that the apparent Km for Na+ for sugar transport is sharply dependent on the magnitude of the membrane potential. When intracellular Na+ is absent, the maximal velocity (Vmax) achieved for sugar influx is the same with or without a potential, although Vmax is obtained at a lower Na+ concentration when a potential is imposed (interior negative). Intracellular Na+ severely inhibits the influx of sugar in the absence of a potential, but this effect is largely overcome when a potential is present. The Vmax obtained when intracellular Na+ is present is a function of the potential. These results are consistent with a transport model in which Na+ binding to the Na+-dependent sugar carrier at the extracellular surface of the membrane and debinding at the inner surface of the membrane are both potential-dependent events.

Purification of a putative Na + /D-glucose cotransporter from pig kidney brush border membranes on a phlorizin affinity column

Phlorizin, a potent inhibitor of the Na + /D-glucose cotransporter, was derivatised to 3-aminophlorizin and subsequently coupled to Affi-Gel 15. Affinity chromatography of pig kidney brush border membranes solubilised in Triton X-100 allowed the purification of a 60 kDa protein on this resin. We consider this protein to be the Na + /D-glucose cotransporter, or part of it, for the following reasons: (i) binding of this protein to Affi-Gel 15 specifically requires phlorizin covalently attached to the resin and is lowered when phlorizin is replaced by phloretin; (ii) binding of the 60 kDa protein to a phlorizin affinity column requires the presence of Na +; (iii) polyclonal as well as monoclonal antibodies against the 60 kDa protein inhibit binding of phlorizin to brush border membranes from rabbit and pig kidney.

High affinity phlorizin binding to the LLC-PK1 cells exhibits a sodium:phlorizin stoichiometry of 2:1.

The phlorizin binding properties of luminal membrane vesicles isolated from the LLC-PK1 cells, a continuous epithelial cell line derived from pig kidney, are studied. Scatchard analysis of this binding indicates the existence of a single high affinity sodium-dependent site with KD = 0.4 microM at 266 mM sodium. The specificity properties of this site indicate that it represents the binding of phlorizin to the hexose binding site of the sodium-dependent D-glucose transporter previously identified in this cell line. Both phlorizin equilibrium binding and the rate of phlorizin binding were found to be sigmoidal functions of sodium concentration. A Hill analysis of these data was consistent with a sodium:phlorizin stoichiometry of 2:1 in good agreement with the sodium:glucose stoichiometry already established in these cells. Phlorizin dissociation was also found to be sodium-dependent. On the basis of the phlorizin binding data presented here, a number of models of the binding of phlorizin and sodium to the transporter can be excluded. An analysis of a random binding model consistent with the data is presented. The significance of the LLC-PK1 sodium-dependent D-glucose transporter as a model system for related renal and intestinal transporters is discussed.

Changes in glucose uptake by and phlorizin binding to brush-border membrane vesicles of small intestine from streptozotocin-induced diabetic rats.

The changes in the intestinal glucose transport in streptozotocin-induced diabetic rats have been demonstrated with brush-border membrane vesicles. When Na+-dependent D-glucose uptake and phlorizin binding activities were compared, both significantly increased either in the jejunum or ileum of diabetic rats compared with those of control. Kinetic studies showed the increases in the Vmax of glucose transport and the maximum binding of phlorizin (Bmax), whereas the Kt and Kd remained unchanged, respectively. These results suggested that the increase in glucose transport in diabetic rat intestine was due to the increase in the number of the intact glucose transporters.

Purification and reconstitution of a 75-kilodalton protein identified as a component of the renal Na+/glucose symporter.

A 75-kilodalton (kDa) protein was purified from solubilized renal brush border membranes by using high-pressure liquid chromatography (HPLC) and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Functional and immunological properties identified the 75-kDa protein as a component of the Na+/glucose symport system. The purified protein was specifically recognized by a monoclonal antibody that functionally interacts with the Na+/glucose symporter. Na+-dependent phlorizin binding activity was associated with fractions containing the 75-kDa protein during HPLC fractionation on the anion exchanger Mono-Q and was greatly increased after reconstitution into egg yolk phosphatidylcholine vesicles. The final purified preparation contained glucosamine and a blocked N-terminus.

Monoclonal antibodies that bind the renal Na+/glucose symport system. 2. Stabilization of an active conformation.

Conformation-dependent fluorescein isothiocyanate (FITC) labeling of the pig renal Na+/glucose symporter was investigated with specific monoclonal antibodies (MAb's). When renal brush border membranes were pretreated with phenyl isothiocyanate (PITC), washed, and then treated at neutral pH with FITC in the presence of transporter substrates Na+ and glucose, most of the incorporated fluorescence was associated with a single peak after resolution by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular mass of the FITC-labeled species ranged from 79 to 92 kDa. Labeling of this peak was specifically reduced by 70% if Na+ and glucose were omitted. Na+ could not be replaced by K+, Rb+, or Li+. FITC labeling of this peak was also stimulated after incubation of membranes with MAb's known to influence high-affinity phlorizin binding, and stimulation was synergistically increased when MAb's were added in the presence of Na+ and glucose. Substrate-induced or MAb-induced labeling correlated with inactivation of Na+-dependent phlorizin binding. MAb's recognized an antigen of 75 kDa in the native membranes whereas substrate-induced FITC labeling was accompanied by loss of antigen recognition and protection from proteolysis. These findings are consistent with a model in which MAb's stabilize a Na+-induced active conformer of the Na+/glucose symport system.

Use of mild detergent gel electrophoresis for isolation and characterization of the kidney brush border d-glucose transporter

Gradient gel electrophoresis was performed under mild detergent conditions to separate pig kidney brush border membrane proteins and to identify the smallest functional molecular protein entity of the d-glucose transporter. The various protein bands obtained from the nondenaturing gel system in a semipreparative scale were eluted by electrodialysis. These proteins were then reintegrated into proteoliposomes and tested for d-glucose-inhibitable [ 3H]phlorizin binding. The d-glucose transporter had a molecular mass of 70 kDa in mild detergent electrophoresis conditions and in subsequent SDS analysis.

Ischemia induces surface membrane dysfunction. Mechanism of altered Na+-dependent glucose transport.

Reversible ischemia reduced renal cortical brush border membrane (BBM) Na+-dependent D-glucose uptake (336 +/- 31 vs. 138 +/- 30 pmol/mg per 2 s, P less than 0.01) but had no effect on Na+-independent glucose or Na+-dependent L-alanine uptake. The effect on D-glucose uptake was present after only 15 min of ischemia and was due to a reduction in maximum velocity (1913 +/- 251 vs. 999 +/- 130 pmol/mg per 2 s; P less than 0.01). This reduction was not due to more rapid dissipation of the Na+ gradient, altered sidedness of the vesicles, or an alteration in membrane potential. Ischemia did, however, reduce the BBM sphingomyelin-to-phosphatidylcholine (SPH/PC) and cholesterol-to-phospholipid ratios and the number of specific high-affinity Na+-dependent phlorizin binding sites (390 +/- 43 vs. 146 +/- 24 pmol/mg; P less than 0.01) without altering the binding dissociation constant (Kd). 20 mM benzyl alcohol also reduced the number of Na+-dependent phlorizin binding sites (418 +/- 65 vs. 117 +/- 46; P less than 0.01) without altering Kd. The reduction in Na+-dependent D-glucose transport correlated with ischemic-induced changes in the BBM SPH/PC and cholesterol-to-phospholipid ratios and membrane fluidity. Taken together these data indicate the cellular site responsible for ischemic-induced reduction in renal cortical transcellular glucose transport is the BBM. We propose the mechanism involves marked alterations in BBM lipids leading to large increases in BBM fluidity which reduces the binding capacity of Na+-dependent glucose carriers. These data indicate that reversible ischemia has profound effects on the surface membrane function of epithelial cells.

Identification of D-glucose-binding polypeptides which are components of the renal Na+-D-glucose cotransporter.

D-Glucose-binding polypeptides in the Na+-D-glucose cotransporter from pig renal cortex were identified by affinity labeling with two D-glucose analogs, 10-N-(N-[4-azido-2-nitrophenyl]-beta-alanyl)amino-1-decyl-beta-D- glucopyranoside (NapADG) and 10-N-(bromoacetyl)amino-1-decyl-beta-D-glucopyranoside (BADG). During short-term incubation in the dark, NapADG and BADG are reversible inhibitors of Na+ gradient-dependent D-glucose uptake and Na+-dependent phlorizin binding with Ki values of about 40 and 400 microM, respectively. Irreversible inhibition of Na+-dependent phlorizin binding, which was prevented by D-glucose or phlorizin, was measured after a 1-h incubation with BADG. Both NapADG and BADG selectively labeled polypeptides with apparent molecular weights of 82,000, 75,000, 64,000, and 47,000. Since labeling of the Mr 82,000 and 75,000 polypeptides by both analogs was partially dependent on the presence of Na+ and was partially protected by D-glucose or phlorizin but not by L-glucose or D-mannose, these polypeptides are thought to be components of the renal Na+-D-glucose cotransporter which contain D-glucose-binding sites. For the Mr 64,000 and 47,000 polypeptides, Na+ dependence and D-glucose protection were not constantly observed. However, also, these polypeptides are thought to be components or proteolytic splitting products of the Na+-D-glucose cotransporter since we observed that three monoclonal antibodies showed cross-reaction with the BADG-labeled Mr 82,000, 64,000, and 47,000 polypeptides (K. Korn, A. Raszeja-Specht, S. Bernotat-Danielowski, and H. Koepsell, manuscript in preparation). When the BADG-labeled Mr 82,000 and 75,000 polypeptides were analyzed after two-dimensional separation by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, three-labeled, D-glucose-protectable polypeptides with the respective molecular weights and isoelectric points of 82,000 and 5.6, 75,000 and 5.4, and 75,000 and 6.9 were distinguished. The data indicate that renal brush-border membranes contain several polypeptides which are components of the Na+-D-glucose cotransporter and contain D-glucose-binding sites.

Diethylpyrocarbonate inhibition of sodium-glucose cotransport in kidney brush-border membrane vesicles

Exposure of kidney brush-border membrane vesicles to the acylating reagent diethylpyrocarbonate resulted in inactivation of the glucose transporter, as demonstrated by inhibition of sodium-coupled d-glucose transport and phlorizin binding. The transport site(s) was protected against inactivation by the simultaneous presence of sodium ions and d-glucose, and were partially protected by phlorizin. Transport activity was not restored by hydroxylamine; this rules out the possibility of diethylpyrocarbonate interaction with histidine, serine or tyrosine transporter residues. Dithiothreitol, a thiol protector, slightly prevented diethylpyrocarbonate inactivation. It is therefore suggested that (an) amino group(s) in the translocation complex is involved, at the level of the sugar transport site and the preferential protection of d-glucose against diethylpyrocarbonate inactivation related to a conformation change caused by the simultaneous binding of sodium and d-glucose to the cotransporter.

Intestinal adaptation to diabetes. Altered Na-dependent nutrient absorption in streptozocin-treated chronically diabetic rats.

To examine the pattern and mechanisms of enhanced intestinal nutrient absorption in diabetes, we measured intestinal transport of 3-O-methylglucose (3OMG), l-alanine (ALA), and SO4 in male Lewis rats made diabetic with streptozocin. Diabetes enhanced 3OMG absorption fivefold in ileum and threefold in jejunum; ALA absorption increased twofold in ileum but not at all in jejunum; ileal SO4 transport was unaffected. Increases in 3OMG and ALA transport were due solely to increases in maximum velocity. The enhancement of ileal glucose absorption was half-maximal in 40-45 d, could be reversed by 10 d of treatment with insulin and did not result from adrenergic denervation. The density of glucose carriers per milligram brush border protein (measured as [3H]phlorizin binding sites) was not altered but there was a sixfold increase in the number of glucose-inhibitable [3H]phlorizin-binding sites in the intact epithelium. Generalized mucosal hypertrophy accounted for less than 30% of this increase. We conclude that the intestine adapts to streptozocin-induced diabetes through recruitment of additional brush border carriers for sugar, probably in the midvillus-to-crypt region.

Phlorizin binding to isolated enterocytes: membrane potential and sodium dependence.

Phlorizin binding is studied in isolated intestinal epithelial cells of the chick. Cells are ATP depleted to allow extensive manipulation of ionic gradients and membrane potential (delta psi). Phlorizin binding is assayed at steady state. Carrier specific phlorizin binding is defined as D-glucose (90 mM) inhibitable binding. Specific binding displays simple Michaelian kinetics as a function of phlorizin, indicating the presence of a single homogeneous binding site. Sodium concentrations and delta psi modify the apparent binding affinity but not the maximum number of binding sites. In contrast, the activation curve as a function of sodium concentrations is sigmoid and the apparent maximum number of binding sites at saturating sodium is phlorizin dependent. The rate of phlorizin association is both delta psi and sodium-concentration dependent. Dissociation is sodium-concentration dependent but not delta psi dependent. Theoretical analysis indicates binding order of substrates is random. In addition, data suggests that the phlorizin/sodium stoichiometry is 2:1. The delta psi dependence can be explained by two models: either translocation is the delta psi-dependent step and the free carrier is anionic, or sodium binding is the delta psi-dependent step.

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.