Conformational Changes in the Na/Proline Cotransporter of Intestinal Brush Borders<sup>a</sup>

Peerce, Brian E.; Wright, Ernest M.

doi:10.1111/j.1749-6632.1985.tb14854.x

Cited by 17 publications

(14 citation statements)

References 5 publications

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“…Controversy exists as to the existence of a second glucose transporter protein in the small intestine similar to that observed in renal brush border membranes (51). A number of investigators have argued for the presence of two distinct transporters based on kinetic evidence (52)(53)(54)(55)(56)(57), whereas others have claimed that differences in Km can be explained by either differences in membrane fluidity (30) or changes in cis-Na+ concentration and resultant conforlnational changes in the transporter (58). In the present study, no firm conclusions could be drawn concerning this matter.…”

Section: Methodscontrasting

confidence: 55%

Epidermal Growth Factor and Postnatal Development of Intestinal Transport and Membrane Structure

1991

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ABSTRACT. The effects of epidermal growth factor (EGF) on postnatal development of intestinal transport and the physical composition of the microvillus membrane were examined. New Zealand White rabbits received EGF (40 pg/kg/d) from d 3 of life to d 17 either intraperitoneally or orogastrically. Intestinal H20, Na' , and glucose absorption expressed per cm of intestine were significantly increased in animals receiving EGF by either route. When EGF was given by the orogastric route, nutrient absorption rates normalized to mucosal DNA were not elevated; thus, increased absorption induced by orogastric EGF appeared to be secondary to mucosal hyperplasia. In contrast, systemic EGF up-regulated cellular nutrient transport. To evaluate at which membrane level these changes occurred, brush border membrane vesicles were isolated from both the jejunum and ileum of control and EGF-treated animals. Rates of Na'-dependent glucose transport into the vesicles revealed that in the ileum systemic EGF up-regulated maximal rates of glucose transport by 54% without affecting the Km. These observations were associated with alterations in the lipid composition and physical properties of the microvillus membrane. EGF-treated animals had significant reductions in membrane cholesterol content and altered ratios of phospholipid subclasses. The net result of these variations was that the microvillus membrane isolated from EGF-treated animals was significantly more fluid than membrane from controls. Thus, these results suggest that EGF modulates development of transport function during the postnatal period both by stimulating mucosal growth and by inducing specific transport processes. Furthermore, these changes are associated with alterations in the physical composition of the microvillus

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Section: Methodscontrasting

confidence: 55%

Epidermal Growth Factor and Postnatal Development of Intestinal Transport and Membrane Structure

1991

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“…In vesicle studies specific binding of phlorizin to the glucose transporter rapidly achieves a maximal value at 2-45 sec (Chesney, Sacktor & Kleinzeller, 1974;Peerce & Wright, 1984), thereafter declining slowly due to dissipation of the initial Na + gradient (Toggenburger et al, 1982) or to reduced binding (Glossmann & Neville, 1972). In the intact intestinal mucosa specific binding is slower, as one would expect from the much greater unstirred layers, but is nevertheless faster than nonspecific binding (Fig.…”

Section: Time Dependence Of Bindingmentioning

confidence: 99%

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

Ferraris¹,

Diamond²

1986

J. Membrain Biol.

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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+.

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“…For Na+-D-glucose cotransport in calf kidney a functional molecular weight of 345,000, and for phlorizin binding in rabbit kidney functional molecular weights of 230,000 and 110,000 have been determined by target size analysis [23,31,34]. With amino acid specific reagents a M~ 75,000 polypeptide has been identified as a component of the intestinal transporter in rabbit [27,28] and with covalently binding D-glucose analogs and monoclonal antibodies 110, 18, 25, 26] M, 75,000 and 47,001) polypeptides have been shown to be components of the renal transporter in pig and rat. Furthermore, a cDNA coding for a M~ 73,080 polypeptide was cloned from rabbit intestine which was able to drastically increase Na+-D-glucose cotransport in oocytes from Xenopus laeuis after injection of the corresponding mRNA [12].…”

Section: Introductionmentioning

confidence: 99%

Two substrate sites in the renal Na+-d-glucose cotransporter studied by model analysis of phlorizin binding and-d-glucose transport measurements

et al. 1990

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Time courses of phlorizin binding to the outside of membrane vesicles from porcine renal outer cortex and outer medulla were measured and the obtained families of binding curves were fitted to different binding models. To fit the experimental data a model with two binding sites was required. Optimal fits were obtained if a ratio of low and high affinity phlorizin binding sites of 1:1 was assumed. Na+ increased the affinity of both binding sites. By an inside-negative membrane potential the affinity of the high affinity binding site (measured in the presence of 3 mM Na+) and of the low affinity binding site (measured in the presence of 3 or 90 mM Na+) was increased. Optimal fits were obtained when the rate constants of dissociation were not changed by the membrane potential. In the presence of 90 mM Na+ on both membrane sides and with a clamped membrane potential, KD values of 0.4 and 7.9 microM were calculated for the low and high affinity phlorizin binding sites which were observed in outer cortex and in outer medulla. Apparent low and high affinity transport sites were detected by measuring the substrate dependence of D-glucose uptake in membrane vesicles from outer cortex and outer medulla which is stimulated by an initial gradient of 90 mM Na+ (out greater than in). Low and high affinity transport could be fitted with identical Km values in outer cortex and outer medulla. An inside-negative membrane potential decreased the apparent Km of high affinity transport whereas the apparent Km of low affinity transport was not changed. The data show that in outer cortex and outer medulla of pig high and low affinity Na(+)-D-glucose cotransporters are present which contain low and high affinity phlorizin binding sites, respectively. It has to be elucidated from future experiments whether equal amounts of low and high affinity transporters are expressed in both kidney regions or whether the low and high affinity transporter are parts of the same glucose transport molecule.

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Conformational Changes in the Na/Proline Cotransporter of Intestinal Brush Borders^a

Cited by 17 publications

References 5 publications

Epidermal Growth Factor and Postnatal Development of Intestinal Transport and Membrane Structure

Epidermal Growth Factor and Postnatal Development of Intestinal Transport and Membrane Structure

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

Two substrate sites in the renal Na+-d-glucose cotransporter studied by model analysis of phlorizin binding and-d-glucose transport measurements

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Conformational Changes in the Na/Proline Cotransporter of Intestinal Brush Bordersa

Cited by 17 publications

References 5 publications

Epidermal Growth Factor and Postnatal Development of Intestinal Transport and Membrane Structure

Epidermal Growth Factor and Postnatal Development of Intestinal Transport and Membrane Structure

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

Two substrate sites in the renal Na+-d-glucose cotransporter studied by model analysis of phlorizin binding and-d-glucose transport measurements

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Conformational Changes in the Na/Proline Cotransporter of Intestinal Brush Borders^a