Distance between substrate sites on the Na-glucose cotransporter by fluorescence energy transfer.

Peerce, Brian E.; Wright, Ernest M.

doi:10.1073/pnas.83.21.8092

Cited by 24 publications

(4 citation statements)

References 19 publications

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“…Recently, the sodium/glucose cotransporter has been identified as a 75-kDa polypeptide (1,2), and some progress has been made in the characterization of this transport protein (3). The facilitated glucose carrier in the basolateral membrane is probably similar, if not identical, to the 55-kDa glucose carrier in human erythrocytes (see refs.…”

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confidence: 99%

Expression of size-selected mRNA encoding the intestinal Na/glucose cotransporter in Xenopus laevis oocytes.

Hediger¹,

Ikeda²,

Coady³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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103

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The expression of the rabbit intestinal brushborder Na/glucose cotransporter has been studied in Xenopus oocytes. Poly(A)I RNA isolated from the intestinal mucosa was injected into oocytes, and the expression of the transporter in the oocyte plasma membrane was assayed by measuring the Na-dependent phlorizin-sensitive uptake of methyl a-D-[1"Clglucopyranoside (MeGlc). Expression of the glucose carrier was detected 3-7 days after mRNA injection, and the rate of glucose transport was proportional to the amount of mRNA injected. mRNA (50 ng) increased the maximum velocity (V.)of MeGlc uptake by as much as 10-fold over background. The total mRNA was fractionated by preparative agarose gel electrophoresis and each fraction was assayed for its ability to induce transport activity. The mRNA encoding the Na/ glucose cotransporter was found in a single fraction of "z2.3 kilobases (kb), which contained 3% of the total mRNA. A similar mRNA fraction (2.0-2.6 kb) isolated from colon did not induce expression of this transporter. In vitro translation of the fractionated intestinal mRNA showed enhanced synthesis of two protein bands at 57 and 63 kDa. The mRNA encoding the cotransporter is smaller (2.3 kb) than that (2.6-2.9 kb) encoding the 55-kDa facilitated glucose carrier in human hepatoma cells and rat brain.Active glucose absorption across the small intestine is performed by enterocytes at the tip of the villus. Transport occurs in two stages: the first is intracellular accumulation of the sugar across the brush-border membrane by a sodium/ glucose cotransporter, and the second is facilitated diffusion of sugar out of the cell across the basolateral membrane.Recently, the sodium/glucose cotransporter has been identified as a 75-kDa polypeptide (1, 2), and some progress has been made in the characterization of this transport protein (3). The facilitated glucose carrier in the basolateral membrane is probably similar, if not identical, to the 55-kDa glucose carrier in human erythrocytes (see refs. 4-6).Intestinal glucose absorption exhibits adaptive regulation with diet, starvation, diabetes, gestation, lactation, and aging (7). For example, in diabetes mellitus, the maximal velocity (Vmax) of sodium-dependent glucose transport increases, and this is probably due to an increase in the number of sodium/glucose cotransporters. There is also a genetic component, and this is best exemplified by the rare hereditary disorder called primary glucose/galactose malabsorption (see ref. 8), which again is probably due to a defect in the cotransporter.As a first step in cloning the gene for the intestinal sodium/glucose cotransporter, we have attempted to express the carrier in Xenopus laevis oocytes and to identify the size of the mRNA encoding the 75-kDa protein. Amphibian oocytes translate foreign mRNAs very efficiently (9) and have been successfully exploited to express functional membrane transport proteins ranging from ion channels to ion exchangers (10-12). Here we demonstrate that Xenopus oocytes successfully translate i...

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mentioning

confidence: 99%

Expression of size-selected mRNA encoding the intestinal Na/glucose cotransporter in Xenopus laevis oocytes.

Hediger¹,

Ikeda²,

Coady³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

103

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“…Evidence suggesting that more than the glucose site is involved in the conformational change is indicated by the existence of an SH residue removed from the active sites that is essential for Na-dependent glucose uptake, phlorizin binding, and Na-induced quenching of FITC fluorescence (Semenza et al, 1984;Peerce & Wright, 1984a,b). This view is strengthened by fluorescence energytransfer experiments (Peerce & Wright, 1986) which indicate that the Na+ and glucose binding sites are separated by some 35 A.…”

Section: Discussionmentioning

confidence: 98%

“…These considerations lead to the conclusion that Na+ produces rather global conformational changes on the intestinal brush border Na+/glucose carrier. Binding of Na+ to the 75-kDa transport protein at a site 30-40 Á away from the glucose binding site (Peerce & Wright, 1986) produces an increase in solvent exposure of the glucose binding site (increase quenching of PYTC by Tl+, Figure 2) and a redistribution of the tryptophan residues 15-30 Á from the glucose binding site (Figures 4 and 5 and Table III). Similar longrange conformational changes in other transport proteins have been reported.…”

Section: Discussionmentioning

confidence: 99%

Examination of the sodium-induced conformational change of the intestinal brush border sodium/glucose symporter using fluorescent probes

Peerce

Wright²

1987

Biochemistry

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The Na+-induced change in conformation of the intestinal brush border glucose carrier has been examined by three procedures. In the first, we have measured the effect of Na+ on the binding of fluorescein isothiocyanate (FITC) to the glucose site; 100 mM Na increased the specific [blocked by D-glucose, p-(chloromercuri)benzenesulfonic acid, and N-acetylimidazole] FITC binding to a 75-kilodalton polypeptide 3-fold. In the second series, we have examined the effect of Na+ on the susceptibility of the fluorescently labeled glucose site [pyrene isothiocyanate (PYTC) labeled] to a hydrophilic quencher (Tl+); 100 mM NaCl increased the fraction of PYTC sites available to Tl+ from 32% to 92% and decreased the apparent quenching constant from 94 to 44 M-1. Finally, in the third series, we probed the distribution of tryptophan residues 15-30 A from the glucose site using a "distant reporter group method", where tryptophan was used an an energy donor to anthracene isothiocyanate bound to the glucose site. Tryptophan quench reagents (I-, Cs+, and acrylamide) were then employed to probe the accessibility of the glucose site tryptophans in the presence and absence of sodium. In the absence of Na+, there were two major classes of glucose tryptophans--exterior surface residues and residues buried in the hydrophobic protein matrix. Na+ caused a redistribution of the donor tryptophans such that a higher percentage were accessible to I- (51% vs. 25%) and fewer were accessible to Cs+ (13% vs. 25%) and acrylamide (27% vs. 57%).(ABSTRACT TRUNCATED AT 250 WORDS)

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“…)T2 _ (ji3/3! )T3] (8) where T is the mean decay rate, and jin is the n-th moment around T of G(F ). (,u 2/T2) is a measure of the dispersion in G(F ).…”

Section: Dynamic Light Scatteringmentioning

confidence: 99%

Elasticity of biomembranes studied by dynamic light scattering

Fujime

Miyamoto²

1991

SPIE Proceedings

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Combination of osmotic swelling and dynamic light scattering makes it possible to measure the elastic modulus of biomembranes. By this technique, we have observed a drastic increase in membrane flexibility on activation of Na/glucose cotransporters in membrane vesicles prepared from brush-borders of rat small intestine, and on activation by micromolar [Ca2] of exocytosis in secretory granules isolated from rat pancreatic acinar cells and bovine adrenal chromaffin cells.

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Distance between substrate sites on the Na-glucose cotransporter by fluorescence energy transfer.

Cited by 24 publications

References 19 publications

Expression of size-selected mRNA encoding the intestinal Na/glucose cotransporter in Xenopus laevis oocytes.

Expression of size-selected mRNA encoding the intestinal Na/glucose cotransporter in Xenopus laevis oocytes.

Examination of the sodium-induced conformational change of the intestinal brush border sodium/glucose symporter using fluorescent probes

Elasticity of biomembranes studied by dynamic light scattering

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