Photoaffinity labeling and identification of (a component of) the small‐intestinal Na<sup>+</sup>, D‐glucose transporter using 4‐azidophlorizin

Hosang, Markus; Gibbs, Michael; Diedrich, Donald F.; Semenza, Giorgio

doi:10.1016/0014-5793(81)81130-1

Cited by 56 publications

(12 citation statements)

References 32 publications

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“…It is, therefore, not unreasonable to conclude that the Na'-dependent conformation change of the FITC binding protein is related to Na' activation of glucose transport. The FITC binding protein (71,000 daltons) is most likely the glucose carrier since it is very similar to the one purified to homogeneity by Semenza's group, using monoclonal antibodies (15), and identified through 4-azidophlorizin photoaffinity labeling (16) …”

Section: Methodsmentioning

confidence: 99%

Conformational changes in the intestinal brush border sodium-glucose cotransporter labeled with fluorescein isothiocyanate.

Peerce¹,

Wright²

1984

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

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Fluorescein isothiocyanate (FITC) was used to label the rabbit intestinal brush border Na+-glucose carrier, identify the carrier protein on sodium dodecyl sulfate/ polyacrylamide gel electrophoresis, and monitor the effect of ions and substrates on fluorescence quenching. Enriched brush border preparations were employed to study both glucose transport and FITC binding. FITC and a nonfluorescent analog (phenyl isothiocyanate, PITC) both inhibited Na+-dependent D-glucose transport irreversibly. Inhibition was blocked completely by the presence of Na+ and D-glucose during labeling. PITC was used to label nonspecific amino groups in the presence of glucose and Na+, and then the glucose carrier was labeled with FITC in the absence of substrates. Fluorescence of FITC bound to the carrier was quenched specifically with Na+ in a saturable fashion, and this indicates a Na+-dependent conformational change in the carrier. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of FITC-labeled membranes revealed specific labeling of a 71,000-dalton peptide. We conclude that Na+ induces a conformational shift in the 71,000-dalton glucose carrier, and this is quite consistent with the kinetics of Na+-dependent glucose transport in these membranes.Sodium-dependent organic solute transport across the brush border membrane of the small intestine plays a primary role in the absorption of sugars and amino acids (1). In these systems Na+ gradients provide the driving force for uphill solute uptake, and kinetic experiments suggest that Nat increases the affinity of the carrier for the solute through conformational changes. However, little direct information is available about either the nature of the conformation changes or even the identity of the carrier. Since site-specific reagents with fluorescent labels have been used successfully to probe conformation changes in other transport proteins (2-7), we have explored this approach with intestinal brush borders. Fluorescein isothiocyanate (FITC), an irreversible group-specific reagent for amino groups, has been used to label the Na+-glucose carrier, identify the carrier on NaDodSO4 gels, and monitor conformation changes. Nonspecific FITC binding was minimized by using enriched membrane preparations and labeling nonspecific sites with nonfluorescent isothiocyanates in the presence of excess Na+ and glucose. We find that Na+ produces a specific, saturable decrease in FITC fluorescence that is consistent with a Nat-dependent conformational change in the glucose camer. METHODSBrush border membrane vesicles (BBMVs) were isolated from rabbit small intestine by the Ca2t differential centrifugation method (8) and further purified by an adaptation of Hopfer's KSCN treatment (9). Briefly, the crude brush borders were resuspended by homogenization in 0.6 M KSCN and 10 mM Hepes/Tris buffer at pH 7.5, incubated at 40C, and then diluted 1:10 with 10 mM Hepes/Tris, pH 7.5-This suspension was centrifuged at 6000 x g for 10 min, and the resultant supernatant was centrifuged at 38,000 x g fo...

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Section: Methodsmentioning

confidence: 99%

Conformational changes in the intestinal brush border sodium-glucose cotransporter labeled with fluorescein isothiocyanate.

Peerce¹,

Wright²

1984

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

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“…Cytochalasin B Plastidic Glucose Translocator 789 (Shanahan, 1982;Klip et al, 1983), derivatives of phlorizin (Hosang et al, 1981), and N -(azidosalicyl)-6-amido-6-deoxyglucopyranose (ASA-Glc; Shanahan et al, 1985) are potent inhibitors of GLUT1 and have been used as affinity probes for the purification of GLUT1 (Baldwin and Lienhard, 1989). In general, these inhibitors had only a small effect on Glc uptake into chloroplasts; for example, uptake of 2 mM D -Glc was inhibited only ‫ف‬ 30% by 10 M cytochalasin B, 40% by 250 M phlorizin, and not at all by 10 M ASA-Glc.…”

Section: Effect Of Inhibitors On the Uptake Of Glc Into Intact Chloromentioning

confidence: 99%

Identification, Purification, and Molecular Cloning of a Putative Plastidic Glucose Translocator

et al. 2000

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During photosynthesis, part of the fixed carbon is directed into the synthesis of transitory starch, which serves as an intermediate carbon storage facility in chloroplasts. This transitory starch is mobilized during the night. Increasing evidence indicates that the main route of starch breakdown proceeds by way of hydrolytic enzymes and results in glucose formation. This pathway requires a glucose translocator to mediate the export of glucose from the chloroplasts. We have reexamined the kinetic properties of the plastidic glucose translocator and, using a differential labeling procedure, have identified the glucose translocator as a component of the inner envelope membrane. Peptide sequence information derived from this protein was used to isolate cDNA clones encoding a putative plastidic glucose translocator from spinach, potato, tobacco, Arabidopsis, and maize. We also present the molecular characterization of a candidate for a hexose transporter of the plastid envelope membrane. This transporter, initially characterized more than 20 years ago, is closely related to the mammalian glucose transporter GLUT family and differs from all other plant hexose transporters that have been characterized to date. INTRODUCTIONIn plants, carbon fixed during the day is exported from the chloroplasts in the form of triose phosphate (trioseP), which is converted in the cytosol to sucrose. Sucrose often serves as the predominant photoassimilate being allocated to sink tissues. The export of trioseP from the chloroplasts is mediated by the trioseP/3-phosphoglycerate/phosphate translocator (TPT; Fliege et al., 1978; Flügge et al., 1989). Rather than being exported, a considerable amount of the fixed carbon is maintained within the chloroplasts and is involved in the biosynthesis of transitory starch, which could amount to approximately one-half of the carbon assimilated by photosynthesis during the day. During the next dark period, transitory starch is mobilized to sustain a continuous supply of carbon (i.e., sucrose) for export to growing sinks as well as for energy metabolism in leaves. Mutants lacking the ability to synthesize (Caspar et al., 1985; Hanson and McHale, 1988;Huber and Hanson, 1992; Geiger et al., 1995) or degrade transitory starch (Zeeman et al., 1998a(Zeeman et al., , 1998bCaspar et al., 1991) show reduced growth under conditions in which photosynthesis is restricted.Starch degradation could follow either the phosphorolytic pathway, yielding trioseP, or the amylolytic pathway, leading to free sugars, glucose (Glc), and maltose. There is evidence that the dominant pathway for the degradation of transitory starch is the amylolytic one. First, trioseP, the end product of the phosphorolytic pathway, must be exported from the chloroplasts and subsequently be converted to hexose phosphate (hexoseP) in the cytosol. This reaction is controlled by the regulatory metabolite fructose 2,6-bisphosphate, which is a strong inhibitor of the cytosolic fructosebisphosphate phosphatase (Stitt, 1990). During the transition from...

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“…On the other hand, LIN et al (1981) showed that one of the bands on SDS-PAGE with molecular weight of 60K-70K daltons was particularly enhanced after the affinity chromatography on a phlorizin-polymer. HosANG et al (1981) have identified the phlorizin-binding proteins as 72K dalton polypeptide by applying the photoaffinity labelling technique. These results together with those of the present study and MALATHI et al (1980) would suggest that the native Na+/ D-glucose cotransporter contains a subunit of around 70K daltons or it has a dimer structure consisting of a subunit of 70K-80K dalton polypeptide.…”

Section: Discussionmentioning

confidence: 99%

Purification and reconstitution of Na+/D-glucose cotransport carriers from guinea pig small intestine.

Kano-Kameyama

Hoshi

1983

Jpn.J.Physiol

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Sodium/D-glucose cotransport carriers solubilized from intestinal brush border membranes were purified and incorporated into liposomes made of soybean phospholipids, and transport properties of the reconstituted system were studied. The brush border membrane vesicles prepared from guinea pig small intestine were first treated with deoxycholate and papain in order to remove unnecessary membrane proteins, and then the remaining membrane proteins were solubilized with Triton X-100. The solubilized proteins were fractionated by gel-filtration according to molecular size and the fractions containing proteins of molecular weight of around 340 K daltons were further separated by chromatofocusing according to isoelectric point. The Na+ gradient-dependent overshoot uptake of D-glucose was seen when the proteins which were finally eluted in the pH range of 5.0-5.5 were incorporated into the liposomes. The proteins purified and incorporated into liposomes could be visualized on sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis as a single band of 160,000 dalton glycoprotein. The proteoliposomes constructed with the purified proteins revealed about 20-fold higher accumulation of D-glucose as compared with those constructed with unpurified membrane protein extracts. Kinetically, however, the reconstituted system revealed somewhat different characteristics from those of the native brush border membrane vesicles, i.e. retarded time course of overshooting uptake and an S-shaped relationship between 1-min uptake of glucose (the quasiinitial rate of glucose uptake) and glucose concentration. The reciprocal of the uptake rate was linearly proportional to the reciprocal of the square of glucose concentration and the estimated Hill coefficient was about 2.

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Photoaffinity labeling and identification of (a component of) the small‐intestinal Na⁺, D‐glucose transporter using 4‐azidophlorizin

Cited by 56 publications

References 32 publications

Conformational changes in the intestinal brush border sodium-glucose cotransporter labeled with fluorescein isothiocyanate.

Conformational changes in the intestinal brush border sodium-glucose cotransporter labeled with fluorescein isothiocyanate.

Identification, Purification, and Molecular Cloning of a Putative Plastidic Glucose Translocator

Purification and reconstitution of Na+/D-glucose cotransport carriers from guinea pig small intestine.

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Photoaffinity labeling and identification of (a component of) the small‐intestinal Na+, D‐glucose transporter using 4‐azidophlorizin

Cited by 56 publications

References 32 publications

Conformational changes in the intestinal brush border sodium-glucose cotransporter labeled with fluorescein isothiocyanate.

Conformational changes in the intestinal brush border sodium-glucose cotransporter labeled with fluorescein isothiocyanate.

Identification, Purification, and Molecular Cloning of a Putative Plastidic Glucose Translocator

Purification and reconstitution of Na+/D-glucose cotransport carriers from guinea pig small intestine.

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Photoaffinity labeling and identification of (a component of) the small‐intestinal Na⁺, D‐glucose transporter using 4‐azidophlorizin