Photoaffinity labeling of the human erythrocyte monosaccharide transporter with an aryl azide derivative of D-glucose.

Shanahan, Michael F.; Wadzinski, Brian E.; Lowndes, Joseph M.; Ruoho, Arnold E.

doi:10.1016/s0021-9258(17)39116-0

Cited by 33 publications

(6 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Both ASA and GalNASA have an absorbance maximum at 272 nm in PBS/Az with a shoulder or broad peak at 310-330 nm, similar to that reported for Af-(4-azidosalicyl)-6-amido-6-deoxyglucopyranose (Shanahan et al, 1985). Upon photolysis, the absorbance at 272 nm decreased, and the solution became slightly yellow, serving as a diagnostic test for the presence of ASA or GalNASA in the solution.…”

Section: Resultssupporting

confidence: 72%

“…The higher concentrations of probe required in these studies frequently result in high levels of nonspecific labeling. In spite of this limitation, photochemical derivatives of monosaccharides have been used successfully to study concanavalin A (Beppu et al, 1975), glyceraldehyde-3-phosphate dehydrogenase (May, 1986), and the glucose transporter of erythrocyte membranes (Shanahan et al, 1985). Recently, Lee and Lee (1986) prepared a glycopeptide in which the photochemical moiety was coupled to the terminal sugar residues involved in high-affinity binding to the Gal/GalNAc receptor of mammalian liver cells, thus overcoming the weaknessess of both earlier approaches.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Affinity labeling of the carbohydrate binding site of the lectin discoidin I using a photoactivatable radioiodinated monosaccharide

Kohnken¹,

Berger²

1987

Biochemistry

View full text Add to dashboard Cite

N-(4-Azidosalicyl)galactosamine (GalNASA), a photoactivatable, radioiodinatable analogue of N-acetylgalactosamine (GalNAc), has been prepared and characterized. We have used this reagent for labeling of the carbohydrate binding site of discoidin I, an endogenous lectin produced by Dictyostelium discoideum. GalNASA behaved as a ligand for discoidin I, as judged by its ability to compete in an assay measuring the carbohydrate binding activity of discoidin I. In this assay, it exhibited a Ki,app of 800 microM, comparable to that of GalNAc. The Ki,app of GalNASA decreased to 40 microM upon prior photolysis with ultraviolet light. In contrast, N-(4-azidosalicyl)ethanolamine produced no inhibition of carbohydrate binding regardless of photolysis. Covalent labeling of discoidin I with 125I-GalNASA was entirely dependent upon ultraviolet light. A portion of the labeling, representing 40-60% of the total, was sensitive to reagents which were known to inhibit carbohydrate binding by discoidin I, including GalNAc, asialofetuin, and ethylenediaminetetraacetic acid. N-Acetylglucosamine, which is not a ligand of discoidin I, was without effect. As a control, no carbohydrate-sensitive labeling was observed upon incubation of 125I-GalNASA with bovine serum albumin. The carbohydrate-sensitive fraction of discoidin I photolabeling with 125I-GalNASA exhibited a Kd of 15-40 microM, in agreement with the Ki,app of prephotolyzed GalNASA observed in the carbohydrate binding assay. Some labeling occurred if 125I-GalNASA was photolyzed prior to incubation with discoidin I, suggesting the involvement of long-lived species in the labeling reaction. Partial proteolytic digestion of photolabeled discoidin I revealed specific fragments whose labeling was completely blocked by GalNAc.(ABSTRACT TRUNCATED AT 250 WORDS)

show abstract

Section: Resultssupporting

confidence: 72%

Section: Discussionmentioning

confidence: 99%

Affinity labeling of the carbohydrate binding site of the lectin discoidin I using a photoactivatable radioiodinated monosaccharide

Kohnken¹,

Berger²

1987

Biochemistry

View full text Add to dashboard Cite

show abstract

“…We next examined whether molecules that had been shown to bind tightly to the human erythrocyte Glc transporter (GLUT1) also inhibited Glc uptake by chloroplasts. Cytochalasin B (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 Chloroplastsmentioning

confidence: 99%

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

Weber¹,

Servaites²,

Geiger³

et al. 2000

The Plant Cell

View full text Add to dashboard Cite

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.

show abstract

“…Although it has been known that ATP transport into RER vesicles occurs in a protein-mediated manner, none of the proteins responsible for ATP transport in the RER have been identified. In order to identify the ATP transporter isolated from RER vesicles, we used the photoaffinity labeling technique, a powerful tool to enable highly specific labeling of membrane proteins (Shanahan et al, 1985;Weber & Eichholz, 1985), and in Vitro reconstitution into artificial membranes as a functional assay (Levy et al, 1992). In addition, to obtain a clear understanding of the ATP transporter, we have partially purified the ATP transporter, sustaining its activity subsequent to its incorporation into liposomes generated from microsomal lipids.…”

mentioning

confidence: 99%

Identification of the ATP Transporter of Rat Liver Rough Endoplasmic Reticulum via Photoaffinity Labeling and Partial Purification

1996

View full text Add to dashboard Cite

In order to identify the ATP transporter in rat liver rough endoplasmic reticulum (RER), a photoreactive azido derivative of ATP, 3'-O-(p-azidobenzoyl)-ATP (AB-ATP), was synthesized by the reaction of ATP with N-hydroxysuccinimido 4-azidobenzoate (NHS-AB). The activity of the ATP transporter was determined by measuring the influx of [8-14C]ATP. The ATP transport had an apparent Km value of 6.5 microM and a Vmax of 1 nmol min-1 (mg of protein)-1. The transport of ATP was specifically inhibited by AB-ATP and 4, 4'-diisothiocyanatostilbene-2', 2'-disulfonic acid (DIDS). Under a dim light, AB-ATP was a competitive inhibitor of the ATP transport with Ki value of 0.19 microM, which indicates that AB-ATP has a high affinity for the ATP transporter, so it can be utilized as a photoaffinity probe for the identification of the ATP transporter in rat liver RER. An SDS--PAGE analysis of RER vesicles photolabeled with [gamma-32P]AB-ATP indicates the presence of a 56-kDa protein. The 56-kDa protein was completely protected from photoaffinity labeling by 10 microM ATP but not by 30 microM GTP. The specific labeling of the 56-kDa protein was sensitive to the anion transport inhibitor DIDS. In order to confirm whether the apparent uptake of ATP was due to the 56-kDa protein, the ATP transporter was partially purified through two successive ion-exchange chromatography steps (DEAE and Mono-S). The fraction showing the high activity of the ATP transporter also contained the 56-kDa protein photolabeled with [gamma-32P]AB-ATP. On the basis of the photoaffinity labeling and reconstitution experiment, we conclude that the 56-kDa protein represents the ATP transporter in rat liver RER.

show abstract

Photoaffinity labeling of the human erythrocyte monosaccharide transporter with an aryl azide derivative of D-glucose.

Cited by 33 publications

References 19 publications

Affinity labeling of the carbohydrate binding site of the lectin discoidin I using a photoactivatable radioiodinated monosaccharide

Affinity labeling of the carbohydrate binding site of the lectin discoidin I using a photoactivatable radioiodinated monosaccharide

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

Identification of the ATP Transporter of Rat Liver Rough Endoplasmic Reticulum via Photoaffinity Labeling and Partial Purification

Contact Info

Product

Resources

About