5-(Pentafluorobenzoylamino)fluorescein: A Selective Substrate for the Determination of Glutathione Concentration and GlutathioneS-Transferase Activity

Arttamangkul, Seksiri; Bhalgat, Mahesh K.; Haugland, Rosaria P.; Diwu, Zhenjun; Liu, Jixiang; Klaubert, Dieter H.; Haugland, Richard P.

doi:10.1006/abio.1999.4044

Cited by 29 publications

(17 citation statements)

References 30 publications

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“…123 An in situ activity assay was also recently developed to measure the rate of lysosomal efflux of the Gaucher PC IFG utilizing the substrate 5-(pentafluorobenzoylamino)fluorescein-di-b-D-glucopyranoside (PFBF-b-glucose). 70 The advantage of this substrate is that the enzymatically liberated PFBF fluorophore is rapidly conjugated to thiol groups 124 and remains trapped inside the cell during the assay period. 113 This trapping permits detection of activity within lysosomes by fluorescence microscopy (Fig.…”

Section: Assays To Measure Lysosomal Enzyme Traffickingmentioning

confidence: 99%

Identification and Characterization of Pharmacological Chaperones to Correct Enzyme Deficiencies in Lysosomal Storage Disorders

Valenzano

Khanna

Powe

et al. 2011

ASSAY and Drug Development Technologies

140

135

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Many human diseases result from mutations in specific genes. Once translated, the resulting aberrant proteins may be functionally competent and produced at near-normal levels. However, because of the mutations, the proteins are recognized by the quality control system of the endoplasmic reticulum and are not processed or trafficked correctly, ultimately leading to cellular dysfunction and disease. Pharmacological chaperones (PCs) are small molecules designed to mitigate this problem by selectively binding and stabilizing their target protein, thus reducing premature degradation, facilitating intracellular trafficking, and increasing cellular activity. Partial or complete restoration of normal function by PCs has been shown for numerous types of mutant proteins, including secreted proteins, transcription factors, ion channels, G protein-coupled receptors, and, importantly, lysosomal enzymes. Collectively, lysosomal storage disorders (LSDs) result from genetic mutations in the genes that encode specific lysosomal enzymes, leading to a deficiency in essential enzymatic activity and cellular accumulation of the respective substrate. To date, over 50 different LSDs have been identified, several of which are treated clinically with enzyme replacement therapy or substrate reduction therapy, although insufficiently in some cases. Importantly, a wide range of in vitro assays are now available to measure mutant lysosomal enzyme interaction with and stabilization by PCs, as well as subsequent increases in cellular enzyme levels and function. The application of these assays to the identification and characterization of candidate PCs for mutant lysosomal enzymes will be discussed in this review. In addition, considerations for the successful in vivo use and development of PCs to treat LSDs will be discussed.

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Section: Assays To Measure Lysosomal Enzyme Traffickingmentioning

confidence: 99%

Identification and Characterization of Pharmacological Chaperones to Correct Enzyme Deficiencies in Lysosomal Storage Disorders

Valenzano

Khanna

Powe

et al. 2011

ASSAY and Drug Development Technologies

140

135

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“…Caco-2 cells were pretreated for 24 h with dl-buthionine-(S,R)-sulfoximine (BSO) (9 mM), a specific irreversible inhibitor of ␥-glutamylcysteine synthetase (Griffith and Meister, 1979), to reduce intracellular glutathione for 24 h (Arttamangkul et al, 1999) before loading with calcein. Apical calcein efflux was unaffected by BSO pretreatment [0.25 Ϯ 0.01 and 0.26 Ϯ 0.01% available substrate/min at 60 min for control and BSO-pretreated cells (n ϭ 12), respectively].…”

Section: Downloaded Frommentioning

confidence: 99%

Differential Multidrug Resistance-Associated Protein 1 through 6 Isoform Expression and Function in Human Intestinal Epithelial Caco-2 Cells

Prime-Chapman

Fearn²,

Cooper³

et al. 2004

J Pharmacol Exp Ther

119

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Multidrug resistance-associated protein (MRP) isoforms 1 through 6 mRNA are expressed in the human intestine and Caco-2 cells. In Caco-2 cells, the rank order for mRNA expression was MRP2 Ն MRP6 Ͼ MRP4 Ն MRP3 Ͼ MRP1 ϭ MRP5. The functional expression of MRP-like activity was quantified as the efflux of the fluorescent probe calcein from confluent, polarized monolayers of Caco-2 cells. Calcein efflux was sensitive to temperature, energy depletion, and the MRP an-Calcein efflux across the apical membrane of Caco-2 cells exceeded that across the basolateral by approximately 2-fold, correlating with the apical localization of MRP2 visualized by immunocytochemical staining. T84 cells do not express MRP2 and show a predominance of basolateral calcein efflux over apical efflux. MRP3 was localized by immunocytochemical staining to the basolateral membrane. MRP1 staining was not localized to either membrane domain and MRP5 staining was not detected. Thus, basolateral calcein efflux may reflect a function of MRP3 or MRP4 and 6 inferred by their basolateral localization in other tissues. Basolateral, but not apical, calcein efflux was sensitive to glutathione depletion with buthioninesulfoximine, indicating that whereas MRP2-mediated apical efflux is independent of glutathione, basolateral efflux is glutathione-dependent. Benzbromarone, probenecid, pravastatin, and diclofenac were able to inhibit both apical and basolateral calcein efflux. The apical calcein efflux in Caco-2 cells was selectively sensitive to indomethacin and propranolol, but not verapamil or erythromycin, whereas the converse was observed for basal efflux. The differential pharmacological sensitivity of apical (MRP2) and basolateral calcein efflux provides tools for dissecting MRP isoform functional roles.

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“…Human recombinant full-length GST A1-1 isolated from overexpressing plasmid in Escherichia coli was purchased from Oxford Biomedical Research (Oxford, MI, USA) [21,22]. We purified it toward >95% using metal affinity column chromatography.…”

Section: Preparation Of Gst A1-1 Proteinmentioning

confidence: 99%

Frequency and significance of anti-glutathione S-transferase autoantibody (anti-GST A1-1) in autoimmune hepatitis

Kato

Miyakawa

Ishibashi

2004

Journal of Autoimmunity

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5-(Pentafluorobenzoylamino)fluorescein: A Selective Substrate for the Determination of Glutathione Concentration and GlutathioneS-Transferase Activity

Cited by 29 publications

References 30 publications

Identification and Characterization of Pharmacological Chaperones to Correct Enzyme Deficiencies in Lysosomal Storage Disorders

Identification and Characterization of Pharmacological Chaperones to Correct Enzyme Deficiencies in Lysosomal Storage Disorders

Differential Multidrug Resistance-Associated Protein 1 through 6 Isoform Expression and Function in Human Intestinal Epithelial Caco-2 Cells

Frequency and significance of anti-glutathione S-transferase autoantibody (anti-GST A1-1) in autoimmune hepatitis

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