A long-wavelength fluorescent substrate for continuous fluorometric determination of α-mannosidase activity: Resorufin α-d-mannopyranoside
| Autor: | Gabriele M. Cook, Daniel J. Coleman, John J. Naleway, David R. Rose, Meenakshi Venkatesan, Douglas A. Kuntz, Staci P. Williamson |
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| Rok vydání: | 2010 |
| Předmět: |
Biophysics
Golgi Apparatus Oxazines 010402 general chemistry 01 natural sciences Biochemistry Article Fluorescence spectroscopy Substrate Specificity law.invention 03 medical and health sciences symbols.namesake chemistry.chemical_compound alpha-Mannosidase law Animals Humans Fluorometry Enzyme Inhibitors Molecular Biology Fluorescent Dyes 030304 developmental biology chemistry.chemical_classification 0303 health sciences Chromatography Swainsonine Substrate (chemistry) Cell Biology Golgi apparatus Fluorescence Recombinant Proteins High-Throughput Screening Assays 0104 chemical sciences 3. Good health Kinetics Enzyme chemistry Mannosides Recombinant DNA symbols Drosophila Lysosomes |
| Zdroj: | Analytical Biochemistry. 399:7-12 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/j.ab.2009.11.039 |
| Popis: | A simple and reliable continuous assay for measurement of alpha-mannosidase activity is described and demonstrated for analysis with two recombinant human enzymes using the new substrate resorufin alpha-d-mannopyranoside (Res-Man). The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585 nm with excitation at 571 nm and has a pK(a) of 5.8, allowing continuous measurement of fluorescence turnover at or near physiological pH values for human lysosomal and Drosophila Golgi alpha-mannosidases. The assay performed using recombinant Drosophila Golgi alpha-mannosidase (dGMII) has been shown to give the kinetic parameters K(m) of 200 microM and V(max) of 11 nmol/min per nmol dGMII. Methods for performing the assay using several concentrations of the known alpha-mannosidase inhibitor swainsonine are also presented, demonstrating a potential for use of the assay as a simple method for high-throughput screening of inhibitors potentially useful in cancer treatment. |
| Databáze: | OpenAIRE |
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