A microtiter-based fluorescence assay for (1,3)-beta-glucan synthases.
A high-throughput assay for UDP-Glc:(1,3)-beta-glucan synthase(EC 2.4.1.34, UDP-glucose:1,3-beta-D-glucan, 3-beta-glucosyltransferase) from fungi and higher plants is described. The assay is performed in microtiter plates and is extremely inexpensive compared to other standard assays for these enzymes. The reduction in price is achieved by replacing the conventional substrate UDP-[14C]Glc with its nonradioactive counterpart, and the nonradioactive glucan produced is quantified as a fluorescent complex following specific interaction with the fluorochrome present in commercial aniline blue. In addition to a > 100-fold reduction in cost, the assay is highly reproducible and nearly as sensitive as radioactive assays and has the additional advantages of increased safety and avoidance of the need for filtration and washing steps to collect the glucan product. As such, the assay is highly suitable for high-throughput screening for inhibitors of these enzymes.
Shedletzky
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Department of Plant Sciences, Hebrew University of Jerusalem, Israel.
Unger
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C
Delmer
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DP
D000814:Aniline Compounds; D004791:Enzyme Inhibitors / Q000032:analysis; D007887:Fabaceae / Q000201:enzymology; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D005964:Glucosyltransferases / Q000032:analysis*; D016014:Linear Models; D008565:Membrane Proteins; D008832:Microchemistry; D010946:Plants, Medicinal; D012015:Reference Standards; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029702:Schizosaccharomyces pombe Proteins; D012680:Sensitivity and Specificity; D017186:Titrimetry
D000814:Aniline Compounds; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D029702:Schizosaccharomyces pombe Proteins; C017006:aniline blue; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase