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Characterization and optimization of in vitro assay conditions for (1,3)β-glucan synthase activity from Aspergillus fumigatus and Candida albicans for enzyme inhibition screening
| Title: | Characterization and optimization of in vitro assay conditions for (1,3)β-glucan synthase activity from Aspergillus fumigatus and Candida albicans for enzyme inhibition screening. |
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| Name(s): |
Wood, R. L., creator Miller, T. K., creator Wright, Amy E., creator McCarthy, Peter J., creator Taft, C. S., creator Pomponi, Shirley A., creator Selitrennikoff, Claude, creator Harbor Branch Oceanographic Institute |
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| Type of Resource: | text | |
| Genre: | Article | |
| Issuance: | single unit | |
| Date Issued: | 1998 | |
| Publisher: | Nature Publishing Group | |
| Extent: | 12 p. | |
| Physical Description: | ||
| Language(s): | English | |
| Identifier: | 3351974 (digitool), FADT3351974 (IID), fau:6523 (fedora), 10.7164/antibiotics.51.665 (doi) | |
| Note(s): |
(1, 3)β-D-Glucan synthase (E.C.2.4.1.34. UDP-glucose: 1, 3-β-D-glucan 3-β-glucosyl transferase) catalyzes the polymerization of glucose ([1-3]-β-linkages) using UDP-glucose as substrate. We have determined optimal in vitro conditions for the assay of (1, 3)β-glucan synthase activity from Aspergillus fumigatus and Candida albicans. These included lysis of cells in the following for C. albicans, 100 mM HEPES, pH 8.0, 10 μM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), 2 mM ethylenediaminetetraacetic acid (EDTA), disodium salt, 5 mM NaF, 250 mM sucrose, and 10 mM NaH2PO4; and for A. fumigatus, 50 mM HEPES, 10 mM EDTA, 750 mM sucrose, 10 mM NaH2PO4, 100 mM cellobiose and 50 μM GTPγS. Resulting low-speed supernatants were used as enzyme sources to determine the optimal in vitro assay conditions. We have characterized the resulting enzyme activities and tested the optimized assays with known (1, 3)β-glucan synthase inhibitors including cilofungin, papulacandin, aculeacin A, and echinocandin B. We have used both optimized assays to screen >1000 extracts of marine macroorganisms and, using bioassay-guided purification, have identified (1, 3)β-glucan synthase inhibitors. This manuscript is an author version with the final publication available at http://www.nature.com/ja/index.html and may be cited as: Wood, R. L., Miller, T. K., Wright, A., McCarthy, P., Taft, C. S., Pomponi, S., & Selitrennikoff, C. P. (1998). Characterization and optimization of in vitro assay conditions for (1,3)β-glucan synthase activity from Aspergillus fumigatus and Candida albicans for enzyme inhibition screening. The Journal of Antibiotics, 51(7), 665-675. doi:10.7164/antibiotics.51.665 Florida Atlantic University. Harbor Branch Oceanographic Institute contribution #1252. |
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| Subject(s): |
Aspergillus fumigatus Candida albicans Glucosyltransferases In vitro Glucan synthase Enzyme inhibitors |
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| Persistent Link to This Record: | http://purl.flvc.org/FCLA/DT/3351974 | |
| Links: | http://doi.org/10.7164/antibiotics.51.665 | |
| Restrictions on Access: | ©1998 Nature Publishing Group | |
| Host Institution: | FAU |
