Associating protein activities with their genes: rapid identification of a gene encoding a methylglyoxal reductase in the yeastSaccharomyces cerevisiae
| Autor: | Georgia Shearer, Ching-Nen Chen, James L. Dover, Daniel H. Kohl, Parag R. Chitnis, Lauren G. Holden, Maja Svrakic, Larysa Porubleva, Mark Johnston |
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| Rok vydání: | 2003 |
| Předmět: |
chemistry.chemical_classification
7-Dehydrocholesterol reductase Methylglyoxal reductase biology Methylglyoxal Saccharomyces cerevisiae Bioengineering biology.organism_classification Applied Microbiology and Biotechnology Biochemistry Alcohol Oxidoreductases Open Reading Frames chemistry.chemical_compound Enzyme chemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Protein purification Genetics Lactaldehyde Gene Biotechnology |
| Zdroj: | Yeast. 20:545-554 |
| ISSN: | 1097-0061 0749-503X |
| DOI: | 10.1002/yea.979 |
| Popis: | Methylglyoxal is associated with a broad spectrum of biological effects, including cytostatic and cytotoxic activities. It is detoxified by the glyoxylase system or by its reduction to lactaldehyde by methylglyoxal reductase. We show that methylglyoxal reductase (NADPH-dependent) is encoded by GRE2 (YOL151w). We associated this activity with its gene by partially purifying the enzyme and identifying by MALDI-TOF the proteins in candidate bands on SDS-PAGE gels whose relative intensities correlated with specific activity through three purification steps. The candidate proteins were then purified using a glutathione-S-transferase tag that was fused to them, and tested for methylglyoxal reductase activity. The advantage of this approach is that only modest protein purification is required. Our approach should be useful for identifying many of the genes that encode the metabolic pathway enzymes that have not been associated with a gene (about 275 in S. cerevisiae, by our estimate). |
| Databáze: | OpenAIRE |
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