Associating protein activities with their genes: rapid identification of a gene encoding a methylglyoxal reductase in the yeast <i>Saccharomyces cerevisiae</i>

Yeast - Tập 20 Số 6 - Trang 545-554 - 2003
Ching‐Nen Nathan Chen1, L. V. Porubleva2, Georgia Shearer1, Maja Svrakic1, Lauren G. Holden1, James L. Dover3, Mark Johnston3, Parag R. Chitnis2, Daniel H. Kohl1
1Department of Biology, Washington University, St. Louis, MO 63130 USA
2Department of Biochemistry and Biophysics, Iowa State University Ames, IA 50011 USA
3Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA

Tóm tắt

AbstractMethylglyoxal 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). Copyright © 2003 John Wiley & Sons, Ltd.

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Yeast

Tập 20 Số 6

545-554

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