Inhibition effect of expression of Cu/Zn superoxide dismutase from rice on synthesis of Glutathione in Saccharomyces cerevisiae

Abstract

The expression of a rice Cu/Zn superoxide dismutase (Cu/Zn-SOD) in Saccharomyces cerevisiae regulated by GAPDH promoter, involved in the inhibition of endogenous Glutathione (GSH) synthesis, and the competitive expression was detected by constructing the expression vector transferred Cu/Zn-SOD gene into wild-type S. cerevisiae. Transcription and expression of the Cu/Zn-SOD gene in S. cerevisiawere were confirmed by northern blot and SDS-PAGE, respectively, and activity of the Cu/Zn-SOD from crude extracts was enzymatically detected based on the effect of nitroblue tetrazolium (NBT) after running a native polyacrylamide gel. The GSH synthesis was also tested by DTNB (5, 5′-Dithiobis (2-nitrobenzoic acid)) method. Results showed that GSH synthesis was evidently suppressed by the expression of Cu/Zn-SOD gene in both control and heat shock strains. It implied that the expression of the Cu/Zn-SOD gene in S. cerevisiae has more potential facility in response to oxidative exposure than that of endogenous GSH, although Cu/Zn-SOD and GSH were both contributed to the function of oxygen radical oxidoreduction.

Keywords

Cu/Zn Superoxide Dismutase / GAPDH promoter / Saccharomyces cerevisiae / glutathione

References

[1] Abarca D., Roldan M., Martin M., Sabater B. Arabidopsis thaliana ecotype Cvi shows an increased tolerance to photo-oxidative stress and contains a new chloroplastic copper/zinc superoxide dismutase isoenzyme J Exp Bot, 2001, 52(360): 1417-1425.

[2] Beauchamp C., Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels Anal Biochem, 1971, 44: 276-287.

[3] Fan X., He X., Guo X., Qu N., Wang C., Zhang B. Increasing glutathione formation by functional expression of the γ-glutamylcysteine synthetase gene in Saccharomyces cerevisiae Biotechnology Letters, 2004, 26: 415-417.

[4] Forman H.J., Fridovich I. Superoxide dismutase: a comparison of rate constants Arch Biochem Biophy., 1973, 158(1): 396-400.

[5] Ishikawa T., Sies H. Dolphin D., Poulson R., Avramovic O. Glutathione as an antioxidant: toxicological aspects Glutathione, part B, 1989 New York: John Willy and Sons 85-110.

[6] He X., Huai W., Tie C., Liu Y., Zhang B. Breeding of high ergosterol-producing yeast strains J Ind Microbiol Biotechnol, 2000, 25: 39-44.

[7] Jamieson D.J. Oxidative stress responses of the Saccharomyces cerevisiae Yeast, 1998, 14(16): 1511-1527.

[8] Jamieson D.J., Rivers S.L., Stephen D.W.S. Analysis of Saccharomyces cerevisiae proteins induced by peroxide and superoxide stress Microbiology, 1994, 140: 3277-3283.

[9] Jung S., Kernodle S.P., Scandalios J.G. Differential antioxidant responses to norflurazon-induced oxidative stress in maize Redox Rep, 2001, 6(5): 311-317.

[10] McCord J.M., Fridovich I. Superoxide dismutase, An enzymatic function for erythrocuprein (hemocuprein) J Biol Chem, 1969, 224: 6049-6055.

[11] Mysore T.B., Shinkel T.A., Collins J. et al. Overexpression of glutathione peroxidase with two isoforms of superoxide dismutase protects mouse islets from oxidative injury and improves islet graft function Diabetes, 2005, 54(7): 2109-16.

[12] Penninckx M. A short review on the role of glutathione in the response of yeasts to nutritional, environmental, and oxidative stresses Enzyme and Microbial Technology, 2000, 26: 737-742.

[13] Reddy D.C., Venkaiah B. Purification and characterization of Cu-Zn superoxide dismutases from mungbean (Vigna radiata) seedlings J Biosci, 1984, 6(1): 115-123.

[14] Schuessel K., Schafer S., Bayer T.A., Czech C., Pradier L., Muller-Spahn F., Muller W.E., Eckert A. Impaired Cu/Zn-SOD activity contributes to increased oxidative damage in APP transgenic mice Neurobiol Dis, 2005, 18(1): 89-99.

[15] Tang X., Mary A.W. Plant Gene Register. Soybean Root Nodule cDNA Encoding Glutathione Reductase Plant Physiol, 1994, 104: 1081-1082.

[16] Wachter A., Wolf S., Steininger H., Bogs J., Rausch T. Differential targeting of GSH1 and GSH2 is achieved by multiple transcription initiation: implications for the compartmentation of glutathione biosynthesis in the Brassicaceae Plant J, 2005, 41(1): 15-30.

[17] Welch W.J., Chabrashvili T., Solis G., Chen Y., Gill P.S., Aslam S., Wang X., Ji H., Sandberg K., Jose P., Wilcox C.S. Role of extracellular superoxide dismutase in the mouse angiotensin slow pressor response Hypertension, 2006, 48(5): 934-941.

[18] Yoo H.Y., Kim S.S., Rho H.M. Overexpression and simple purification of human superoxide dismutase (SOD1) in Saccharomyces cerevisiae and its resistance to oxidative stress Journal of Biotechnology, 1999, 68: 29-35.