高渗、低温和氧化条件下酵母细胞应激产生活性物质的研究
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摘要
本论文对酵母细胞在高渗、低温和氧化条件下的应激反应进行了研究,筛选了耐高渗酵母突变株,通过低剂量刺激酵母细胞产生活性衍生物—LYCD,开辟了制备LYCD的新途径,考察了LYCD对氧化损伤的酵母细胞的保护作用。
采用对数期正常酵母细胞为研究对象,对其进行低剂量高渗(KCl)和氧化(H_2O_2)预处理后发现细胞对更高剂量产生了抗性,并且这种抗性具有自我适应性和交叉适应性。此外,在低温和氧化应激反应之间也存在交叉适应性。这种交叉适应性说明,高渗和氧化应激、低温和氧化应激对细胞的损伤在一定程度上具有相似性,预处理后可能诱导出相似的保护物质。
测定了酵母经高渗、低温和氧化预处理前后细胞内几种重要抗氧化剂(GSH、SOD、CAT)和总抗氧化能力(T-AOC)的变化,结果表明:预处理都可以使正常酵母细胞中抗氧化剂含量提高,总抗氧化能力增强。并分析了正常酵母预处理前后细胞内脂质过氧化物—丙二醛(MDA)的含量,发现预处理后,再置于氧化致死条件下,MDA的含量降低,细胞的存活率也升高。这不仅说明预处理使细胞抗氧化水平提高,减少了MDA的积累,细胞增加了对这几种不良环境的抗性,更直接说明高渗、低温与氧化关系密切。
将经过高渗和低温预处理后的酵母进行破壁,分别得到LYCD_(KCl)和LYCD_(LT),发现两者都对细胞抗氧化具有保护活性。分别对其处理条件进行优化,确定了LYCDK_(KCl)的最佳处理条件为:1%KCl预处理90min,LYCD_(LT)的最佳处理条件为:4℃预处理60min。
通过紫外诱变筛选了耐高渗酵母突变株(CYK),与正常酵母相比,发现耐高渗突变株对氧化具有很强的抗性,而且耐高渗酵母提取物(LYCDK)也具有保护细胞抵抗氧化的能力。分析了正常酵母和耐高渗酵母以及预处理对耐高渗酵母细胞内GSH、SOD、CAT、T-AOC和MDA的差异,发现耐高渗酵母胞内抗氧化水平高于正常酵母菌株,且预处理增加了LYCDK中抗氧化剂的含量,降低了MDA水平。
对正常酵母细胞预处理前后蛋白的变化进行了电泳分析,结果表明,一些蛋白是H_2O_2和KCl都可诱导合成的,另一些蛋白是由H_2O_2或KCl特异性诱导合成的。
探讨了H_2O_2和紫外线条件下,LYCD对酵母细胞氧化损伤的保护作用。在H_2O_2(10mmol/L)和紫外线(20W紫外灯下30cm处照射60s)氧化损伤酵母细胞模型中添加LYCD后,细胞存活率显著上升,且有剂量依赖性。LYCD提高了紫外线氧化损伤细胞上清液中GSH、SOD和CAT含量,降低了MDA水平。
This study was based on stress response of yeast cell under hyperosmosis, low temperature and oxidative condition. Live Yeast Cell Derivative (LYCD) was produced by yeast cell, which was pretreated by low dose hyperosmosis, temperature and oxidative condition. The experiment setup the new method producing LYCD by selecting hyperosmotic mutant. And protective effects of LYCD on yeast cells damaged by H_2O_2 and UV were studied.After being pretreated with a sublethal dose of either oxidative or osmotic stress, yeast cells could withstand a subsequent higher dose of the two stress. And cross adaptation also existed between the low temperature and oxidative stress. The cross adaptation indicated that damage mechanism of hperosmotic and low temperature stress were similar with the one of oxidative stress, some similar substance was produced by yeast cells under the three stresses.The following conclusions were drawn by testing the change of antioxidants and MDA in yeast cell : pretreating of low dose could increase the content of GSH , the activity of SOD and CAT, and the total anti-oxidative capability(T-AOC), could reduce the accumulation of MDA. And these induced the resistance to lethal concentration of H_2O_2. Furthermore study showed that hyperosmotic, low temperatue and oxidative stress may share a common mechanism of damage.LYCD_(KCl) and LYCD_(LT) were produced by breaking up the yeast cells ,which were pretreated with hyperosmosis and low temperatue. The results showed that LYCD_(KCl) and LYCD_(LT) could protect cell to resist oxidative condition caused by H_2O_2 and UV. The optimum conditions of producing LYCD_(KCl) and LYCD_(LT) were as follow: the concentration of KCl is 1%, and the treating time is 90min;the temperature is 4℃, and the treating time is 60min.After UV treating, the hyperosmotic mutant was selected. And it had the more ability for resistance to lethal dose of oxidative condition. The extract of mutant had almost biological activities. At the same time, the activities of GSHL SOD and CAT in mutant were higher than the initial strain, and pretreated also increase content of antioxidants and decrease the level of MDA.Through protein electrophoresis experiment, the yeast pretreated with low dose H_2O_2 and hyperosmosis were analysed. Some changes were induced by H_2O_2 and KCl, which were not made in untreated cells. Some other changes were induced by H_2O_2 or KCl, indicating that the existence of a set of unique peroxide-inducible proteins and unique hyperosmotic proteins.
Protective effects of LYCD on yeast cells damaged by H2O2 and UV were studied. Oxidative damaged models of yeast cells damaged by UV (20W, 30cm, 60s) and H2O2 (lOmmol/L) were established. The survival rates of yeast cells were increased by addition of LYCD in the models. And LYCD could also enhance the activities of GSH, SOD, CAT, and decrease the level of MDA in supernates of UV damaged yeast cell model.
采用对数期正常酵母细胞为研究对象,对其进行低剂量高渗(KCl)和氧化(H_2O_2)预处理后发现细胞对更高剂量产生了抗性,并且这种抗性具有自我适应性和交叉适应性。此外,在低温和氧化应激反应之间也存在交叉适应性。这种交叉适应性说明,高渗和氧化应激、低温和氧化应激对细胞的损伤在一定程度上具有相似性,预处理后可能诱导出相似的保护物质。
测定了酵母经高渗、低温和氧化预处理前后细胞内几种重要抗氧化剂(GSH、SOD、CAT)和总抗氧化能力(T-AOC)的变化,结果表明:预处理都可以使正常酵母细胞中抗氧化剂含量提高,总抗氧化能力增强。并分析了正常酵母预处理前后细胞内脂质过氧化物—丙二醛(MDA)的含量,发现预处理后,再置于氧化致死条件下,MDA的含量降低,细胞的存活率也升高。这不仅说明预处理使细胞抗氧化水平提高,减少了MDA的积累,细胞增加了对这几种不良环境的抗性,更直接说明高渗、低温与氧化关系密切。
将经过高渗和低温预处理后的酵母进行破壁,分别得到LYCD_(KCl)和LYCD_(LT),发现两者都对细胞抗氧化具有保护活性。分别对其处理条件进行优化,确定了LYCDK_(KCl)的最佳处理条件为:1%KCl预处理90min,LYCD_(LT)的最佳处理条件为:4℃预处理60min。
通过紫外诱变筛选了耐高渗酵母突变株(CYK),与正常酵母相比,发现耐高渗突变株对氧化具有很强的抗性,而且耐高渗酵母提取物(LYCDK)也具有保护细胞抵抗氧化的能力。分析了正常酵母和耐高渗酵母以及预处理对耐高渗酵母细胞内GSH、SOD、CAT、T-AOC和MDA的差异,发现耐高渗酵母胞内抗氧化水平高于正常酵母菌株,且预处理增加了LYCDK中抗氧化剂的含量,降低了MDA水平。
对正常酵母细胞预处理前后蛋白的变化进行了电泳分析,结果表明,一些蛋白是H_2O_2和KCl都可诱导合成的,另一些蛋白是由H_2O_2或KCl特异性诱导合成的。
探讨了H_2O_2和紫外线条件下,LYCD对酵母细胞氧化损伤的保护作用。在H_2O_2(10mmol/L)和紫外线(20W紫外灯下30cm处照射60s)氧化损伤酵母细胞模型中添加LYCD后,细胞存活率显著上升,且有剂量依赖性。LYCD提高了紫外线氧化损伤细胞上清液中GSH、SOD和CAT含量,降低了MDA水平。
This study was based on stress response of yeast cell under hyperosmosis, low temperature and oxidative condition. Live Yeast Cell Derivative (LYCD) was produced by yeast cell, which was pretreated by low dose hyperosmosis, temperature and oxidative condition. The experiment setup the new method producing LYCD by selecting hyperosmotic mutant. And protective effects of LYCD on yeast cells damaged by H_2O_2 and UV were studied.After being pretreated with a sublethal dose of either oxidative or osmotic stress, yeast cells could withstand a subsequent higher dose of the two stress. And cross adaptation also existed between the low temperature and oxidative stress. The cross adaptation indicated that damage mechanism of hperosmotic and low temperature stress were similar with the one of oxidative stress, some similar substance was produced by yeast cells under the three stresses.The following conclusions were drawn by testing the change of antioxidants and MDA in yeast cell : pretreating of low dose could increase the content of GSH , the activity of SOD and CAT, and the total anti-oxidative capability(T-AOC), could reduce the accumulation of MDA. And these induced the resistance to lethal concentration of H_2O_2. Furthermore study showed that hyperosmotic, low temperatue and oxidative stress may share a common mechanism of damage.LYCD_(KCl) and LYCD_(LT) were produced by breaking up the yeast cells ,which were pretreated with hyperosmosis and low temperatue. The results showed that LYCD_(KCl) and LYCD_(LT) could protect cell to resist oxidative condition caused by H_2O_2 and UV. The optimum conditions of producing LYCD_(KCl) and LYCD_(LT) were as follow: the concentration of KCl is 1%, and the treating time is 90min;the temperature is 4℃, and the treating time is 60min.After UV treating, the hyperosmotic mutant was selected. And it had the more ability for resistance to lethal dose of oxidative condition. The extract of mutant had almost biological activities. At the same time, the activities of GSHL SOD and CAT in mutant were higher than the initial strain, and pretreated also increase content of antioxidants and decrease the level of MDA.Through protein electrophoresis experiment, the yeast pretreated with low dose H_2O_2 and hyperosmosis were analysed. Some changes were induced by H_2O_2 and KCl, which were not made in untreated cells. Some other changes were induced by H_2O_2 or KCl, indicating that the existence of a set of unique peroxide-inducible proteins and unique hyperosmotic proteins.
Protective effects of LYCD on yeast cells damaged by H2O2 and UV were studied. Oxidative damaged models of yeast cells damaged by UV (20W, 30cm, 60s) and H2O2 (lOmmol/L) were established. The survival rates of yeast cells were increased by addition of LYCD in the models. And LYCD could also enhance the activities of GSH, SOD, CAT, and decrease the level of MDA in supernates of UV damaged yeast cell model.
引文
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[4] 赵保路.氧自由基和天然抗氧剂[M].北京:科学出版社,1999,1-20.
[5] 薛丽君,金中初.真核细胞氧化应激及其适应机制[J].国外医学卫生学分册,1997,24(4),207-210
[6] 李芳红.线粒体DNA的氧化损伤[J].国外医学卫生学分册,2000,27(1),33-36
[7] Kanner J, German JB. Initition of lipid peroxidation in biological systems. CRC Crit Rev Food Sci Nrtr, 25, 1997, 317-325
[8] Scandalios. Response of plant antioxidant defense genes to environment stress [J]. Advance in Genetics, 1990, 28:2-35.
[9] Christman, M.F. Morgan, R.W. Positive control of a regulon for defense against oxidative stress and some heat shock protein in Salmondla typhimuriumm [J]. Cell,1985,41:753-7626
[10] Cohen, G., Rapatz, W. And Ruis, G. Sequence of the S. cerevisiae CTA1 gene and amino acid sequence of catalase A derived from it [J]. Gen. Microbiol , 1988,176, 159-163
[11] Hartig, A. And Ruis, H. Nucleotide sequence of the S. cerevisiae CTT1 gene and deduced amino-acid sequence of yeast catalase T [J]. Eur. J. Biochem, 1986,160:487-490
[12] Marcos DDP, and Anita D. Panc. Acquisition of tolerance against oxidative damage in S. cerevisiae [J]. Microbiology ,2001,2:1-11
[13] Jin Sook Jeong, Soo Jin Kwon, Sang Won Kang, Sue Goo Rhee, and Kanghua Kim. Purification and characterization of a second Type Thioredoox Peroiddase from S. cerevisiae [J]. Biochemistry ,1999,38:776-783
[14] Angela M. Avery and Simon V. Avery. JBC S. cerevisiae Expresses Three Phospholipid Hydroperoxide Glutathione peroxidases. Papers in press .July 9,2001 M105672200
[15] Marchler, g., et al. A S. cerevisiae USAelement controlled by protein kinase A activates transcription in response to a variety of stress conditions [J]. EMBO , 1993,12:1997-2003
[16] 贾耀勇.人类健康与美的使者——金属硫蛋白[J].北京日化,2001,3(64),15-18
[17] Culotta, V.C., Howard, W.R. and Liu, X. F. CRS5 encodes a metallothionein-lide protein in S. cerevisiae[J]. Biol. Chem. 269, 25295-25302
[18] Tamai, K.T., Gralla, E.B., Ellerby, L.M. Yeast and mammalian metallothions functionally substitute for yeast copper-zinc superoxide[J]. Proc. Natl Acad. Sci, USA, 1993, 90,8013-8017
[19] Zhao, X.J., Raitt, D., Burke, P.V. Function and expression of flavohemoglobin in S. cerevisiae. [J]. Biol. Chem. 1996,271,25131-25138
[20] Muller, E. G. D. Thioredoxin deficiency in yeast prolongs S phase and shortens the G1 interval of the cell cycle [J]. Bio. Chem. 1991, 266, 9194-9202
[21] Muller, E. G. D. A glutathione reductase mutant of yeast accumulates high levels of oxidized glutathione and requires thiredoxin for growth. [J]. Mol. Biol. Cell. 1996, 7, 1805-1813
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