Park J I, Grant C M, Davies M J, Dawes I W
School of Biochemistry & Molecular Genetics and Cooperative Research Center for Food Industry Innovation, University of New South Wales, Sydney, New South Wales 2052, Australia.
J Biol Chem. 1998 Sep 4;273(36):22921-8. doi: 10.1074/jbc.273.36.22921.
The involvement of oxidative stress in freeze-thaw injury to yeast cells was analyzed using mutants defective in a range of antioxidant functions, including Cu,Zn superoxide dismutase (encoded by SOD1), Mn superoxide dismutase (SOD2), catalase A, catalase T, glutathione reductase, gamma-glutamylcysteine synthetase and Yap1 transcription factor. Only those affecting superoxide dismutases showed decreased freeze-thaw tolerance, with the sod1 mutant and the sod1 sod2 double mutant being most affected. This indicated that superoxide anions were formed during freezing and thawing. This was confirmed since the sod1 mutant could be made more resistant by treatment with the superoxide anion scavenger MnCl2, or by freezing in the absence of oxygen, or by the generation of a rho0 petite. Increased expression of SOD2 conferred freeze-thaw tolerance on the sod1 mutant indicating the ability of the mitochondrial superoxide dismutase to compensate for the lack of the cytoplasmic enzyme. Free radicals generated as a result of freezing and thawing were detected in cells directly using electron paramagnetic resonance spectroscopy with either alpha-phenyl-N-tert-butylnitrone or 5, 5-dimethyl-1-pyrroline-N-oxide as spin trap. Highest levels were formed in the sod1 and sod1 sod2 mutant strains, but lower levels were detected in the wild type. The results show that oxidative stress causes major injury to cells during aerobic freezing and thawing and that this is mainly initiated in the cytoplasm by an oxidative burst of superoxide radicals formed from oxygen and electrons leaked from the mitochondrial electron transport chain.
利用一系列抗氧化功能存在缺陷的突变体,分析了氧化应激在酵母细胞冻融损伤中的作用,这些突变体包括铜锌超氧化物歧化酶(由SOD1编码)、锰超氧化物歧化酶(SOD2)、过氧化氢酶A、过氧化氢酶T、谷胱甘肽还原酶、γ-谷氨酰半胱氨酸合成酶和Yap1转录因子。只有那些影响超氧化物歧化酶的突变体表现出冻融耐受性降低,其中sod1突变体和sod1 sod2双突变体受影响最大。这表明在冷冻和解冻过程中形成了超氧阴离子。这一点得到了证实,因为用超氧阴离子清除剂MnCl2处理、在无氧条件下冷冻或产生rho0小菌落可使sod1突变体更具抗性。SOD2表达的增加赋予了sod1突变体冻融耐受性,表明线粒体超氧化物歧化酶能够补偿细胞质酶的缺乏。使用α-苯基-N-叔丁基硝酮或5,5-二甲基-1-吡咯啉-N-氧化物作为自旋捕获剂,通过电子顺磁共振光谱直接在细胞中检测冷冻和解冻产生的自由基。在sod1和sod1 sod2突变菌株中形成的自由基水平最高,但在野生型中检测到的水平较低。结果表明,氧化应激在需氧冷冻和解冻过程中对细胞造成严重损伤,这主要是由线粒体电子传递链泄漏的氧气和电子形成的超氧自由基的氧化爆发在细胞质中引发的。