Chang E C, Kosman D J
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo 14214.
J Biol Chem. 1989 Jul 25;264(21):12172-8.
Three Cu,Zn superoxide dismutase (SOD-1)-deficient Saccharomyces cerevisiae mutants do not grow in 100% O2 in rich medium and require Met and Lys when grown in air (Bilinski, T., Krawiec, Z., Liczmanski, A., and Litwinska, J. (1985) Biochem. Biophys. Res. Commun. 130, 533-539). We show herein that medium manganese (II) accumulated by the mutants rescues these O2-sensitive phenotypes; 2 mM medium Mn2+ represented the threshold required for cell growth. The accumulation of Mn2+ was not oxygen-inducible since mutants grown aerobically and anaerobically accumulated the same amount of Mn2+. Mn2+ accumulation is not unique to these mutants since wild type accumulated almost twice as much Mn2+ as did mutant. ESR spectra of the cell extracts and whole cells loaded with Mn2+ were typical of free Mn(II) ion. These spectra could not account quantitatively for the total cellular Mn2+, however. A screen for soluble antioxidant activities in the Mn2+-supplemented cells detected O2- (superoxide) scavenging activity, with no change in catalase or peroxidase activities. This O2- scavenging activity was CN- and heat-resistant. No achromatic bands were revealed in nondenaturing gels of Mn2+- containing cell extracts stained for O2- scavenging activity. The Mn2+-dependent O2- scavenging activity in the cell extracts was quenched by EDTA and dialyzable. More than 60% of both the intracellular Mn2+ and the O2- scavenging activity was removed by 2-h dialysis. Dialyzed cells were not viable in air unless resupplemented with either Met or Mn2+. Although Mn2+ supported the aerobic growth of these mutants, excess Mn2+, which correlated with an elevated O2- scavenging activity, was toxic to both mutant and wild type. The results indicate that free or loosely bound Mn2+ ion protects the mutants against oxygen stress by providing an intracellular, presumably cytosolic, O2- scavenging activity which replaces the absent SOD-1.
三种铜锌超氧化物歧化酶（SOD-1）缺陷型酿酒酵母突变体在富含培养基的100%氧气环境中无法生长，在空气中生长时需要甲硫氨酸和赖氨酸（Bilinski, T., Krawiec, Z., Liczmanski, A., and Litwinska, J. (1985) Biochem. Biophys. Res. Commun. 130, 533 - 539）。我们在此表明，突变体积累的培养基中的锰（II）挽救了这些对氧气敏感的表型；2 mM的培养基Mn2+是细胞生长所需的阈值。Mn2+的积累不是由氧气诱导的，因为在需氧和厌氧条件下生长的突变体积累的Mn2+量相同。Mn2+的积累并非这些突变体所特有，因为野生型积累的Mn2+几乎是突变体的两倍。加载了Mn2+的细胞提取物和完整细胞的电子顺磁共振光谱是游离Mn（II）离子的典型光谱。然而，这些光谱无法对细胞内的总Mn2+进行定量分析。对补充了Mn2+的细胞中的可溶性抗氧化活性进行筛选，检测到超氧阴离子（O2-）清除活性，而过氧化氢酶或过氧化物酶活性没有变化。这种O2-清除活性对氰化物和热具有抗性。在用于检测O2-清除活性的含Mn2+细胞提取物的非变性凝胶中未显示无色带。细胞提取物中依赖于Mn2+的O2-清除活性可被EDTA淬灭且可透析。2小时的透析可去除细胞内超过60%的Mn2+和O2-清除活性。除非重新补充甲硫氨酸或Mn2+，透析后的细胞在空气中无法存活。尽管Mn2+支持这些突变体的需氧生长，但过量的Mn2+与升高的O2-清除活性相关，对突变体和野生型均有毒性。结果表明，游离或松散结合的Mn2+离子通过提供一种细胞内（可能是胞质中的）O2-清除活性来保护突变体免受氧应激，这种活性替代了缺失的SOD-1。
