Juan J Y, Keeney S N, Gregory E M
Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg 24060.
Arch Biochem Biophys. 1991 Apr;286(1):257-63. doi: 10.1016/0003-9861(91)90038-k.
Deinococcus radiodurans, a radiation-resistant aerobe, synthesized a 43,000 Mr dimeric superoxide dismutase. The holoenzyme, sp act 3300 U/mg, contained 1.5 g-atoms Mn, 0.6 g-atom Fe, and 0.1 g-atom Zn per mole dimer. Apoprotein, prepared by dialysis of the holoenzyme in denaturant plus chelator and then renatured in chelex-treated Tris chloride buffer, rapidly regained superoxide dismuting activity upon incubation in 1 mM MnCl2. Reconstitution was dependent on Mn concentration and pH. The Mn-reconstituted protein, sp act 3560 U/mg, contained 1.7 g-atoms Mn per mole dimer. The holoenzyme and Mn-reconstituted apoprotein migrated with the same patterns in 10% acrylamide gels and focused to the same pattern upon isoelectric focusing. Fluorescence emission maxima of the holoenzyme, Mn-reconstituted apoprotein, and the renaturated apoprotein were 329 +/- 1 nm but differed from the denatured apoprotein (352 nm). Apoprotein bound 1.7 g-atoms Zn and from 3-7 g-atoms Fe per mole dimer on incubation with 1 mM ZnSO4 and Fe(NH4)2(SO4)2, respectively. Although neither Zn nor Fe restored superoxide dismuting activity, the ferrous and the zinc salt inhibited reconstitution of the apoprotein with manganese. Metal addition to renatured aposuperoxide dismutase offers a novel approach to reconstitution of procaryote superoxide dismutases.
耐辐射球菌是一种抗辐射需氧菌，它能合成一种分子量为43000的二聚体超氧化物歧化酶。该全酶的比活性为3300 U/mg，每摩尔二聚体含有1.5克原子锰、0.6克原子铁和0.1克原子锌。通过在变性剂加螯合剂中对全酶进行透析，然后在经螯合树脂处理的氯化 Tris 缓冲液中复性制备的脱辅基蛋白，在1 mM MnCl2中孵育后能迅速恢复超氧化物歧化活性。重组依赖于锰浓度和pH值。锰重组蛋白的比活性为3560 U/mg，每摩尔二聚体含有1.7克原子锰。全酶和锰重组脱辅基蛋白在10%丙烯酰胺凝胶中迁移模式相同，在等电聚焦时聚焦到相同模式。全酶、锰重组脱辅基蛋白和复性脱辅基蛋白的荧光发射最大值为329±1 nm，但与变性脱辅基蛋白（352 nm）不同。脱辅基蛋白在分别与1 mM ZnSO4和Fe(NH4)2(SO4)2孵育时，每摩尔二聚体结合1.7克原子锌和3 - 7克原子铁。虽然锌和铁都不能恢复超氧化物歧化活性，但亚铁盐和锌盐会抑制脱辅基蛋白与锰的重组。向复性的脱辅基超氧化物歧化酶中添加金属为原核超氧化物歧化酶的重组提供了一种新方法。
