Isolation and reconstitution of iron- and manganese-containing superoxide dismutases from Bacteroides thetaiotaomicron.
Isolation and reconstitution of iron- and manganese-containing superoxide dismutases from Bacteroides thetaiotaomicron.
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从多形拟杆菌中分离和重建含铁和锰的超氧化物歧化酶。
DOI：
10.1128/jb.166.2.528-532.1986
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发表时间：
1986
影响因子：
3.2
通讯作者：
Gregory,EM
中科院分区：
文献类型：
--
作者：
Pennington,CD;Gregory,EM
来自厌氧维持的多形拟杆菌提取物的超氧化物歧化酶 (SOD) 是由同等大小的 23,000 分子量单体非共价连接而成的二聚体。比活性为 1,200 U/mg 的制剂每摩尔二聚体含有 1.1 g 原子的 Fe、0.6 g 原子的 Zn 和少于 0.05 g 原子的 Mn。通过在 5 M 氯化胍-20 mM 8-羟基喹啉中透析铁-SOD 制备的脱辅基蛋白在没有外源金属复性时不具有超氧化物清除活性。通过在 20 mM Tris (pH 7.0) 中对 1 mM Fe(NH4)2 或 1 mM MnCl2 进行透析，使变性的脱辅基蛋白恢复酶活性。 Fe 重构酶和天然酶被 0.2 mM NaN3 抑制约 50%，而 Mn 重构酶被 10 mM NaN3 抑制 60%。厌氧细胞的通气导致抗叠氮化物 SOD 的诱导四倍。从充气细胞中分离出来的酶（分子量为 43,000）是大小相同的亚基的二聚体。金属含量为每摩尔二聚体1.0克原子的Mn、0.55克原子的Fe和0.3克原子的Zn。添加铁或锰后，该酶的变性脱辅基蛋白的酶活性也得到恢复。单独和组合测试的组成型 Fe-SOD 和 O2 诱导的 Mn-SOD 在丙烯酰胺凝胶上的迁移相同，具有相似的氨基酸组成，并且丙氨酸作为唯一的 N 末端氨基酸。这些数据与厌氧维持或充氧细胞中单个脱辅基蛋白的合成一致。我们已经观察到来自脆弱拟杆菌的 SOD 具有类似的现象（E.M.Gregory，Arch.Biochem.Biophys.238：83-89，1985）。
Superoxide dismutase (SOD) from extracts of anaerobically maintained Bacteroides thetaiotaomicron was a dimer of equally sized 23,000-molecular-weight monomers joined noncovalently. A preparation with a specific activity of 1,200 U/mg contained 1.1 g-atom of Fe, 0.6 g-atom of Zn, and less than 0.05 g-atom of Mn per mol of dimer. The apoprotein, prepared by dialysis of iron-SOD in 5 M guanidinium chloride-20 mM 8-hydroxyquinoline, had no superoxide-scavenging activity when renatured without exogenous metal. Enzymatic activity was restored to the denatured apoprotein by dialysis against either 1 mM Fe(NH4)2 or 1 mM MnCl2 in 20 mM Tris (pH 7.0). The Fe-reconstituted enzyme and the native enzyme were inhibited approximately 50% by 0.2 mM NaN3, whereas the Mn-reconstituted enzyme was inhibited 60% by 10 mM NaN3. Aeration of the anaerobic cells resulted in a fourfold induction of an azide-resistant SOD. The enzyme (43,000 molecular weight) isolated from aerated cells was a dimer of equally sized subunits. The metal content was 1.0 g-atom of Mn, 0.55 g-atom of Fe, and 0.3 g-atom of Zn per mol of dimer. Enzymatic activity of the denatured apoprotein from this enzyme was also restored on addition of either iron or manganese. The constitutive Fe-SOD and the O2-induced Mn-SOD, tested alone and in combination, migrated identically on acrylamide gels, had similar amino acid compositions, and had alanine as the sole N-terminal amino acid. These data are consistent with the synthesis of a single apoprotein in either anaerobically maintained or oxygenated cells. We have observed a similar phenomenon with SOD from Bacteroides fragilis (E. M. Gregory, Arch. Biochem. Biophys. 238:83-89, 1985).