Barkley K B, Gregory E M
Department of Biochemistry, Virginia Polytechnic Institute, Blacksburg.
Arch Biochem Biophys. 1990 Jul;280(1):192-200. doi: 10.1016/0003-9861(90)90535-7.
Superoxide dismutase was isolated from each of the anaerobically grown organisms Actinomyces naeslundii, Actinomyces strain E1S.25D, and Actinomyces odontolyticus. The enzymes were 100,000-110,000 mol wt acidic proteins (pI 4.3-4.6) and contained Mn and Zn, but no detectable Fe. The Mn and Zn content varied with the enzyme source. A. naeslundii superoxide dismutase, specific activity 2200 U/mg, contained 2.3 g atoms Mn and 1.4 g atoms Zn per mole tetramer whereas A. odontolyticus SOD, specific activity 700 U/mg, contained 1.4 g atoms Mn and 1.8 g atoms Zn per mole tetramer. Actinomyces strain E1S.25D, specific activity 1300 U/mg, contained 1.8 g atoms Mn and 1.2 g atoms Zn per mole tetramer. The amino acid compositions of the enzymes were comparable except for arginine, lysine, and tryptophan content. The enzymatic activity of each enzyme was stable in 5 mM H2O2 at 23 degrees C for 2 h. The enzymes were only modestly inhibited by 20 mM NaN3. The enzymatic activity was increased at low ionic strength but was markedly decreased at increased ionic strength with each salt tested except sodium perchlorate, which caused marked inhibition even at low ionic strength. Polyclonal antibodies to A. naeslundii and Actinomyces strain E1S.25D precipitated and inactivated their respective antigens whereas the precipitated A. odontolyticus superoxide dismutase-antibody complex retained virtually full catalytic activity. Immunological studies revealed that the native A. naeslundii and Actinomyces strain E1S.25D MnSODs share common epitopes and cross-reacted with precipitin lines of complete identity in Ouchterlony double diffusion gels. Antibody to the A. odontolyticus enzyme displayed only partial cross-reactivity with superoxide dismutase from the two other Actinomyces. Western blotting of the denatured antigens revealed reactivities of the antibodies that differed only slightly from the results of the Ouchterlony gels.
超氧化物歧化酶是从厌氧生长的内氏放线菌、放线菌E1S.25D菌株和溶牙放线菌中分别分离得到的。这些酶是分子量为100,000 - 110,000的酸性蛋白质（等电点4.3 - 4.6），含有锰和锌，但未检测到铁。锰和锌的含量因酶源而异。内氏放线菌超氧化物歧化酶的比活性为2200 U/mg，每摩尔四聚体含有2.3克原子锰和1.4克原子锌，而溶牙放线菌超氧化物歧化酶的比活性为700 U/mg，每摩尔四聚体含有1.4克原子锰和1.8克原子锌。放线菌E1S.25D菌株的比活性为1300 U/mg，每摩尔四聚体含有1.8克原子锰和1.2克原子锌。除精氨酸、赖氨酸和色氨酸含量外，这些酶的氨基酸组成具有可比性。每种酶的酶活性在23℃下于5 mM过氧化氢中稳定2小时。这些酶仅受到20 mM叠氮化钠的适度抑制。酶活性在低离子强度下增加，但在离子强度增加时显著降低，所测试的每种盐（除高氯酸钠外）均如此，高氯酸钠即使在低离子强度下也会导致显著抑制。针对内氏放线菌和放线菌E1S.25D菌株的多克隆抗体沉淀并使各自的抗原失活，而沉淀的溶牙放线菌超氧化物歧化酶 - 抗体复合物几乎保留了全部催化活性。免疫学研究表明，天然的内氏放线菌和放线菌E1S.25D菌株的锰超氧化物歧化酶具有共同的表位，在双向免疫扩散凝胶中与完全相同的沉淀线发生交叉反应。针对溶牙放线菌酶的抗体与另外两种放线菌的超氧化物歧化酶仅表现出部分交叉反应。变性抗原的蛋白质印迹显示抗体的反应性与双向免疫扩散凝胶的结果仅略有不同。
