Nikonova L V, Beletsky I P, Umansky S R
Laboratory of Radiation Biochemistry, Russian Academy of Sciences, Pushchino, Moscow Region.
Eur J Biochem. 1993 Aug 1;215(3):893-901. doi: 10.1111/j.1432-1033.1993.tb18107.x.
Three nuclease activities have been found and characterized in rat thymocyte nuclear extracts. A Mn(2+)-dependent nuclease is loosely bound to nuclear components and can be extracted with 0.35 M NaCl. The enzyme is activated by Mn2+ but not by Mg2+, Ca2+, or both. Its molecular mass is 36-40 kDa when measured by gel filtration and 37 kDa by SDS/PAGE. An acidic nuclease is independent of divalent ions, produces DNA strand breaks with 5'-OH ends, its molecular mass is about 37 kDa. Two fractions of Ca2+/Mn(2+)-dependent nuclease, differing in binding to CM-Sepharose but identical in other respects, are active in the presence of Mn2+ but can be additionally activated by Ca2+. They are inactive in the presence of Mg2+ or Ca2+ but cleave DNA in Ca2+/Mg(2+)-containing medium. The molecular mass of the enzyme is 22 kDa as determined by both gel filtration and electrophoresis. The dependence of nuclease activities on pH, ions, and sulfhydryl reagents is described. Cycloheximide injection to both control and irradiated animals strongly inhibits the activities of Ca2+/Mn(2+)-dependent nuclease from thymocyte nuclei separated by chromatography on CM-Sepharose and does not change the activities of Mn(2+)-dependent and acidic nucleases. Nuclease activity in thymocyte nuclei from irradiated rats is increased in Ca2+/Mg(2+)-containing and Ca2+/Mn(2+)-containing media whereas there is no change in the activity of acidic nuclease. Ca2+/Mn(2+)-dependent nuclease is extracted from thymocyte nuclei of irradiated rats with 0.35 M NaCl but from control nuclei only with 0.5 M NaCl. Possible reasons of labilization of Ca2+/Mn(2+)-dependent-nuclease binding to the nuclear structures in dying thymocytes are discussed.
在大鼠胸腺细胞核提取物中发现并鉴定了三种核酸酶活性。一种依赖锰离子（Mn²⁺）的核酸酶与核成分松散结合，可用0.35M氯化钠提取。该酶由Mn²⁺激活，但不受镁离子（Mg²⁺）、钙离子（Ca²⁺）或两者激活。通过凝胶过滤测定其分子量为36 - 40 kDa，通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS/PAGE）测定为37 kDa。一种酸性核酸酶不依赖二价离子，产生5'-羟基末端的DNA链断裂，其分子量约为37 kDa。两部分依赖钙离子/锰离子（Ca²⁺/Mn²⁺）的核酸酶，与羧甲基琼脂糖凝胶（CM - Sepharose）的结合不同但在其他方面相同，在Mn²⁺存在时具有活性，但可被Ca²⁺进一步激活。它们在Mg²⁺或Ca²⁺存在时无活性，但在含Ca²⁺/Mg²⁺的介质中可切割DNA。通过凝胶过滤和电泳测定该酶的分子量为22 kDa。描述了核酸酶活性对pH、离子和巯基试剂的依赖性。向对照动物和受辐照动物注射放线菌酮强烈抑制通过CM - Sepharose层析分离的胸腺细胞核中依赖Ca²⁺/Mn²⁺的核酸酶活性，而不改变依赖Mn²⁺的核酸酶和酸性核酸酶的活性。在含Ca²⁺/Mg²⁺和含Ca²⁺/Mn²⁺的介质中，受辐照大鼠胸腺细胞核中的核酸酶活性增加，而酸性核酸酶活性无变化。依赖Ca²⁺/Mn²⁺的核酸酶用0.35M氯化钠可从受辐照大鼠的胸腺细胞核中提取，但从对照细胞核中仅用0.5M氯化钠才能提取。讨论了依赖Ca²⁺/Mn²⁺的核酸酶与垂死胸腺细胞核结构结合不稳定的可能原因。
