缺失nifE的棕色固氮菌突变种MoFe蛋白的纯化及体外激活
결실nifE적종색고담균돌변충MoFe단백적순화급체외격활
Purification and Activation In Vitro of MoFe Protein from a nifE Deleted Mutant Strain of Azotobacter vinelandii
  • 万方数据
    植物学报 식물학보
    ACTA BOTANICA SINICA
    2003年 7期 815-819 ,共5页

    赵剑峰%赵颖%汪志平%吕玉兵%潜中兴%黄巨富

    조검봉%조영%왕지평%려옥병%잠중흥%황거부

    △nifE Av1%纯化%激活和体外组装%FeMoco和含Mn重组液 △nifE Av1%純化%激活和體外組裝%FeMoco和含Mn重組液
    △nifE Av1%순화%격활화체외조장%FeMoco화함Mn중조액
    △ nif E Av1%purification%activation and assembly in vitro%FeMoco and reconstituent solution containing Mn
    经DEAE纤维素、Sephacryl S-300和Q-Sepharose柱层析分离纯化,从缺失nifE的棕色固氮菌(Azotobactervinelandii Lipmann)突变种(DJ35)的无细胞粗提物中得到△nifE MoFe蛋白(△nifE Av1).SDS凝胶电泳分析表明,△nifE Av1的亚单位种类和分子量分别与棕色固氮菌野生型(OP)MoFe蛋白(Av1)的α和β亚单位相似.当与固氮酶Fe蛋白(Av2)活性互补时,△nifE Av1不具有还原质子的能力,但从OP Av1中抽提的FeMoco却可使其激活.经过量的邻菲啰啉(o-phen)厌氧处理并经Sephadex G-25柱层析分离后,便得到△nifE Av1 .在同时存在Av2和MgATP发生系统的条件下,△nifE Av1 ,而不是△nifE Av1,可为由KMnO4、高柠檬酸铁、Na2S、Na2S2O4和二硫苏糖醇组成的含Mn重组液(RS-Mn)显著激活.但在缺少MgATP或Av2的条件下,RS-Mn则不能激活△nifE Av1 .这就表明,RS-Mn对△nifE Av1 的激活需要o-phen的预先处理及同时存在Av2和MgATP的这二个条件.
    경DEAE섬유소、Sephacryl S-300화Q-Sepharose주층석분리순화,종결실nifE적종색고담균(Azotobactervinelandii Lipmann)돌변충(DJ35)적무세포조제물중득도△nifE MoFe단백(△nifE Av1).SDS응효전영분석표명,△nifE Av1적아단위충류화분자량분별여종색고담균야생형(OP)MoFe단백(Av1)적α화β아단위상사.당여고담매Fe단백(Av2)활성호보시,△nifE Av1불구유환원질자적능력,단종OP Av1중추제적FeMoco각가사기격활.경과량적린비라람(o-phen)염양처리병경Sephadex G-25주층석분리후,편득도△nifE Av1 .재동시존재Av2화MgATP발생계통적조건하,△nifE Av1 ,이불시△nifE Av1,가위유KMnO4、고저몽산철、Na2S、Na2S2O4화이류소당순조성적함Mn중조액(RS-Mn)현저격활.단재결소MgATP혹Av2적조건하,RS-Mn칙불능격활△nifE Av1 .저취표명,RS-Mn대△nifE Av1 적격활수요o-phen적예선처리급동시존재Av2화MgATP적저이개조건.
    The △ nif E MoFe protein ( △ nif EAv1) was obtained by a chromatography on DE52, SephacrylS-300 and Q-Sepharose columns from the unheated crude extract of nifE-deleted mutant strain (DJ35)of Azotobacter vinelandii Lipmann. The analysis by SDS-PAGE showed that the △ nifEAv1 was similar toOP MoFe protein (Av1) of A. vinelandii in the kinds and molecular weights of subunits (α and β subunit).When complemented with nitrogenase Fe protein (Av2), the △ nif EAv1 had hardly any proton-reductionactivity, but could be significantly activated by FeMoco extracted from OP Av1. After the △ nif E Av1 wastreated with an excess o-phenanthroline (o-phen) and chromatographied on Sephadex G-25 column underatmosphere of Ar, △ nif EAv1 was obtained. In the presence of both Av2 and MgATP regenerationsystem, the △ nif E Av1 , rather than △ nif E Av1, was significantly activated in vitro by a reconstituentsolution containing Mn which composed of KMnO4, ferric homocitrate, Na2S, Na2S2O4 (DT) and dithiothreitol(DTT). But in the absence of MgATP or Av2, the activation of △nifE Av1 did not happen. It indicates thatactivation of △ nif E Av1 by RS-Mn requires the pretreatment with o-phen and the simultaneous presenceof Av2 and MgATP.
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