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Glutathione reductase was purified from chicken liver and some characteristics of the enzyme were investigated. The purification
procedure was composed of four steps: preparation of homogenate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity
chromatography, and Sephadex G-200 gel filtration chromatography. Owing to the four consecutive procedures, the enzyme was
purified 1714-fold, with a yield of 38%. Specific activity at the final step was 120 enzyme unit (EU)/mg of protein. The purified
enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight
of the enzyme was found to be 100 kDa by Sephadex G-200 gel filtration chromatography, and the subunit molecular weight was
found to be 43 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, and optimum temperature were 7.0, 7.4, 0.75
M Tris-HCl buffer including 1 mM EDTA, and 50°C, respectively. K
M and V
max values for NADPH and glutathione disulfide (GSSG) substrates were also determined for the enzyme. 相似文献
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Group II introns are large metallo-ribozymes that use divalent metal ions in folding and catalysis. The 3'-terminal domain 6 (D6) contains a conserved adenosine whose 2'-OH group acts as the nucleophile in the first splicing step. In the hierarchy of folding, D6 binds last into the active site. In order to investigate and understand the folding process to the catalytically active intron structure, it is important to know the individual binding affinities of Mg2+ ions to D6. We recently studied the solution structure of a 27 nucleotide long D6 (D6-27) from the mitochondrial yeast group II intron Sc.ai5gamma, also identifying five Mg2+ binding sites including the one at the 5'-terminal phosphate residues. Mg2+ coordination to the 5'-terminal di- and triphosphate groups is strongest (e.g., log KA,TP = 4.55 +/- 0.10) and is evaluated here in detail for the first time. The other four binding sites within D6-27 are filled simultaneously (e.g., log KA,BR = 2.38 +/- 0.06) and thus compete for the free Mg2+ ions in solution, having a distinct influence on the individual affinities of the various sites. For the first time, we take this competition into account to obtain the intrinsic binding constants, describing a method that is generally applicable. Our data illustrates that any RNA molecule undergoing tertiary contacts to a second RNA molecule first needs to be loaded evenly and specifically with metal ions to compensate for the repulsion between the negatively charged RNA molecules. 相似文献
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Chen D Albuquerque IF Baublis VV Bondar NF Carrigan RA Cooper PS Lisheng D Denisov AS Dobrovolsky AV Dubbs T Endler AM Escobar CO Foucher M Golovtsov VL Goritchev PA Gottschalk H Gouffon P Grachev VT Khanzadeev AV Kubantsev MA Kuropatkin NP Lach J Lang Pengfei Lebedenko VN Li Chengze Li Yunshan Mahon JR McCliment E Morelos A Newsom C Pommot Maia MC Samsonov VM Schegelsky VA Shi Huanzhang Smith VJ Sun CR Tang Fukun Terentyev NK Timm S Tkatch II Uvarov LN Vorobyov AA Yan Jie Zhao Wenheng 《Physical review letters》1992,69(23):3286-3289
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Foucher M Albuquerque IF Bondar NF Carrigan R Chen D Li Chengze Cooper PS Denisov AS Dobrovolsky AV Dubbs T Endler AM Escobar CO Tang Fukun Golovtsov VL Goritchev PA Gottschalk H Gouffon P Grachev VT Shi Huanzhang Yan Jie Khanzadeev AV Kubantsev MA Kuropatkin NP Lach J Luksys M Lebedenko VN Dai Lisheng Mahon JR McCliment E Morelos A Newsom C Lang Pengfei Pommot Maia MC Samsonov VM Zheng Shuchen Smith VJ Terentyev NK Timm S Tkatch II Uvarov LN Vorobyov AA Zhao Wenheng Zhong Yuanyuan 《Physical review letters》1992,68(20):3004-3007