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Purification of a stable form of human aromatase in the absence of substrate: Effect of neuronal physiological pH changes on its substrate binding and activity

机译:在没有基质的情况下纯化稳定形式的人芳族芳香酶:神经元生理pH变化对其基底结合和活性的影响

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Recombinant human aromatase has been expressed in E. coli and for the first time purified in a stable form in the absence of any substrate or inhibitor: the active purified protein shows a Soret peak at 418 nm that completely shifts to 450 nm in the reduced CO-bound form. The protein retained the same activity of the one purified in the presence of the substrate androstenedione. The ability of the recombinant protein to bind the substrate androstenedione in the pH range 5.5-10 was investigated by UV-vis spectroscopy. The typical low-to-high spin transition, reflecting the displacement of the water ligand by substrate, was measured and a pH-dependency in substrate binding was found. Furthermore, a change of pH within 6.5 and 7.4, observed in physiological conditions in some active neurons, resulted in an increase of the K_D and K_M by 3 and 1.5 folds, respectively.
机译:已经在大肠杆菌中表达了重组人芳族糖糖糖,并且首次在没有任何基材或抑制剂的情况下以稳定形式纯化的第一次纯化:活性纯化的蛋白质显示在418nm处的Soret峰,其在减少的CO中完全转移到450nm - 行为。 蛋白质保留了在底甾酮化硫基存在下纯化的相同活性。 通过UV-Vis光谱研究重组蛋白在pH范围5-10中结合底物和rostenione的能力。 测量典型的低至高旋转过渡,反射水配体通过衬底的位移,并发现了底物结合的pH依赖性。 此外,在一些活性神经元的生理条件下观察到的6.5和7.4内的pH的变化,分别增加了K_D和K_M的增加3和1.5倍。

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