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首页> 美国卫生研究院文献>Plant Physiology >d-Ribulose-5-Phosphate 3-Epimerase: Cloning and Heterologous Expression of the Spinach Gene and Purification and Characterization of the Recombinant Enzyme
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d-Ribulose-5-Phosphate 3-Epimerase: Cloning and Heterologous Expression of the Spinach Gene and Purification and Characterization of the Recombinant Enzyme

机译：d-核糖-5-磷酸3-表异构酶：克隆和 菠菜基因的异源表达纯化和 表征 重组酶

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摘要

We have achieved, to our knowledge, the first high-level heterologous expression of the gene encoding d-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by dl-α-glycerophosphate or ethanol and destabilized by d-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.
机译：就我们所知，我们已经从任何来源获得了编码d-核糖5-磷酸3-表异构酶的基因的第一个高水平异源表达，从而可以分离和表征光合生物中的差向异构酶。极不稳定的重组菠菜（Spinacia oleracea L.）酶被dl-α-甘油磷酸或乙醇稳定，并被d-核糖-5-磷酸或2-巯基乙醇破坏。尽管存在这种不稳定性，但纯化材料前所未有的高比活性表明，在整个分离过程中都保持了酶的结构完整性。乙二胺四乙酸盐和二价金属阳离子不影响差向异构酶活性，因此排除了后者在催化中的需要。从克隆的菠菜基因的序列和纯化的重组酶在变性条件下的电泳迁移率推导，其25-kD亚基大小与酵母和哺乳动物的相应差向异构酶的大小大致相同。但是，与 在其他物种中，重组菠菜酶为八聚体 通过凝胶过滤和聚丙烯酰胺评估，而不是二聚体 非变性条件下进行凝胶电泳。西方印迹 用针对纯化的重组酶的抗体进行分析 从菠菜叶中提取的差向异构酶也是八聚体。

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