Enolase from the ectomycorrhizal fungus Tuber borchii Vittad.: biochemical characterization, molecular cloning, and localization
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- 作者：Polidori ; Emanuela ; Saltarelli ; Roberta ; Ceccaroli ; Paola ; Buffalini ; Michele ; Pierleoni ; Raffaella ; et. al.
- 关键词：Ectomycorrhizal fungus ; Enolase ; Gene cloning ; Glycolysis ; Protein purification
- 刊名：Fungal Genetics and Biology
- 出版年：2004
- 出版时间：February, 2004
- 年：2004
- 卷：41
- 期：2
- 页码：157-167
- 全文大小：485 K
文摘
Enolase from Tuber borchii mycelium was purified to electrophoretical homogeneity using an anion-exchange and a gel permeation chromatography. Furthermore, the corresponding gene (eno-1) was cloned and characterized. The purified enzyme showed a higher affinity for 2-PGA (0.26mM) with respect to PEP; the stability and activity of enolase were dependent of the divalent cation Mg2+. T. borchii eno-1 has an ORF of 1323bp coding for a putative protein of 440 amino acids and Southern blotting analysis revealed that the gene is present as a single copy in T. borchii. The enzymatic activity and the mRNA expression level evaluated in mycelia grown either in different carbon sources, in pyruvate or during starvation were the same in all the conditions tested, while biochemical and Northern blotting analyses performed with mycelia at different days of growth showed T. borchii eno-1 regulation in response to the growth phase. Finally, Western blotting analysis demonstrated that enolase is localized only in the cytosolic fraction confirming its important role in glycolysis.