Purification and Characterization of an Alkaline Phosphatase Induced by Phosphorus Starvation in Common Bean (Phaseolus vulgaris L.) Roots
Purification and Characterization of an Alkaline Phosphatase Induced by Phosphorus Starvation in Common Bean (Phaseolus vulgaris L.) Roots
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DOI：
10.29356/jmcs.v56i1.279
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发表时间：
2017-10
期刊：
影响因子：
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通讯作者：
Luisa Mola Morales;N. Gutierrez;Vanessa Maya;C. Parra;E. Martínez‐Barajas;P. Coello
中科院分区：
文献类型：
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作者：
Luisa Mola Morales;N. Gutierrez;Vanessa Maya;C. Parra;E. Martínez‐Barajas;P. Coello
普通豆（Phaseolus vulgaris L.）根系的两种磷酸酶同工型在磷缺乏的条件下表现出活性增加的特征。选择其中一种APIII，通过离子交换色谱和制备电泳进一步纯化。通过SDS-PAGE和凝胶过滤分析，估计APIII的分子质量为35 kDa，表明该酶为单体形式。根据pH值为8的要求，将该磷酸酶分类为碱性磷酸酶。它不仅具有广泛的底物特异性，对焦磷酸盐的活性最高，而且能有效地催化水解多磷酸、葡萄糖-1-磷酸和磷酸烯醇-丙酮酸酯。钼酸盐、钒酸盐和磷酸盐均能完全抑制活性，而氟化物仅能部分抑制活性。虽然二价阳离子对该酶的焦磷酸酶活性不是必需的，但在mg2 +存在下，焦磷酸盐的水解显著增加。
Two phosphatase isoforms from roots of the common bean (Phaseolus vulgaris L.) showed an increase in activity in response to phosphate deficiency. One of them (APIII) was chosen for further purification through ionic exchange chromatography and preparative electrophoresis. The estimated molecular mass of APIII was 35 kDa by both SDS-PAGE and gel filtration analyses, suggesting a mono- meric form of the active enzyme. The phosphatase was classified as an alkaline phosphatase based on the requirement of pH 8 for optimum catalysis. It not only exhibited broad substrate specificity, with the most activity against pyrophosphate, but also effectively catalyzed the hydrolysis of polyphosphate, glucose-1-phosphate and phosphoenol- pyruvate. Activity was completely inhibited by molybdate, vanadate and phosphate but was only partially inhibited by fluoride. Although divalent cations were not essential for the pyrophosphatase activity of this enzyme, the hydrolysis of pyrophosphate increased substantially in the presence of Mg 2+ .