Raso María José, Pineda Manuel, Piedras Pedro
Departamento de Botánica, Ecología y Fisiología Vegetal, Campus Rabanales, Edif. Severo Ochoa, 1 Planta, Universidad de Córdoba, 14071-Córdoba, Spain.
Physiol Plant. 2007 Nov;131(3):355-66. doi: 10.1111/j.1399-3054.2007.00969.x.
Allantoinase (allantoin amidohydrolase, EC 3.5.2.5) catalyses the hydrolysis of allantoin to allantoic acid, a key reaction in the biosynthesis and degradation of ureides. This activity was determined in different tissues of French bean plants (Phaseolus vulgaris L.) which were grown under nitrogen-fixing conditions. Allantoinase activity was detected in all tissues analysed, but the highest levels of specific activity were found in developing fruits, from which allantoinase has been purified to electrophoretic homogeneity and further characterized. After diethylaminoethyl (DEAE)-Sephacel chromatography, two peaks showing allantoinase activity were obtained in the chromatographic profile and the corresponding proteins were independently purified. Total allantoinase activity was purified 200-fold, indicating the relevance of this enzymatic activity in French bean developing fruits, with allantoinase representing 0.5% of total soluble protein. Both proteins with allantoinase activity are monomeric with molecular masses of 45 and 42 kDa. The specific activities of the purified proteins were 560 and 295 units mg(-1), which correspond to turnover numbers of 25,200 and 12,100 min(-1), respectively. The two proteins have very similar biochemical properties showing Michaelis-Menten kinetics for allantoin with K(m) values of about 60 mM, with high optimal temperatures; are metalloenzymes; are inhibited by compounds reacting with sulphydryl groups; and are unaffected by reducing agents. All analysed tissues exhibited the two activities responsible for allantoin degradation, although one of them was the main form in leaves (the most photosynthetic tissue) and the other protein was the main form in roots (non-photosynthetic tissue). The allantoinase activity and distribution of both proteins have been analysed during fruit development. For both proteins, the allantoinase activity and distribution pattern were the same in plants growing either under nitrogen-fixing conditions or fertilized with nitrate.
尿囊素酶（尿囊素酰胺水解酶，EC 3.5.2.5）催化尿囊素水解生成尿囊酸，这是脲类生物合成和降解中的关键反应。在固氮条件下生长的菜豆植株（菜豆）的不同组织中测定了该活性。在所分析的所有组织中均检测到尿囊素酶活性，但在发育中的果实中发现了最高水平的比活性，已从其中将尿囊素酶纯化至电泳纯并进行了进一步表征。经二乙氨基乙基（DEAE）-葡聚糖凝胶柱层析后，在层析图谱中获得了两个显示尿囊素酶活性的峰，并分别纯化了相应的蛋白质。尿囊素酶的总活性纯化了200倍，表明该酶活性在菜豆发育中的果实中具有重要意义，尿囊素酶占总可溶性蛋白的0.5%。两种具有尿囊素酶活性的蛋白质均为单体，分子量分别为45 kDa和42 kDa。纯化后的蛋白质比活性分别为560和295单位mg⁻¹，对应的转换数分别为25,200和12,100 min⁻¹。这两种蛋白质具有非常相似的生化特性，对尿囊素表现出米氏动力学，Kₘ值约为60 mM，具有较高的最适温度；是金属酶；被与巯基反应的化合物抑制；不受还原剂影响。所有分析的组织均表现出两种负责尿囊素降解的活性，尽管其中一种在叶片（光合最强的组织）中是主要形式，另一种蛋白质在根（非光合组织）中是主要形式。在果实发育过程中分析了两种蛋白质的尿囊素酶活性和分布。对于这两种蛋白质，在固氮条件下生长或用硝酸盐施肥的植物中，尿囊素酶活性和分布模式相同。
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