Islas-Flores Ignacio, Villanueva Marco A
Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos 62250, México.
Biochim Biophys Acta. 2007 Apr;1770(4):543-50. doi: 10.1016/j.bbagen.2006.12.001. Epub 2006 Dec 9.
A phosphate-hydrolyzing activity from Glycine max embryo axes was purified by a series of chromatographic steps and electroelution from activity gels, and demonstrated to be an inositol-1 (or 4)-monophosphatase by partial internal amino acid sequence. This enzyme hydrolyzed ATP, sodium pyrophosphate (NaPPi), inositol hexakisphosphate, and inositol 1-monophosphate, but not p-nitrophenyl phosphate, ADP, AMP or glucose 6-P. Using NaPPi as substrate, the highly purified protein hydrolyzed up to 0.4 mmol phosphate min(-1) mg(-1) protein and had a Km(avg) of 235 microM for NaPPi. Since NaPPi is relatively inexpensive and readily available, we used this as substrate for the subsequent characterization. We observed the following: (a) specific inhibition by Li and NaF but not by butanedione monoxime, or orthovanadate; (b) activation by Cu(2+) and Mg(2+); (c) optimum activity at pH 7.4; and (d) temperature stability after 1-h incubations at 37-80 degrees C, with maximum activity at 37 degrees C. The partially purified protein was detected by in-gel activity assays and the band was electroeluted to yield a highly purified protein. Analysis by SDS-PAGE and native IEF-PAGE yielded a single major polypeptide of 29 kDa and pI approximately 5.9, respectively. In addition, in-gel activity from embryo axes and whole hypocotyls at early germination times revealed one high and one intermediate molecular weight isoform, but only the intermediate one corresponded to IMPase. Throughout the post-imbibition period, the activity of the high molecular weight isoform disappeared and IMPase increased, indicating an increasing expression of the enzyme as germination and growth proceeded. These data indicate that the inositol-1 (or 4)-monophosphatase present in the embryo axis of G. max has a wide phosphate substrate specificity, and may play an important role in phosphate metabolism during the germination process.
通过一系列色谱步骤和从活性凝胶中进行电洗脱，对来自大豆胚胎轴的一种磷酸水解活性进行了纯化，并通过部分内部氨基酸序列证明其为肌醇-1（或4）-单磷酸酶。该酶可水解ATP、焦磷酸钠（NaPPi）、肌醇六磷酸和肌醇-1-单磷酸，但不能水解对硝基苯磷酸、ADP、AMP或葡萄糖6-磷酸。以NaPPi为底物时，高度纯化的蛋白质水解磷酸盐的速率高达0.4 mmol·min⁻¹·mg⁻¹蛋白质，对NaPPi的Km（平均值）为235 μM。由于NaPPi相对便宜且易于获得，我们将其用作后续表征的底物。我们观察到以下几点：（a）受到Li和NaF的特异性抑制，但不受丁二酮单肟或原钒酸盐的抑制；（b）被Cu²⁺和Mg²⁺激活；（c）在pH 7.4时具有最佳活性；（d）在37 - 80℃孵育1小时后具有温度稳定性，在37℃时活性最高。通过凝胶内活性测定法检测到部分纯化的蛋白质，然后将条带电洗脱以获得高度纯化的蛋白质。SDS-PAGE和天然IEF-PAGE分析分别得到一条29 kDa的主要多肽和pI约为5.9的结果。此外，在早期萌发时，胚胎轴和整个下胚轴的凝胶内活性显示出一种高分子量同工型和一种中等分子量同工型，但只有中等分子量的同工型对应于肌醇单磷酸酶。在整个吸胀后时期，高分子量同工型的活性消失，而肌醇单磷酸酶增加，表明随着萌发和生长的进行，该酶的表达增加。这些数据表明，大豆胚胎轴中存在的肌醇-1（或4）-单磷酸酶具有广泛的磷酸底物特异性，并且可能在萌发过程中的磷酸盐代谢中发挥重要作用。
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