Department of Biochemistry, Warsaw University of Life Sciences - SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland.
Plant Physiol Biochem. 2011 Nov;49(11):1342-9. doi: 10.1016/j.plaphy.2011.05.008. Epub 2011 May 31.
Prolyl aminopeptidase (PAP) was isolated from the shoots of three-day-old triticale seedlings and was purified using a five-step purification procedure (acid precipitation, gel filtration, anion-exchange chromatography, hydrophobic chromatography and rechromatography). The enzyme was purified 460-fold with a recovery of 6%. Prolyl aminopeptidase appears to be a tetramer consisting of four subunits, each with a molecular weight of approximately 54kDa. Its pH and temperature optimum are pH 7.5 and 37°C, respectively. The enzyme prefers substrates with Pro and Hyp at the N-terminus, but is also capable of hydrolysing β-naphthylamides (β-NA) of Ala, Phe, and Leu. The K(m) value of PAP against Pro-β-NA was the lowest among the substrates tested and it was 1.47×10(-5)M. The activity of PAP was not inhibited by EDTA, 1,10-phenantroline, or pepstatin A. The most effective inhibitors were DFP, Pefabloc, and PMSF, which are serine protease inhibitors. However, significant inhibition was also observed in the presence of E-64, which modifies sulfhydryl groups. A significant increase of the aminopeptidase activity against Pro-β-NA was observed in shoots of triticale plants grown under salinity, drought stress, and in the presence of cadmium and aluminium ions in the nutrient solution.
脯氨酰氨基肽酶（PAP）从三天大的黑小麦幼苗的芽中分离出来，并通过五步纯化程序（酸沉淀、凝胶过滤、阴离子交换层析、疏水层析和再层析）进行纯化。该酶的纯度提高了 460 倍，回收率为 6%。脯氨酰氨基肽酶似乎是由四个亚基组成的四聚体，每个亚基的分子量约为 54kDa。其最适 pH 和温度分别为 pH7.5 和 37°C。该酶优先水解 N 端为 Pro 和 Hyp 的底物，但也能够水解 Ala、Phe 和 Leu 的β-萘酰胺（β-NA）。PAP 对 Pro-β-NA 的 K(m)值是所有测试底物中最低的，为 1.47×10(-5)M。EDTA、1,10-菲咯啉和 Pepstatin A 均不能抑制 PAP 的活性。DFP、Pefabloc 和 PMSF 是最有效的抑制剂，它们是丝氨酸蛋白酶抑制剂。然而，在存在修饰巯基的 E-64 的情况下，也观察到明显的抑制作用。在盐胁迫、干旱胁迫以及营养液中存在镉和铝离子的条件下，黑小麦植株芽中脯氨酰氨基肽酶对 Pro-β-NA 的活性显著增加。
