Study of relationship between development of embryo specific endopeptidase activites and induction of seed germination in maize plant.

在玉米（Zea mays L dentcorn cv. W64A）干种子中观察到非常高的蛋白酶活性。主要活性显示为活性染色后在天然 PAGE 上具有 0.45 Rf 值的一条带。该活性特异地存在于干种子的胚中，但不在胚乳和盾片中。该酶在发芽早期可能具有重要作用，因为活性直到48小时才消失，尽管种子在吸胀后72小时后才会发芽。该酶的作用在于诱导种子发芽，因为该酶活性的发生时间与胚胎中 LEA 蛋白的降解同时发生。在粗提物中稳定检测该酶活性非常困难。许多效应物影响粗提物的活性。例如，一价阳离子，尤其是钠离子，活性急剧消失。并且，经EDTA处理后，活性显着增加。然而，活化可能不是由于螯合金属离子所致。特异性蛋白酶抑制剂的作用表明该酶是一种类胰蛋白酶丝氨酸内肽酶。粗提物经硫酸铵沉淀可有效使其活性稳定。通过使用离子交换层析、凝胶过滤、疏水层析和非变性 PAGE 尝试纯化酶。这些层析的组合总是给出相同的 15-20 个肽，其在 SDS-PAGE 上的分子量为 45-65 kD。亲和层析也给出相同模式的肽。这些结果表明该酶至少在体外可以与这些肽形成复合物。

Very high proteinase activity was observed in maize (Zea mays L dentcorn cv. W64A) dry seeds. Major activity was shown as one band which has 0.45 of Rf value on a native PAGE after activity staining. The activity located specifically in embryo from dry seed, but not in endosperm and scutelum. The enzyme may have an important role during early stage of germination, because of the activity disappeared until 48 hours though the seed will germinate after 72 hours after imbibition. The role of this enzyme is interested in induction of seed germination, because timing of development of the activity is concomitant with degradation of LEA proteins in the embryo.It was very hard to detect the activity stably in crude extract. Many effectors affected the activity in crude extract. For example, monovalent cation, especially, sodium ion, disappeared the activity dramatically. And, the activity increased significantly by treatment of EDTA.However, the activation may not due to the chelate metallo-ion. The effect of specific inhibitors for protease showed the enzyme is a trypsin-like serine-endopeptidase.Sedimentation by Ammonium sulfate from crude extract was effectively to have it's stabale activity. Purification of the enzyme was attempted by using ion-exchange chromatography, gel-filtration, hydrophobic chromatography, and native PAGE.Combination of these chromatography always gave the same 15-20 peptides which have 45-65 kD of molecular masses on SDS-PAGE.Affinity chromatography also gave the same pattern of peptides. These results indicate that the enzyme may make a complex with these peptides at least in vitro.