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

玉米（Zea mays L dentcorn cv）的蛋白酶活性非常高。W64A)干种子。活性染色后，主要活性为1条，在天然PAGE上的Rf值为0.45。这种活性只存在于干种子的胚中，而不存在于胚乳和鳞甲中。该酶在萌发初期可能起重要作用，但在吸胀后72小时后萌发，但在48小时后活性消失。这种酶在诱导种子萌发方面的作用很重要，因为其活性的发育时间与胚胎中LEA蛋白的降解有关。粗提物中活性的稳定检测非常困难。影响粗提物活性的因素很多。例如，一价阳离子，尤其是钠离子，其活性急剧下降。经EDTA处理后，其活性显著增加。然而，活化可能不是由于螯合金属离子。特异性抑制剂对蛋白酶的作用表明该酶是一种胰蛋白酶样丝氨酸内肽酶。粗提物经硫酸铵沉淀有效，具有稳定的活性。通过离子交换层析、凝胶过滤、疏水层析和天然PAGE对酶进行纯化。这些色谱组合在SDS-PAGE上总是得到相同的15-20个分子量为45- 65kd的肽。亲和层析也给出了相同的多肽模式。这些结果表明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.