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

在玉米(Zea Mays L)中发现了很高的蛋白酶活性。W64A)干燥种子。活性染色后在PAGE上显示一条Rf值为0.45的条带。该活性仅定位于干种子的胚中，而不存在于胚乳和盾片中。该酶可能在种子萌发的早期阶段起着重要的作用，因为种子吸胀后72小时才萌发，但直到48小时后酶活性才消失。这种酶在诱导种子萌发方面的作用是有兴趣的，因为它的活性发展的时间是伴随着胚胎中LEA蛋白的降解的，在粗提液中很难稳定地检测到这种活性。粗提物中多种效应物对活性有影响。例如，一价阳离子，特别是钠离子，显著地消除了这种活性。EDTA处理能显著提高酶的活性，但可能不是由于金属离子的络合作用所致。对酶的专一性抑制作用表明，该酶为类胰酶丝氨酸内切酶，用硫酸铵沉淀粗提物可有效地保持其稳定的活性。用离子交换层析、凝胶过滤、疏水层析和天然PAGE对该酶进行了纯化，结合这些层析得到的多肽在SDS-PAGE上的相对分子质量为45-65kD，亲和层析也得到了相同的多肽图谱。这些结果表明，至少在体外，该酶可能与这些多肽形成复合体。

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.