Abstract Plant proteolytic enzymes belonging to the S8 family of serine proteases are known as subtilases or subtilisin-like proteases. Subtilases compose the largest family of plant serine peptidases and have been associated to a wide range of physiological responses and developmental stages. We here describe a simple and fast method to reveal subtilase activity in vivo in germinating seeds of different crops. The method is based on the hydrolysis of N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-AAPF-pNA), a synthetic peptide highly specific for subtilases, and subsequent diazotization of liberated p-nitroaniline giving an intense pink dye. The method was tested in germinating seeds of four cereals (wheat, maize, sorghum and rice). In order to verify subtilase activity in such materials, soluble extracts were prepared and analyzed by a standard protocol for in vitro measurement of Suc-AAPF-pNA degradation. Results revealed subtilase activity in vivo in all the cereal grains tested, showing strong correlation with spectrophotometric measurement of subtilase activity in soluble extracts. The present work not only describes for the first time a method to asses for subtilase activity in vivo, but also reveals the ubiquity of active subtilases during the germination of different plant species.

中文翻译：

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发芽种子中枯草杆菌酶活性的体内测定方法

摘要 属于丝氨酸蛋白酶 S8 家族的植物蛋白水解酶被称为枯草杆菌蛋白酶或枯草杆菌蛋白酶样蛋白酶。枯草杆菌酶是最大的植物丝氨酸肽酶家族，与广泛的生理反应和发育阶段有关。我们在这里描述了一种简单快速的方法来揭示体内枯草杆菌酶在不同作物种子发芽中的活性。该方法基于水解 N-琥珀酰-Ala-Ala-Pro-Phe 对硝基苯胺 (Suc-AAPF-pNA)，这是一种对枯草杆菌酶具有高度特异性的合成肽，随后重氮化释放出的对硝基苯胺，产生强烈的粉红色染料。该方法在四种谷物（小麦、玉米、高粱和水稻）的发芽种子中进行了测试。为了验证这些材料中的枯草杆菌酶活性，可溶性提取物通过标准方案制备和分析，用于体外测量 Suc-AAPF-pNA 降解。结果揭示了所有测试谷物的体内枯草杆菌酶活性，表明与可溶性提取物中枯草杆菌酶活性的分光光度法测量有很强的相关性。目前的工作不仅首次描述了一种在体内评估枯草杆菌酶活性的方法，而且揭示了在不同植物物种萌发过程中活性枯草杆菌酶的普遍存在。