Laboratório de Biotecnologia, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, 28013-602 Campos dos Goytacazes, RJ, Brazil.
Phytochemistry. 2011 Nov;72(16):1955-61. doi: 10.1016/j.phytochem.2011.06.009. Epub 2011 Jul 29.
In order to better understand the physiological functions of protease inhibitors (PIs) the PI activity in buds and flower organs of passion fruit (Passiflora edulis Sims) was investigated. Trypsin and papain inhibitory activities were analyzed in soluble protein extracts from buds at different developmental stages and floral tissues in anthesis. These analyses identified high levels of inhibitory activity against both types of enzymes at all bud stages. Intriguingly, the inhibitory activity against both proteases differed remarkably in some floral tissues. While all organs tested were very effective against trypsin, only sepal and petal tissues exhibited strong inhibitory activity against papain. The sexual reproductive tissues (ovary, stigma-style and stamen) showed either significantly lower activity against papain or practically none. Gelatin-SDS-PAGE assay established that various trypsin inhibitors (TIs) homogenously accumulated in developing buds, although some were differentially present in floral organs. The N-terminal sequence analysis of purified inhibitors from stamen demonstrated they had homology to the Kunitz family of serine PIs. Western-blot analysis established presence of a ∼60 kDa cystatin, whose levels progressively increased during bud development. A positive correlation between this protein and strong papain inhibitory activity was observed in buds and floral tissues, except for the stigma-style. Differences in temporal and spatial accumulation of both types of PIs in passion fruit flowers are thus discussed in light of their potential roles in defense and development.
为了更好地了解蛋白酶抑制剂（PIs）的生理功能，研究了西番莲（Passiflora edulis Sims）芽和花器官中的 PI 活性。在不同发育阶段的芽和开花期花器官的可溶性蛋白提取物中分析了胰蛋白酶和木瓜蛋白酶抑制活性。这些分析表明，在所有芽阶段都对这两种酶具有高水平的抑制活性。有趣的是，一些花器官中两种蛋白酶的抑制活性差异显著。虽然所有测试的器官对胰蛋白酶都非常有效，但只有萼片和花瓣组织对木瓜蛋白酶具有强烈的抑制活性。有性生殖组织（卵巢、柱头和雄蕊）对木瓜蛋白酶的活性较低或几乎没有。凝胶 SDS-PAGE 分析表明，各种胰蛋白酶抑制剂（TIs）均匀地在发育中的芽中积累，尽管在花器官中存在一些差异。从雄蕊中纯化的抑制剂的 N 端序列分析表明它们与丝氨酸 PI 的 Kunitz 家族具有同源性。Western-blot 分析表明存在一种约 60 kDa 的半胱氨酸蛋白酶抑制剂，其水平在芽发育过程中逐渐增加。在芽和花器官中观察到该蛋白与强烈的木瓜蛋白酶抑制活性之间存在正相关，柱头除外。因此，讨论了西番莲花中两种类型的 PI 在时间和空间上的积累差异，以及它们在防御和发育中的潜在作用。
