Ramos M V, Souza D P, Gomes M T R, Freitas C D T, Carvalho C P S, Júnior P A V R, Salas C E
Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Campus do Pici, Cx. Postal 6033, Fortaleza, CE, CEP 60451-970, Brazil,
Protein J. 2014 Apr;33(2):199-209. doi: 10.1007/s10930-014-9551-4.
A 24,118 Da (MALDI-TOF) cysteine peptidase (EC 3.4.22.16) was purified from the latex extract of Cryptostegia grandiflora by two chromatographic steps involving ion exchange and Reverse-phase HPLC. The purified protein (Cg24-I) exhibits a single band profile following reduced SDS-PAGE and a unique amino terminal sequence, 1VPASIDWREKGTVL14, that is similar to other plant cysteine peptidases. Cg24-I displayed optimal proteolytic activity at pH 8.0 with 25 mM phosphate buffer. The proteolytic activity was inhibited with 0.2 mM E-64 and 1 mM iodoacetamide and was stimulated with 1 mM DTT. Cg24-I fully inhibited spore germination of phytopathogenic fungi Fusarium solani at a dose of 28.1 μg/mL. Its toxicity involves the membrane permeabilization of spores as probed by propidium iodide uptake. These results show that latex peptidases are part of the plant's defensive strategy against phytopathogens and that they most likely act synergistically with other recognized defensive proteins.
通过离子交换和反相高效液相色谱两步色谱法，从大花橡胶紫茉莉的乳胶提取物中纯化出一种分子量为24,118道尔顿（基质辅助激光解吸电离飞行时间质谱法）的半胱氨酸肽酶（EC 3.4.22.16）。纯化后的蛋白质（Cg24-I）在还原型十二烷基硫酸钠聚丙烯酰胺凝胶电泳后呈现单一蛋白条带图谱，并且具有独特的氨基末端序列1VPASIDWREKGTVL14，该序列与其他植物半胱氨酸肽酶相似。Cg24-I在含有25 mM磷酸盐缓冲液、pH值为8.0的条件下表现出最佳蛋白水解活性。其蛋白水解活性受到0.2 mM E-64和1 mM碘乙酰胺的抑制，并受到1 mM二硫苏糖醇的刺激。Cg24-I在剂量为28.1 μg/mL时能完全抑制植物病原真菌茄病镰刀菌的孢子萌发。通过碘化丙啶摄取实验表明，其毒性涉及孢子的膜通透性。这些结果表明，乳胶肽酶是植物抵御植物病原体防御策略的一部分，并且它们很可能与其他公认的防御蛋白协同作用。
