A novel Ca²⁺-activated protease from germinating Vigna radiata seeds and its role in storage protein mobilization

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• 作者：Sadaf Khan ; Giti Verma ; Samir Sharma
• 关键词：Ca²⁺ ; Protease ; Protein mobilization ; Vicilin ; Vigna radiata
• 刊名：Journal of Plant Physiology
• 出版年：2010
• 出版时间：15 July 2010
• 年：2010
• 卷：167
• 期：11
• 页码：855-861
• 全文大小：643 K

文摘

Calcium (Ca²⁺)-dependent/activated proteases make decisive cleavages in proteins, affecting their further degradation/activation. Few such Ca²⁺-dependent proteases have been reported from plants, and none during germination-related events. Seeds are woken up from their quiescent state upon imbibition of water. The subsequent process of germination is strongly influenced by hormones (mainly gibberellins) and light, with both resulting in change in intracellular Ca²⁺. We have investigated the effect of Ca²⁺ on protease activity in extracts prepared from dry Vigna radiata (L.) Wilczec seeds and cotyledons 4, 24, 48 and 72 h post-imbibition. Ca²⁺-activated protease activity is present at a very low level in dry seeds, rises with imbibition and peaks 24 h post-imbibition. Subsequently, the activity rapidly declines, even as total protease activity continues to rise. Calcium activation of proteolysis was reversed by ethylene diamine tetraacetic acid (EDTA), ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 1,10, phenanthroline, chlorpromazine and by β-mercaptoethanol in a concentration-dependent manner. Protease activity was also inhibited by para chloro mercuribenzoate (pCMB) and l-trans-epoxysuccinyl-leucylamido(4-guanidino) butane (E 64), while phenyl methyl sulfonyl fluoride (PMSF) and pepstatin did not effect Ca²⁺ activation. The protease could be separated from the calmodulin fraction by size-exclusion chromatography, while retaining its ability for Ca²⁺ activation, excluding the possibility of activation through calmodulin-based pathways. The presence of a Ca²⁺-activated protease in the cotyledons suggests its role in a predetermined program of germination involving elevation of cytosolic Ca²⁺ levels during germination. This protease could be an important enzyme interfacing cytoplasmic signaling events and initiation of storage protein mobilization during seed germination.