Development of an assay for Mg-dechelatase of oilseed rape cotyledons, using chlorophyllin as the substrate
Development of an assay for Mg-dechelatase of oilseed rape cotyledons, using chlorophyllin as the substrate
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以叶绿素为底物开发油菜子叶镁脱螯合酶测定方法
DOI：
10.1111/j.1399-3054.1995.tb00784.x
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发表时间：
1995
影响因子：
6.4
通讯作者：
P. Matile
中科院分区：
文献类型：
--
作者：
Fabrizio Vicentini;F. Iten;P. Matile
以从叶绿素(Chl)中提取的镁-叶绿素(Chlin)为底物，进行了镁脱氢酶的研究。镁离子的释放伴随着吸收峰从644 nm向687 nm的移动。以687 nm处的吸收变化作为油菜类囊体制剂中镁脱氢酶活力的量度。吸收变化与酶剂量呈线性相关。CHLIN释放镁的最适pH为9左右，最适pH为7，反应符合饱和动力学，表观Km值为17 nm左右。半胱胺或还原型谷胱甘肽的存在抑制了该酶的活性。硫醇试剂N-乙基马来酰亚胺不起作用。脱氢酶的大部分活性与叶绿体膜有关。这种酶是部分潜伏性的，要表现出完全的活性，需要用洗涤剂溶解类囊体体。在成熟的绿色油菜子叶中检测到最高活性。在黑暗诱导的衰老期间，活性下降的速度与叶组织中Chl的丧失速度大致相同
Chlorophyllin (Chlin), the Mg-chlorin obtained from chlorophyll (Chl) was employed as substrate of Mg-dechelatase. The release of Mg 2+ was associated with a shift of absorption from 644 to 687 nm. Changes of absorption at 687 nm were taken as a measure of Mg-dechelatase activity present in preparations of oilseed rape thylakoids. Absorption changes were correlated linearly with enzyme dose. The pH optimum of Mg release from Chlin was ca 9 with a broad flank down to pH 7. The reaction showed saturation kinetics with an apparent k m value of ca 17 nM. The activity was inhibited in the presence of cysteamine or reduced glutathion. There was no effect of the thiol reagent N-ethyl maleimide. The bulk of dechelatase activity was associated with the chloroplast membranes. The enzyme is partially latent and the appearance of full activity requires the solubilization of thylakoids with detergent. The highest activities were detected in mature green rape cotyledons. During dark-induced senescence the activity declined at roughly the same rate as Chl was lost in the leaf tissue