Abstract
Homogalacturonans, the most abundant pectins of the plant cell wall, can be methylesterified at the C-6 position of the galacturonic acid residues. Demethylesterification of cell wall pectins is catalyzed by apoplastic pectin methylesterases (PMEs). Several plant developmental processes and plant-environment interactions involve PME-mediated cell wall modification, as it promotes the formation of Ca2+cross-links along the stretches of the demethylesterified galacturonic acid residues (Wolf et al., 2009; Müller et al., 2013), and thus influences the biophysical properties of plant cell walls. Here, we describe a protocol that can be used to estimate the activity of PMEs in a total soluble protein extract from plant or seed tissues. Soluble protein is extracted from the plant/seed materials, and a coupled enzyme assay is performed, according to a procedure modified from Grsic-Rausch and Rausch (2004). The methanol released from methylesterified pectins as a result of PME activity is oxidized to formaldehyde by alcohol oxidase. The formaldehyde is then used as an electron donor by formaldehyde dehydrogenase to reduce NAD+ to NADH. The formation of NADH from NAD+ is followed spectrophotometrically, and used to estimate the PME activity in the protein extract., 同聚半乳糖醛酸,植物细胞壁中最丰富的果胶,可以在半乳糖醛酸残基的C-6位置被甲基酯化。细胞壁果胶的脱甲基酯化由外切纤维果胶甲酯酶(PME)催化。几种植物发育过程和植物 - 环境相互作用涉及PME介导的细胞壁修饰,因为其促进沿着脱甲基酯化的半乳糖醛酸残基的延伸形成Ca 2+ 2 + et al。,2009;Mülleret al。,2013),因此影响植物细胞壁的生物物理性质。在这里,我们描述一个协议,可以用于估计PMEs在植物或种子组织的总可溶性蛋白质提取物的活性。从植物/种子材料中提取可溶性蛋白质,并根据从Grsic-Rausch和Rausch(2004)修改的程序进行偶联酶测定。作为PME活性的结果从甲基酯化果胶释放的甲醇被醇氧化酶氧化成甲醛。然后通过甲醛脱氢酶将甲醛用作电子供体,以将NAD +还原为NADH。通过分光光度法跟踪从NAD +形成NADH,并用于估计蛋白质提取物中的PME活性。
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