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Pectin Methylesterase Activity Assay for Plant Material
植物中果胶甲基酯酶(PME)活性测定实验   

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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.

Materials and Reagents

  1. Arabidopsis thaliana plant or seed materials
  2. Liquid nitrogen
  3. 100 mM sodium phosphate buffer (pH 7.5)
  4. 0.5% (w/v) Pectin (in dH2O) (Sigma-Aldrich, catalog number: P-9135 )
  5. 0.1 U/μl Alcohol oxidase (in 100 mM phosphate buffer) (pH 7.5) (Sigma-Aldrich, catalog number: A2404 )
  6. 0.5 U/μl Formaldehyde dehydrogenase (in 100 mM phosphate buffer) (pH 7.5) (Sigma-Aldrich, catalog number: F1879 )
  7. 0.4 mM NAD+ (in 100 mM phosphate buffer) (pH 7.5) (Sigma-Aldrich, catalog number: N8410 )
  8. PME from orange peel (Sigma-Aldrich, catalog number: P5400 )
  9. Protease inhibitor cocktail (1x) (contains 100 mM PMSF, 2 mM Bestatin, 0.3 mM Pepstatin A, and 0.3 mM E-64) (abmGood, catalog number: G135 )
  10. Protein extraction buffer (see Recipes)
  11. Master mix (see Recipes)

Equipment

  1. Eppendorf tubes
  2. Mortar and pestle
  3. Vortexer
  4. Centrifuge with cooling function
  5. 96 well microplates
  6. Microplate reader

Procedure

  1. Plant or seed tissue (Arabidopsis thaliana) is weighed. Use about 100 mg per extraction.
  2. Tissues are ground to a fine powder in liquid nitrogen using a mortar and pestle. The tissue must be kept frozen during grinding.
  3. Twice the fresh weight (w/v) of extraction buffer is added to the powder, and the powder allowed to thaw in the buffer.
  4. Vortex for 10 sec.
  5. Extracts are rotated at 4 °C for 30 min and centrifuged at 11,500 x g at 4 °C for 20 min.
  6. The supernatant is the soluble protein extract. Use fresh supernatants immediately for the PME enzyme assay, as the activity can be affected by freezing.
  7. Four replicates of each sample (10 μl each) are pipetted into microplate wells. For the negative control, use protein extraction buffer only. For a positive control, use a solution of commercially available PME in protein extraction buffer.
  8. Master mix (180 μl) is added to each well and mixed by pipetting up and down. Avoid the formation of bubbles.
  9. To start the reaction, add 10 μl of the pectin solution to the samples, the negative and the positive control, but not to the background controls. Mix well by pipetting up and down.
  10. Immediately put the plate into the microplate reader. If bubbles have formed during the mixing process, shake plate for 5 sec. Record the changes in absorption at 340 nm over 15 min at room temperature.
  11. The change in absorption per unit time over the linear part of the reaction is calculated for each well, and used to calculate the increase in concentration of NADH. The NADH concentration is calculated using Lambert-Beer's law with the extincion coefficient ε340 for NADH (6,220 M-1cm-1). One nkat PME activity is defined as 1 nmol NADH formed per second.
  12. The activities of the triplicates are averaged.

Recipes

  1. Protein extraction buffer
    100 mM Tris-HCl (pH 7.5)
    500 mM NaCl
    1x protease inhibitor cocktail
  2. Master Mix (per sample)
    20 μl pectin solution
    2 μl alcohol oxidase solution
    2 μl formaldehyde dehydrogenase solution
    156 μl NAD+ solution

Acknowledgments

This work was supported by the Swiss National Science Foundation (grant 31003A_127563; to TB) and by stipends to SB from the European Molecular Biology Organisation (EMBO: ALTF 61-2010) and the Leopoldina Fellowship Programme of the National Academy of Science Leopoldina (LPDS 2009-35).

References

  1. Grsic-Rausch, S. and Rausch, T. (2004). A coupled spectrophotometric enzyme assay for the determination of pectin methylesterase activity and its inhibition by proteinaceous inhibitors. Anal Biochem 333(1): 14-18.
  2. Mueller, K., Levesque-Tremblay, G., Bartels, S., Weitbrecht, K., Wormit, A., Usadel, B., Haughn, G. and Kermode, A. R. (2013). Demethylesterification of cell wall pectins in Arabidopsis plays a role in seed germination. Plant Physiol 161(1): 305-316.
  3. Wolf, S., Mouille, G. and Pelloux, J. (2009). Homogalacturonan methyl-esterification and plant development. Mol Plant 2(5): 851-860.

简介

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。

材料和试剂

  1. 拟南芥植物或种子材料
  2. 液氮
  3. 100mM磷酸钠缓冲液(pH7.5)
  4. 0.5%(w/v)果胶(在dH 2 O中)(Sigma-Aldrich,目录号:P-9135)
  5. 0.1U /μl醇氧化酶(在100mM磷酸盐缓冲液中)(pH7.5)(Sigma-Aldrich,目录号:A2404)
  6. 0.5U /μl甲醛脱氢酶(在100mM磷酸盐缓冲液中)(pH7.5)(Sigma-Aldrich,目录号:F1879)
  7. 0.4mM NAD +(在100mM磷酸盐缓冲液中)(pH7.5)(Sigma-Aldrich,目录号:N8410)
  8. PME(Sigma-Aldrich,目录号:P5400)
  9. 蛋白酶抑制剂混合物(1x)(含有100mM PMSF,2mM Bestatin,0.3mM Pepstatin A和0.3mM E-64)(abmGood,目录号:G135)
  10. 蛋白质提取缓冲液(参见配方)
  11. 主混合(见配方)

设备

  1. Eppendorf管
  2. 砂浆和杵
  3. Vortexer
  4. 带冷却功能的离心机
  5. 96孔微孔板
  6. 酶标仪

程序

  1. 称重植物或种子组织(拟南芥)。 每次提取使用约100mg。
  2. 使用研钵和杵将组织在液氮中研磨成细粉末。 在研磨过程中组织必须保持冷冻。
  3. 向粉末中加入两倍新鲜重量(w/v)的提取缓冲液,并使粉末在缓冲液中解冻。
  4. 涡旋10秒。
  5. 将提取物在4℃下旋转30分钟,并在4℃下以11,500xg离心20分钟。
  6. 上清液是可溶性蛋白质提取物。立即使用新鲜的上清液进行PME酶测定,因为活性会受到冻结的影响
  7. 将每个样品的四个重复(每个10μl)移液到微孔板孔中。对于阴性对照,仅使用蛋白质提取缓冲液。对于阳性对照,使用市售PME在蛋白质提取缓冲液中的溶液。
  8. 将主混合物(180μl)加入到每个孔中,通过上下吹吸混合。避免形成气泡。
  9. 为了开始反应,将10μl果胶溶液加入样品,阴性和阳性对照,但不加入背景对照。通过上下吹吸混匀
  10. 立即将板放入酶标仪。如果在混合过程中形成气泡,摇动板5秒。记录在340nm的吸光度在室温下15分钟的变化。
  11. 对每个孔计算反应的线性部分每单位时间的吸收的变化,并用于计算NADH浓度的增加。 使用Lambert-Beer定律计算NADH浓度,NADH(6,220M -1 cm -1 )的外切系数ε<340。 一个nkat PME活性定义为每秒形成1nmol NADH。
  12. 一式三份的活动平均值。

食谱

  1. 蛋白提取缓冲液
    100mM Tris-HCl(pH7.5) 500 mM NaCl
    1x蛋白酶抑制剂混合物
  2. 主混合(每个样品)
    20μl果胶溶液
    2μl醇氧化酶溶液
    2μl甲醛脱氢酶溶液
    156微升NAD +溶液

致谢

这项工作得到瑞士国家科学基金会(授予31003A_127563;结核病)和欧洲分子生物学组织(EMBO:ALTF 61-2010)和国家科学院Leopoldina Leopoldina奖学金计划(LPDS 2009-35)。

参考文献

  1. Grsic-Rausch,S。和Rausch,T。(2004)。 用于测定果胶甲酯酶活性的偶联分光光度酶测定法及其由蛋白质抑制剂的抑制作用。 a> Anal Biochem 333(1):14-18。
  2. Mueller,K.,Levesque-Tremblay,G.,Bartels,S.,Weitbrecht,K.,Wormit,A.,Usadel,B.,Haughn,G.and Kermode,A.R。 拟南芥中细胞壁果胶的脱甲基酯化在种子萌发中起作用。 Plant Physiol 161(1):305-316。
  3. Wolf,S.,Mouille,G。和Pelloux,J。(2009)。 Homogalacturonan methyl-esterification and plant development。 Mol Plant 2(5):851-860。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Mueller, K., Bartels, S. and Kermode, A. R. (2013). Pectin Methylesterase Activity Assay for Plant Material. Bio-protocol 3(17): e894. DOI: 10.21769/BioProtoc.894.
  2. Mueller, K., Levesque-Tremblay, G., Bartels, S., Weitbrecht, K., Wormit, A., Usadel, B., Haughn, G. and Kermode, A. R. (2013). Demethylesterification of cell wall pectins in Arabidopsis plays a role in seed germination. Plant Physiol 161(1): 305-316.
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