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Separation of Plant 6-Phosphogluconate Dehydrogenase (6PGDH) Isoforms by Non-denaturing Gel Electrophoresis
通过非变性凝胶电泳法分离植物6-磷酸葡萄糖酸脱氢酶(6PGDH)同工型   

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本实验方案简略版
Physiologia Plantarum
Feb 2009

Abstract

6-Phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) catalyzes the third and irreversible reaction of the pentose phosphate pathway (PPP). It carries out the oxidative decarboxylation of the 6-phosphogluconate to yield ribulose-5-phosphate, carbon dioxide and NADPH. In higher plants, 6PGDH has several subcellular localizations including cytosol, chloroplast, mitochondria and peroxisomes (Corpas et al., 1998; Krepinsky et al., 2001; Mateos et al., 2009; Fernández-Fernández and Corpas, 2016; Hölscher et al., 2016). Using Arabidopsis thaliana as plant model and sweet pepper (Capsicum annuum L.) fruits as a plant with agronomical interest, this protocol illustrates how to prepare the plant extracts for the separation of the potential 6PGDH isoforms by electrophoresis on 6% polyacrylamide non-denaturing gels. Thus, this method allows detecting three 6PGDH isoforms in Arabidopsis seedlings and two 6PGDH isoforms in sweet pepper fruits.

Keywords: NADPH (NADPH), Non-denaturing polyacrylamide gel electrophoresis (非变性聚丙烯酰胺凝胶电泳), Pentose phosphate pathway (磷酸戊糖途径), 6-Phosphogluconate dehydrogenase (6-磷酸葡萄糖酸脱氢酶)

Background

Non-denaturing gel electrophoresis is a powerful technique that allows separating native proteins. Their mobility depends of protein size, shape and net charge. In these analytical conditions, the protein preserves its activity and in combination with a specific staining method, it allows to separate the presence of potential isoforms. This approach has been widely used in the case of the family of superoxide dismutases. However, to our knowledge, there are not many papers that analyze the presence of different isoforms of 6PGDH activity in plant tissues (Corpas et al., 1998; Mateos et al., 2009). This straight method may be very useful for researchers working with plant 6PGDHs.

Materials and Reagents

  1. 10-cm-diameter Petri dishes
  2. Arabidopsis thaliana ecotype Columbia seeds (originally obtained from NASC, Nottingham Arabidopsis Stock Center)
  3. Sweet green pepper fruits were provided by Syngenta Seeds S.A. (El Ejido, Spain)
  4. 70% (v/v) ethanol
  5. 0.1% (w/v) SDS
  6. Commercial Bleach
  7. Murashige and Skoog medium (Sigma-Aldrich, catalog number: M5524 )
  8. Phyto-agar (Duchefa Biochemie, catalog number: P1001 )
  9. Sucrose (Sigma-Aldrich, catalog number: 84097 )
  10. Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 5000006 )
  11. Bovine serum albumin (BSA) Fraction V (Roche Diagnostics, catalog number: 10735078001 )
  12. Tris (AMRESCO, catalog number: 0497 )
  13. Ethylenediaminetetraacetic acid, disodium salt, dihydrate (Na2-EDTA) (Sigma-Aldrich, catalog number: E5134 )
  14. Triton X-100 (AMRESCO, catalog number: 0694 )
  15. Glycerol (AMRESCO, catalog number: E520 )
  16. Dithiothreitol (DTT) (Roche Diagnostics, catalog number: 10708984001 )
  17. 30% acrylamide/Bis solution, 19:1 (Bio-Rad Laboratories, catalog number: 1610154 )
  18. 4x gel buffer
  19. Glycine (AMRESCO, catalog number: 0167 )
  20. Ammonium persulfate
  21. TEMED
  22. β-Nicotinamide adenine dinucleotide phosphate, sodium salt, hydrate (NADP) (Sigma-Aldrich, catalog number: N0505 )
  23. Magnesium chloride, hexahydrate (MgCl2·7H2O) (EMD Millipore, catalog number: 442615 )
  24. Nitroblue tetrazolium (NBT) (AMRESCO, catalog number: 0329-1G )
  25. Phenazine methosulfate (PMS) (Sigma-Aldrich, catalog number: P9625 )
  26. 6-Phosphogluconic acid, trisodium salt (6PG) (Sigma-Aldrich, catalog number: P7877 )
  27. Glacial acetic acid (Fisher Scientific)
  28. Grinding buffer (see Recipes)
  29. Non-denaturing polyacrylamide gel electrophoresis (PAGE) on 6% acrylamide gel (see Recipes)
  30. Staining solution (see Recipes)
  31. Stop solution (see Recipes)

Equipment

  1. Plant Growth cabinet (Panasonic Biomedical, model: MLR-352-PE )
  2. Porcelain mortar and pestle
  3. Refrigerated centrifuge
  4. Vertical Slab gels Electrophoresis System (Bio-Rad Laboratories, model: Mini-PROTEAN® Electrophoresis Cell )

Software

  1. ImageJ program (https://imagej.nih.gov/ij/)

Procedure

  1. Plant extracts
    1. Surface-sterilize Arabidopsis thaliana ecotype Columbia seeds for 5 min in 70% (v/v) ethanol containing 0.1% (w/v) SDS at room temperature and with a slight shaking. Then, place for 20 min in sterile water containing 20% (w/v) commercial Bleach and 0.1% (w/v) SDS and then wash four times in sterile water.
    2. Grow seeds on Petri plates over commercial Murashige and Skoog medium (Sigma-Aldrich) with a pH of 5.5, containing 1% (w/v) sucrose and 0.8% (w/v) phyto-agar (Corpas and Barroso, 2014). Then, keep the seedlings for 14 days with a photoperiod of 16 h (light intensity of 100 μE m-2 sec-1) and 8 h dark, with a temperature of 22 °C and 18 °C, respectively.
    3. Sweet green pepper fruits were provided by Syngenta Seeds S.A. (El Ejido, Spain) from plants grown in experimental greenhouses.
    4. Homogenize plant samples in a mortar and pestle with grinding buffer (see Recipes) in a ratio 1:3 (w/v) for Arabidopsis and ratio 1:1 for pepper fruits. Perform these operations at 0-4 °C (on ice).
    5. Centrifuge extracts at 27,000 x g at 4 °C for 20 min.
    6. Use the supernatants for the enzymatic assays. Determine protein concentration with the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA) using bovine serum albumin as standard.

  2. 6PGDH activity staining
    1. Separate plant proteins by non-denaturing polyacrylamide gel electrophoresis (PAGE) on 6% acrylamide gels at 4-7 °C. Usually, it takes around 2 h and 30 min.
    2. After electrophoresis, incubate gels in staining solution (see Recipes) at room temperature (RT). Keep in the dark with shaking until blue-purple bands appear over a colorless background. It takes around 15 min (Figure 1).
    3. Remove the staining solution and add stop solution (see Recipes) to cover gels.


      Figure 1. Analysis of 6-phosphogluconate dehydrogenase (6PGDH) isoforms by non-denaturing gel electrophoresis. A. 6PGDH isoforms activity were separated by native PAGE (6%) and identified by activity staining. Lane 1, Arabidopsis thaliana 14-day-old seedlings. Lane 2, sweet green pepper fruits. B. Densitometric scans of 6PGDH isoforms and its relative quantification (%) made by the ImageJ program.

    Note: It is recommended to use fresh vegetable extracts because they provide better defined bands of activity.

Data analysis

Quantification of the activity of each 6PGDH isoform can be done by densitometric analysis (Figure 1B), for example, using the ImageJ program.

Recipes

  1. Grinding buffer
    50 mM Tris-HCl, pH 7.8 containing 0.1 mM EDTA
    0.2% (v/v) Triton X-100
    10% (v/v) glycerol
    2 mM DTT
  2. Non-denaturing polyacrylamide gel electrophoresis (PAGE) on 6% acrylamide gel
    30% acrylamide/Bis solution, 19:1
    4x gel buffer (1.5 M Tris-HCl, pH 8.9)
    Electrode buffer (38 mM glycine plus 5 mM Tris, pH 8.2)
    10% (w/v) ammonium persulfate (AP)
    TEMED
    The follow table shows the needed volume of each reagent to prepare two 1.5 mm thick gels to be used in a Bio-Rad Mini-PROTEAN Electrophoresis System

    *Note: Added right before each use. The mix of the reagents should be done at 4 °C (on ice).

    The standard running conditions are 15 mA per gel for 30 min and then increase to 25 mA per gel for approximately 90 min. The electrophoresis should be run in a cold room (5-7 °C)
  3. Staining solution
    50 mM Tris-HCl, pH 7.6 containing 0.8 mM NADP
    5 mM EDTA
    2 mM MgCl2
    0.24 mM NBT
    65 mM PMS
    10 mM 6PG
    Note: Freshly prepared before use and protected from the light.
  4. Stop solution
    7% (v/v) acetic acid

Acknowledgments

This work has been supported by the ERDF-cofinanced grant AGL2015-65104-P from the Ministry of Economy and Competitiveness, Spain.

References

  1. Corpas, F. J. and Barroso, J. B. (2014). Peroxynitrite (ONOO-) is endogenously produced in Arabidopsis peroxisomes and is overproduced under cadmium stress. Ann Bot 113(1): 87-96.
  2. Corpas, F. J., Barroso, J. B., Sandalio, L. M., Distefano, S., Palma, J. M., Lupiáñez, J. A. and del Río, L. A. (1998). A dehydrogenase-mediated recycling system of NADPH in plant peroxisomes. Biochem J 330 (Pt 2): 777-784.
  3. Fernández-Fernández, A. D. and Corpas, F. J. (2016). In silico analysis of Arabidopsis thaliana peroxisomal 6-phosphogluconate dehydrogenase. Scientifica (Cairo) 2016: 3482760.
  4. Hölscher, C., Lutterbey, M. C., Lansing, H., Meyer, T., Fischer, K. and von Schaewen, A. (2016). Defects in peroxisomal 6-phosphogluconate dehydrogenase isoform PGD2 prevent gametophytic interaction in Arabidopsis thaliana. Plant Physiol 171(1): 192-205.
  5. Krepinsky, K., Plaumann, M., Martin, W. and Schnarrenberger, C. (2001). Purification and cloning of chloroplast 6-phosphogluconate dehydrogenase from spinach. Cyanobacterial genes for chloroplast and cytosolic isoenzymes encoded in eukaryotic chromosomes. Eur J Biochem 268(9): 2678-2686.
  6. Mateos, R. M., Bonilla-Valverde, D., del Río, L. A., Palma, J. M. and Corpas, F. J. (2009). NADP-dehydrogenases from pepper fruits: effect of maturation. Physiol Plant 135(2): 130-139.

简介

6-磷酸葡萄糖酸脱氢酶(6PGDH; EC 1.1.1.44)催化戊糖磷酸途径(PPP)的第三次和不可逆反应。它进行6-磷酸葡萄糖酸盐的氧化脱羧,得到核酮糖-5-磷酸,二氧化碳和NADPH。在高等植物中,6PGDH具有几种亚细胞定位,包括细胞质,叶绿体,线粒体和过氧化物酶体(Corpas et al。,1998; Krepinsky等人,2001; Mateos et al。,2009;Fernández-Fernándezand Corpas,2016;Hölscher等人,2016)。使用拟南芥作为植物模型和甜椒(Capsicum annuum L.)作为具有农业兴趣的植物的水果,该方案说明如何制备植物提取物用于分离通过在6%聚丙烯酰胺非变性凝胶上电泳可能的6PGDH同种型。因此,该方法允许检测在拟南芥幼苗中的三种6PGDH同种型和甜椒果实中的两种6PGDH同种型。
【背景】非变性凝胶电泳是一种强大的技术,可以分离天然蛋白质。 它们的移动性取决于蛋白质的大小,形状和净电荷。 在这些分析条件下,蛋白质保留了其活性,并结合特定的染色方法,可以分离潜在同种型的存在。 在超氧化物歧化酶家族的情况下,广泛应用这种方法。 然而,据我们所知,没有多少论文分析植物组织中6PGDH活性的不同同工型的存在(Corpas等人,1998; Mateos等人。 ,2009)。 这种直接的方法可能对使用植物6PGDHs的研究人员非常有用。

关键字:NADPH, 非变性聚丙烯酰胺凝胶电泳, 磷酸戊糖途径, 6-磷酸葡萄糖酸脱氢酶

材料和试剂

  1. 10厘米直径的培养皿
  2. 拟南芥生态型哥伦比亚种子(最初从NASC,诺丁汉拟南芥股票中心获得)
  3. 甜食青椒水果由先正达种子S.A.(El Ejido,西班牙)提供
  4. 70%(v / v)乙醇
  5. 0.1%(w / v)SDS
  6. 商业漂白剂
  7. Murashige和Skoog培养基(Sigma-Aldrich,目录号:M5524)
  8. 植物琼脂(Duchefa Biochemie,目录号:P1001)
  9. 蔗糖(Sigma-Aldrich,目录号:84097)
  10. Bio-Rad蛋白测定(Bio-Rad Laboratories,目录号:5000006)
  11. 牛血清白蛋白(BSA)组分V(Roche Diagnostics,目录号:10735078001)
  12. Tris(AMRESCO,目录号:0497)
  13. 乙二胺四乙酸,二钠盐,二水合物(Na 2 -EDTA)(Sigma-Aldrich,目录号:E5134)
  14. Triton X-100(AMRESCO,目录号:0694)
  15. 甘油(AMRESCO,目录号:E520)
  16. 二硫苏糖醇(DTT)(Roche Diagnostics,目录号:10708984001)
  17. 30%丙烯酰胺/ Bis溶液,19:1(Bio-Rad Laboratories,目录号:1610154)
  18. 4x凝胶缓冲液
  19. 甘氨酸(AMRESCO,目录号:0167)
  20. 过硫酸铵
  21. TEMED
  22. β-烟酰胺腺嘌呤二核苷酸磷酸钠,钠盐,水合物(NADP)(Sigma-Aldrich,目录号:N0505)
  23. 氯化镁,六水合物(MgCl 2·7H 2 O)(EMD Millipore,目录号:442615)
  24. Nitroblue四唑(NBT)(AMRESCO,目录号:0329-1G)
  25. 异硫氰酸苯酯(PMS)(Sigma-Aldrich,目录号:P9625)
  26. 6-磷酸葡萄糖酸,三钠盐(6PG)(Sigma-Aldrich,目录号:P7877)
  27. 冰醋酸(Fisher Scientific)
  28. 研磨缓冲液(见配方)
  29. 6%丙烯酰胺凝胶上的非变性聚丙烯酰胺凝胶电泳(PAGE)(参见食谱)
  30. 染色溶液(参见食谱)
  31. 停止解决方案(请参阅食谱)

设备

  1. 植物生长箱(Panasonic Biomedical,型号:MLR-352-PE)
  2. 瓷砂浆和杵
  3. 冷藏离心机
  4. 垂直平板凝胶电泳系统(Bio-Rad Laboratories,型号:Mini-PROTEAN 电泳池)

软件

  1. ImageJ程式( https://imagej.nih.gov/ij/

程序

  1. 植物提取物
    1. 表面消毒拟南芥在室温和轻微摇动下在含有0.1%(w / v)SDS的70%(v / v)乙醇中生殖型Columbia种子5分钟。然后,在含有20%(w / v)商业漂白剂和0.1%(w / v)SDS的无菌水中放置20分钟,然后在无菌水中洗涤四次。
    2. 在含有1%(w / v)蔗糖和0.8%(w / v)植物琼脂(Corpas and Barroso,2014)的pH 5.5的商业Murashige和Skoog培养基(Sigma-Aldrich)上培养Petri板上的种子。然后,将幼苗保持14天,光照时间为16小时(光强度为100μEm -2至 sec -1),黑暗8小时,温度为22°C和18°C。
    3. 甜瓜青椒水果由先正达种子S.A.(El Ejido,西班牙)从实验温室种植的植物提供。
    4. 用研磨缓冲液(参见食谱)将植物样品以拟南芥为1:3(w / v)的比例均匀化,胡椒水果的比例为1:1。在0-4°C(冰上)进行这些操作。
    5. 离心机在4℃下以27,000 x g离心提取20分钟
    6. 使用上清液进行酶测定。使用牛血清白蛋白作为标准,用Bio-Rad蛋白测定(Bio-Rad Laboratories,Hercules,CA)测定蛋白质浓度。

  2. 6PGDH活性染色
    1. 通过非变性聚丙烯酰胺凝胶电泳(PAGE)在4-7℃下在6%丙烯酰胺凝胶上分离植物蛋白。通常需要2小时30分钟。
    2. 电泳后,在室温(RT)下,在染色溶液中孵育凝胶(参见食谱)。保持在黑暗中摇动,直到蓝紫色的乐队出现在无色的背景。大约需要15分钟(图1)
    3. 去除染色溶液并加入终止溶液(参见食谱)以覆盖凝胶。


      图1.通过非变性凝胶电泳分析6-磷酸葡萄糖酸脱氢酶(6PGDH)同种型。 :一种。通过天然PAGE(6%)分离6PGDH同工型活性,并通过活性染色鉴定。第1号,拟南芥拟南芥 14日龄幼苗。车道2,甜青椒水果。 B. 6PGDH同种型的光密度扫描及其由ImageJ程序进行的相对定量(%)。

    注意:建议使用新鲜蔬菜提取物,因为它们提供更好的定义的活动范围。

数据分析

每个6PGDH同种型的活性的定量可以通过光密度分析(图1B)进行,例如使用ImageJ程序。

食谱

  1. 研磨缓冲区
    含有0.1mM EDTA的50mM Tris-HCl,pH 7.8 0.2%(v / v)Triton X-100
    200新新200新200新新200新新200新新200新新200新新200新新200新新200新新200新200新新200新新200新200人200 200“ 2 mM DTT
  2. 6%丙烯酰胺凝胶上的非变性聚丙烯酰胺凝胶电泳(PAGE) 30%丙烯酰胺/双溶液,19:1
    4倍凝胶缓冲液(1.5M Tris-HCl,pH8.9) 电极缓冲液(38mM甘氨酸加5mM Tris,pH 8.2) 10%(w / v)过硫酸铵(AP)
    TEMED
    下表显示了要在Bio-Rad Mini-PROTEAN电泳系统中使用的两个1.5毫米厚凝胶的每种试剂所需的体积

    注意:每次使用之前都会添加。试剂的混合物应在4℃(冰上)进行。

    标准运行条件为每凝胶15mA 30分钟,然后每凝胶增加至25mA约90分钟。电泳应在寒冷的房间(5-7°C)内运行
  3. 染色溶液
    含有0.8mM NADP的50mM Tris-HCl,pH 7.6 5 mM EDTA
    2mM MgCl 2
    0.24 mM NBT
    65 mM PMS
    10 mM 6PG
    注意:使用前准备好并保护光线。
  4. 停止解决方案
    7%(v / v)乙酸

致谢

这项工作得到西班牙经济与竞争力部的ERDF-cofinanced grant AGL2015-65104-P的支持。

食谱

  1. 研磨缓冲区
    含有0.1mM EDTA的50mM Tris-HCl,pH 7.8 0.2%(v / v)Triton X-100
    200新新200新200新新200新新200新新200新新200新新200新新200新新200新新200新200新新200新新200新200人200 200“ 2 mM DTT
  2. 6%丙烯酰胺凝胶上的非变性聚丙烯酰胺凝胶电泳(PAGE) 30%丙烯酰胺/双溶液,19:1
    4倍凝胶缓冲液(1.5M Tris-HCl,pH8.9) 电极缓冲液(38mM甘氨酸加5mM Tris,pH 8.2) 10%(w / v)过硫酸铵(AP)
    TEMED
    下表显示了要在Bio-Rad Mini-PROTEAN电泳系统中使用的两个1.5毫米厚凝胶的每种试剂所需的体积

    注意:每次使用之前都会添加。试剂的混合物应在4℃(冰上)进行。

    标准运行条件为每凝胶15mA 30分钟,然后每凝胶增加至25mA约90分钟。电泳应在寒冷的房间(5-7°C)内运行
  3. 染色溶液
    含有0.8mM NADP的50mM Tris-HCl,pH 7.6 5 mM EDTA
    2mM MgCl 2
    0.24 mM NBT
    65 mM PMS
    10 mM 6PG
    注意:使用前准备好并保护光线。
  4. 停止解决方案
    7%(v / v)乙酸

致谢

这项工作得到西班牙经济与竞争力部的ERDF-cofinanced grant AGL2015-65104-P的支持。

参考

  1. Corpas,FJ和Barroso,JB(2014)。  过氧亚硝酸盐(ONOO - )是在拟南芥过氧化物酶体内产生的,并且在镉胁迫下过度产生。 Ann Bot 113(1):87 -96。
  2. 公司,FJ,Barroso,JB,Sandalio,LM,Distefano,S.,Palma,JM,Lupiáñez,JA和delRío,LA(1998)。  植物过氧化物酶体中NADPH的脱氢酶介导的再循环系统。生物化学J 330(Pt 2 ):777-784。
  3. Fernández-Fernández,AD和Corpas,FJ(2016)。 分析拟南芥过氧化物酶体6-磷酸葡萄糖酸脱氢酶。科学(开罗) 2016 :3482760.
  4. Hölscher,C.,Lutterbey,MC,Lansing,H.,Meyer,T.,Fischer,K.and von Schaewen,A.(2016)。  过氧化物酶体六磷酸葡萄糖酸脱氢酶同种型PGD2中的缺陷阻止拟南芥中的配子体相互作用。 >植物生理学 171(1):192-205。
  5. Krepinsky,K.,Plaumann,M.,Martin,W.and Schnarrenberger,C.(2001)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov / pubmed / 11322889“target =”_ blank“>从菠菜纯化和克隆叶绿体6-磷酸葡萄糖酸脱氢酶。在真核染色体中编码的叶绿体和细胞溶质同功酶的蓝细菌基因。 Eur J Biochem 268(9):2678-2686。
  6. Mateos,RM,Bonilla-Valverde,D.,delRío,LA,Palma,JM and Corpas,FJ(2009)。  来自胡椒水果的NADP-脱氢酶:成熟的作用。生理植物135(2):130-139。 />
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引用:Corpas, F. J., de Freitas-Silva, L., García-Carbonero, N., Contreras, A., Terán, F., Ruíz-Torres, C. and Palma, J. M. (2017). Separation of Plant 6-Phosphogluconate Dehydrogenase (6PGDH) Isoforms by Non-denaturing Gel Electrophoresis. Bio-protocol 7(14): e2399. DOI: 10.21769/BioProtoc.2399.
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