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In vitro Colorimetric Method to Measure Plant Glutamate Dehydrogenase Enzyme Activity
体外比色法测定植物谷氨酸脱氢酶的活性   

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参见作者原研究论文

本实验方案简略版
Journal of Experimental Botany
Nov 2014

Abstract

Glutamate dehydrogenase (GDH) is an NAD(H) dependent enzyme that catalyzes, in vitro, the reversible amination of glutamate. Here we describe how to determine spectrophotometrically GDH activity monitoring NADH evolution. This protocol is described here for Arabidopsis thaliana (A. thaliana) although it is also valid for other plant species. GDH protein is a hexamer composed, in the case of Arabidopsis, of a combination of GDHα, GDHβ and GDHγ subunits. Every combination of subunits is possible; however, it is still barely known whether different combinations affect the enzymatic properties of the hexamers. In other species, hexamers are a combination of GDHα and GDHβ but it cannot be discarded the existence of other genes since for instance GDHγ subunit in Arabidopsis was described in Fontaine et al. (2012).

Glutamate + NAD
+ + H+ → 2-Oxoglutarate + NADH + NH4+

Keywords: Glutamate dehydrogenase (谷氨酸脱氢酶), Enzyme activity (酶的活性), Spectrophotometry (分光光度法)

Materials and Reagents

  1. 4-weak old Arabidopsis thaliana leaves and roots
  2. Ultrapure water
  3. Liquid N2
  4. Ice
  5. Flat bottom microplates (Deltalab, catalog number: 900011.1 )
  6. 4 mm diameter glass beads (Glaswarenfabrik Karl Hecht) (VWR International, catalog number: 201-0278 )
  7. 1.2 ml deep well storage plate (Thermo Fisher Scientific, catalog number: AB-0564 )
  8. EVA Capband for capping 8 tubes (Micronic, catalog number: MP227B1 )
  9. Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, AbD Serotec®, catalog number: 500-006 )
  10. NADH (Sigma-Aldrich, catalog number: 43420 )
  11. PVPP (Sigma-Aldrich, catalog number: 77627 )
  12. Glycerol (EMD Millipore Corporation, catalog number: 104092 )
  13. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
  14. Triton X-100 (Sigma-Aldrich, catalog number: T-9284 )
  15. HEPES (Sigma-Aldrich, catalog number: H3375 )
  16. KOH (EMD Millipore Corporation, catalog number: 105029 )
  17. MnCl2 (Sigma-Aldrich, catalog number: M8266 )
  18. EDTA (Sigma-Aldrich, catalog number: ED255 )
  19. EGTA (Sigma-Aldrich, catalog number: E4378 )
  20. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  21. PMSF (Sigma-Aldrich, catalog number: P7626 )
  22. Delta-aminocaproic acid (Sigma-Aldrich, catalog number: A2504 )
  23. Leupeptin (Sigma-Aldrich, catalog number: L2884 )
  24. Tricine (Sigma-Aldrich, catalog number: T0377 )
  25. CaCl2 (Panreac Applichem, catalog number: 131232 )
  26. 2-Oxogutarate (Merck KGaA, catalog number: 5194 )
  27. (NH4)2SO4 (Panreac Applichem, catalog number: 131140 )
  28. Extraction buffer (see Recipes)
  29. Reaction buffer (see Recipes)
  30. Bradford solution (see Recipes)

Equipment

  1. TissueLyser (RETSCH, model: MM400 )
  2. Plate centrifuge (Sigma, model: 2-16K )
  3. Absorbance microplate reader (Biotek Powerwave X 384 Microplate spectrophotometer)
  4. TissueLyser Adapter Set 2 x 96 (QIAGEN, catalog number: 69984 )

Procedure

  1. Material harvest and homogenization
    1. In a 96 deepwell (1.2 ml) plate, place 2 glass beads (4 mm diameter) into each well.
    2.  Weigh approximately 20 mg of fresh leaves or roots and place them in a 96-deepwell plate, half-submerged in liquid nitrogen. Store sealed with EVA Capband at -80 °C until use.
    3. Grind frozen tissue two times with a TissueLyser for 60 sec at 27 Hz frequency. Plates are coupled to the TissueLyser with a TissueLyser Adapter Set 2 x 96. (Ensure sealing of the wells during homogenization).

  2. Extraction
    1. Add around 3-5 mg PVPP per well (this approximately corresponds to a level micro spatula).
    2.  Add 500 µl of cold extraction buffer to each well and rapidly mix by inverting the plate (avoid thawing the samples prior to adding the buffer).
    3. Homogenize the samples again with TissueLyser two times for 60 sec at 27 Hz frequency.
    4. Centrifuge the plate at 4,000 rpm and 4 °C for 30 min in a plate centrifuge.
    5. Recover the supernatants (400 µl) in a new 96 deepwell plate and keep the samples on ice.

  3. Determination of protein content by Bradford method
    1. In a 96-well spectrophotometer plate, place 1 µl of extract per well and add ultrapure water up to 10 µl. Make a blank with 1 µl of extraction buffer.
    2. For the calibration curve place BSA standards in a range of 0 to 6 µg of BSA/well using a 1 µg/µl BSA stock and complete volume up to 10 µl.
    3. Add in every well 240 µl of Bradford solution and incubate for 5 min at room temperature in the dark.
    4. Read absorbance at 595 nm.

  4. Enzyme activity measurement
    1. Place 20 µl of supernatant per well.
    2. Add 280 µl of reaction buffer. This step should be performed rapidly since the reaction starts as soon as the buffer is added. Thus, if measuring 96 samples the use of a multichannel pipette is strongly advised to ensure reproducibility.
    3. Immediately monitor NADH evolution kinetic at 340 nm in the spectrophotometer for 10 min at 30 °C (Figure 1). Ensure spectrophotometer is already at 30 °C before adding the reaction buffer to the sample.


      Figure 1. Graph showing NADH consumption by GDH aminating activity

    4. For NADH calibration curve, place 300 µl NADH dilutions from 0-0.2 mM in a microplate and read at 340 nm. (NADH dilutions must be prepared just prior to the measurement of the calibration curve to avoid the oxidation of NADH).

Data analysis

  1. To determine protein concentration in the extracts, subtract from the absorbance of every sample the absorbance of the blank (done with extraction buffer) and calculate the protein concentration with the calibration curve performed with BSA standards.
  2. Enzyme activity is calculated with the maximum speed (Vmax) obtained from NADH oxidation monitoring at 340 nm (don’t take the two first minutes of the measurement into account and ensure linearity of the activity). Subtract the blank (made with extraction buffer) from every sample and calculate GDH activity with NADH calibration curve. Finally express the activity per µmol of NADH consumed per minute and per µg of protein (obtained from Bradford assay) or per mg of fresh/dry weight.

Notes

  1. This protocol can be easily adapted to eppendorf tubes and spectrophotometer cuvettes instead of 96-well plates just scaling the volumes. Similarly the homogenization of the samples might be done with a mortar and pestle instead of the TissueLyser.
  2. If possible, we advice to do every 96-well plate step using multi-channel pipettes to save time and thus to gain reproducibility among samples.
  3. This protocol is for Arabidopsis thaliana but it can be used for every species. For some species desalting of protein extracts supernatants might be considered.

Recipes

Note: All solutions should be made in ultrapure water.

  1. Extraction buffer
    Glycerol 10%
    BSA 0.05%
    Triton X-100 0.1%
    50 mM HEPES (pH 7.5, adjusted with KOH)
    10 mM MgCl2
    1 mM EDTA
    1 mM EGTA
    1 mM PMSF
    10 mM DTT
    1 mM Delta-aminocaproic acid
    1 µM Leupeptin
  2. Reaction buffer (pH 8)
    100 mM Tricine (pH 8, adjusted with KOH)
    1 mM CaCl2
    50 mM (NH4)2SO4
    0.25 mM NADH
    13 mM 2-Oxoglutarate
  3. Bradford solution
    Dilute Bio-Rad Protein Assay Dye Reagent Concentrate 1:5 with ultrapure water
    Stored in a dark bottle at 4 °C

Acknowledgments

This protocol is adapted from Sarasketa et al. (2014) and based on the methodology reported by Groat and Vance (1981). This work was supported by the Basque Government (IT526-10), the UPV/EHU (EHUA14/14), the People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program (FP7/2007–2013) under REA grant agreement number 334019 and MINECO (BIO2014-56271-R).

References

  1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  2. Fontaine, J. X., Terce-Laforgue, T., Armengaud, P., Clement, G., Renou, J. P., Pelletier, S., Catterou, M., Azzopardi, M., Gibon, Y., Lea, P. J., Hirel, B. and Dubois, F. (2012). Characterization of a NADH-dependent glutamate dehydrogenase mutant of Arabidopsis demonstrates the key role of this enzyme in root carbon and nitrogen metabolism. Plant Cell 24(10): 4044-4065.
  3. Groat, R. G. and Vance, C. P. (1981). Root nodule enzymes of ammonia assimilation in alfalfa (Medicago sativa L.): DEVELOPMENTAL PATTERNS AND RESPONSE TO APPLIED NITROGEN. Plant Physiol 67(6): 1198-1203.
  4. Sarasketa, A., Gonzalez-Moro, M. B., Gonzalez-Murua, C. and Marino, D. (2014). Exploring ammonium tolerance in a large panel of Arabidopsis thaliana natural accessions. J Exp Bot 65(20): 6023-6033.   

简介

谷氨酸脱氢酶(GDH)是NAD(H)依赖性酶,其在体外催化谷氨酸盐的可逆胺化。在这里我们描述如何确定分光光度法GDH活动监测NADH进化。此协议在这里描述为拟南芥( A thaliana ),虽然它也适用于其他植物物种。 GDH蛋白是在拟南芥属的情况下由GDHα,GDHβ和GDHγ亚基的组合组成的六聚体。每个亚基的组合是可能的;然而,仍然几乎不知道不同的组合是否影响六聚体的酶性质。在其他物种中,六聚体是GDHα和GDHβ的组合,但不能丢弃其它基因的存在,因为例如在Fontaine等人(2012)中描述了拟南芥中的GDHγ亚基。

谷氨酸盐+ NAD + + H + →2-氧代戊二酸+ NADH + NH4 +

关键字:谷氨酸脱氢酶, 酶的活性, 分光光度法

材料和试剂

  1. 4弱的拟南芥叶和根
  2. 超纯水
  3. 液体N <2>

  4. 平底微量培养板(Deltalab,目录号:900011.1)
  5. 4mm直径的玻璃珠(Glaswarenfabrik Karl Hecht)(VWR International,目录号:201-0278)
  6. 1.2ml深孔存储板(Thermo Fisher Scientific,目录号:AB-0564)
  7. 用于封盖8根管的EVA帽带(Micronic,目录号:MP227B1)
  8. Bio-Rad蛋白测定染料试剂浓缩物(Bio-Rad Laboratories,AbD Serotec ,目录号:500-006)
  9. NADH(Sigma-Aldrich,目录号:43420)
  10. PVPP(Sigma-Aldrich,目录号:77627)
  11. 甘油(EMD Millipore Corporation,目录号:104092)
  12. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906)
  13. Triton X-100(Sigma-Aldrich,目录号:T-9284)
  14. HEPES(Sigma-Aldrich,目录号:H3375)
  15. KOH(EMD Millipore Corporation,目录号:105029)
  16. MnCl 2(Sigma-Aldrich,目录号:M8266)
  17. EDTA(Sigma-Aldrich,目录号:ED255)
  18. EGTA(Sigma-Aldrich,目录号:E4378)
  19. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
  20. PMSF(Sigma-Aldrich,目录号:P7626)
  21. δ-氨基己酸(Sigma-Aldrich,目录号:A2504)
  22. 亮肽素(Sigma-Aldrich,目录号:L2884)
  23. Tricine(Sigma-Aldrich,目录号:T0377)
  24. CaCl 2(Panreac Applichem,目录号:131232)
  25. 2-氧代戊二酸酯(Merck KGaA,目录号:5194)
  26. (NH 4)2 SO 4(Panreac Applichem,目录号:131140)
  27. 提取缓冲液(参见配方)
  28. 反应缓冲液(参见配方)
  29. Bradford解决方案(参见配方)

设备

  1. TissueLyser(RETSCH,型号:MM400)
  2. 板式离心机(Sigma,型号:2-16K)
  3. 吸光度酶标仪(Biotek Powerwave X 384微孔板分光光度计)
  4. TissueLyser Adaptor Set 2×96(QIAGEN,目录号:69984)

程序

  1. 材料收获和均质化
    1. 在96深孔(1.2ml)板中,将2个玻璃珠(直径4mm)置于每个孔中。
    2.  称重约20毫克的新鲜叶子或根,并将它们放在 96深孔板,半浸没在液氮中。 存放密封 EVA帽带在-80°C,直到使用
    3. 研磨冷冻组织两次 用TissueLyser在27Hz频率下持续60秒。 板被耦合到 TissueLyser带有TissueLyser适配器套装2 x 96.(确保密封 的均质化)。

  2. 提取
    1. 每孔加入约3-5mg PVPP(这大约相当于微量刮铲水平)。
    2.  向每个孔中加入500μl冷提取缓冲液,并快速混合 倒置板(避免解冻样品之前添加 缓冲)。
    3. 使用TissueLyser再次将样品均匀化两次,以27Hz频率持续60秒
    4. 在板式离心机中在4,000rpm和4℃下将板离心30分钟。
    5. 回收在新的96深孔板中的上清液(400μl),并保持在冰上的样品。

  3. 通过Bradford方法测定蛋白质含量
    1. 在96孔分光光度计板中,每孔放置1μl提取物 并加入超纯水至10μl。 用1μl的空白 提取缓冲区。
    2. 对于校准曲线放置BSA标准品 在0至6μgBSA /孔的范围内,使用1μg/μlBSA原液和 完全体积可达10μl。
    3. 在每孔中加入240μlBradford溶液,并在室温下在黑暗中孵育5分钟
    4. 读取595 nm处的吸光度。

  4. 酶活性测量
    1. 每孔放置20μl的上清液。
    2. 加入280μl反应物 缓冲。 该步骤应该自反应开始后快速进行 一旦添加了缓冲区。 因此,如果测量96个样品的使用   建议多通道移液器确保重现性
    3. 立即监测NADH进化动力学在340 nm的 分光光度计在30℃下10分钟(图1)。 确保 分光光度计已经在30°C,然后加入反应缓冲液 到样品

      图1.显示GDH胺化活性NADH消耗的图表

    4. 对于NADH校准曲线,将300μlNADH稀释液从0-0.2mM,   并在340nm读数。 (NADH稀释液必须准备好 在测量校准曲线之前避免氧化   的NADH)

数据分析

  1. 为了测定提取物中的蛋白质浓度,从中减去 每个样品的吸光度为空白的吸光度 提取缓冲液)并计算蛋白质浓度 校准曲线。
  2. 用获得的最大速度(Vmax)计算酶活性 从NADH氧化监测在340 nm(不要采取两个第一 分钟的测量考虑并确保线性 活动)。 从每个中减去空白(用提取缓冲液制成) 采用NADH标准曲线计算GDH活性。 最后 表达每分钟和每μg消耗的每μmolNADH的活性 蛋白质(从Bradford测定获得)或每mg新鲜/干重。

笔记

  1. 这个协议可以很容易适应eppendorf管和 分光光度计比色皿代替96孔板只是缩放 卷。 类似地,样品的均质化可以用a   砂浆和杵,而不是TissueLyser。
  2. 如果可能,我们建议做每一个96孔板步骤使用 多通道移液器,以节省时间,从而获得可重复性 。
  3. 此协议是拟南芥,但它可以用于每个物种。 对于某些物种,可以考虑对蛋白质提取物进行脱盐。

食谱

注意:所有溶液都应在超纯水中制备。

  1. 提取缓冲区
    甘油10%
    BSA 0.05%
    Triton X-100 0.1%
    50mM HEPES(pH7.5,用KOH调节) 10mM MgCl 2/
    1mM EDTA
    1 mM EGTA
    1mM PMSF
    10 mM DTT
    1mM的δ-氨基己酸
    1μM亮抑蛋白酶
  2. 反应缓冲液(pH 8)
    100mM Tricine(pH8,用KOH调节) 1mM CaCl 2
    50mM(NH 4)2 SO 4 4
    0.25mM NADH 13 mM 2-氧代戊二酸盐
  3. Bradford解决方案
    稀释Bio-Rad蛋白测定染料试剂用超纯水浓缩1:5 储存在4°C的黑暗瓶中

致谢

该协议改编自Sarasketa等人(2014)并基于Groat和Vance(1981)报道的方法。这项工作得到了欧盟第七框架计划(FP7/2007-2013)的巴斯克政府(IT526-10),UPV/EHU(EHUA14/14),人民计划(Marie Curie Actions)编号334019和MINECO(BIO2014-56271-R)。

参考文献

  1. Bradford,M.M。(1976)。 利用蛋白质染料结合原理的快速灵敏的微克量蛋白定量方法。 Anal Biochem 72:248-254。
  2. Fontaine,JX,Terce-Laforgue,T.,Armengaud,P.,Clement,G.,Renou,JP,Pelletier,S.,Catterou,M.,Azzopardi,M.,Gibon,Y.,Lea,PJ,Hirel ,B.和Dubois,F。(2012)。 拟南芥的NADH依赖性谷氨酸脱氢酶突变体的表征证明了该酶在根碳中的关键作用 和氮代谢。植物细胞 24(10):4044-4065。
  3. Groat,R.G。和Vance,C.P。(1981)。 苜蓿中氨同化的根瘤酶( Medicago sativa L.) :开发植物病原体和对应用氮源的反应。


    67(6):1198-1203。
  4. Sarasketa,A.,Gonzalez-Moro,M.B.,Gonzalez-Murua,C.and Marino,D。(2014)。 在大面板的拟南芥自然种质中探索铵耐受性。 J Exp Bot 65(20):6023-6033。   
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sarasketa, A., Vega-Mas, I. and Marino, D. (2015). In vitro Colorimetric Method to Measure Plant Glutamate Dehydrogenase Enzyme Activity. Bio-protocol 5(16): e1560. DOI: 10.21769/BioProtoc.1560.
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Anindita Roy
Institute of Agricultural Scienece
Please can you tell me in which proportion I will mix the extraction and reaction buffer agents in the method of In vitro Colorimetric Method to Measure Plant Glutamate Dehydrogenase Enzyme Activity ? Please reply me ASAP its urgent.
1/26/2018 2:38:17 AM Reply
Daniel Marino
Department of Plant Biology and Ecology, University of the Basque Country (UPV/EHU), Spain

As described in Procedure, point D. It is usual to determine the activity out of 20 microliters of extract adding 280 microliters of reaction buffer. However, in function of the species, the tissue and the developmental stage GDH activity is different. Thus, if the activity is very high you may need to dilute the sample to obtain a linear kinetic. On the contrary, if it is too low you may need to concentrate the sample.

1/26/2018 3:03:04 AM