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Measurement of NADPH Oxidase Activity in Plants
植物中NADPH氧化酶活性的测定   

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Abstract

NADPH oxidase is a membrane-bound enzyme that generates (O2-) by transferring electrons from NADPH to molecular oxygen O2. O2-is spontaneously dismasted to the more stable form H2O2. Both O2-and H2O2 are forms ofreactive oxygen species (ROS), which are involved in regulation of many cellular activities such as transcription, intracellular signaling, and host defense. The NADPH oxidase - dependent generation of O2- in total membrane fraction of plant tissue has been determined by the reduction of the tetrazolium salt XTT by O2-. In the presence of O2-, XTT generates a soluble yellow formazan that can be quantified spectrophotometrically.

Materials and Reagents

  1. Sucrose
  2. HEPES
  3. EDTA
  4. DTT
  5. L-cysteine
  6. MgCl2
  7. PVP
  8. Complete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets (F. Hoffmann-La Roche, catalog number: 04693159001 )
  9. BSA
  10. Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 500-0006 )
  11. Tris-HCl
  12. Sodium 3,3'-( -[(phenylamino)carbonyl] -3,4-tetrazolium)-bis (4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) (Sigma-Aldrich, catalog number: X4626 )
  13. NADPH (Sigma-Aldrich, catalog number: N1630 )
  14. Protein extraction working solution (see Recipes)

Equipment

  1. Microtiter plate reader (Infinite M200 Pro, Tecan)
  2. Microcentrifuge (AqquSpin Micro R) (Thermo Fisher Scientific)
  3. Ultracentrifuge ( Optima TLX , Beckman)
  4. Microtiter plate (BD Biosciences, catalog number: 353075 )

Procedure

  1. Protein extraction and separation of membrane fraction from plant tissues
    1. Harvest tissue in liquid nitrogen. If not used immediately, keep at -80 °C until processing.
    2. Grind tissue in liquid nitrogen and weigh out 0.5 g of the ground tissues in empty Falcon tube that has been pre-chilled in liquid nitrogen and used to tare the scale.
    3. Add 6 ml of ice-cold protein extraction buffer to ground tissues on ice.
    4. Vortexat room temperature to mix thoroughly.
    5. Filter homogenized tissue through four layers of cheese cloth and transfer filtrate (flow-through) to 2-ml microcentrifuge tubes, on ice.
    6. Centrifuge at 10,000 x g for 45 min at 4 °C and transfer supernatant to ultra- centrifuge tube.
    7. Separate total membrane fractions by ultra-centrifugation at 203,000 x g for 60 min at 4 °C.
    8. Discard supernatant and resuspend pellet in 1 ml ice-cold 10 mM Tris-HCl (pH 7.4).

  2. Protein estimation using Bradford microassay (160 μl)
    1. Prepare BSA standards ranging from 5 μg-25 μg/ml as follows:



      These standards will be used to generate a standard curve.
    2. Use 96 well microtiter plate to prepare reaction mix.
    3. Prepare blank byadding 160 μl of water to one well in triplicates.
    4. Prepare test samples by adding 2 μl of supernatant (from section 1) to 158 μl of water.
    5. Add 40 μl of Bradford Assay reagent to BSA standards, blank and test samples.
    6. Mix and incubate at room temperature for 5 min and read absorbance at 595 nm (A595) on plate reader spectrophotometer.
      Note: If spectrophotometer does not include a software to generate standard curve to automatically estimate protein content, generate a BSA standard curve by plotting known protein concentration (X-axis) vs. Absorbance (in Y-axis). Protein concentration for a given unknown sample is estimated by plotting the A595 absorbance of the unknown (in the y-axis) and determining the intersection point with the BSA standard curve and then find the concentration associated with that particular point (in the x-axis). If using excel, after plotting concentration vs A595, obtain the trendline and use the equation for the line and the A595 of the unknown to resolve the unknown concentration.

  3. NADPH oxidase activity assay
    1. Prepare fresh solution of 1 mM XTT and 1 mM NADPH.
    2. Prepare two different assay solutions A and B:


      Note: Because membrane fraction can spontaneously reduce XTT, even in the absence of substrate (NADPH), it is necessary to prepare two blanks, one without NADPH, to correct for this background levels of activity.
    3. Prepare blanks:
      Blank 1: Add 10 μl of water to 240 μl of Assay solution A.
      Blank 2: Add 10 μl of membrane fraction to 240 μl Assay solution B.
    4. Prepare samples by adding 10 μl of membrane fraction (from section 1) to 240 μl of Assay solution A. Read the absorbance at 492 nm (A492) at 0 min and then 20 min intervals for one hour or until saturation point reached.
    5. To get final A492 Blank reading, subtracts A492 Blank1 and A492 Blank 2.
    6. Calculate rate of O2– generation by using an extinction coefficient 2.16 x 104 cm-(Jiang and Zhang 2002).
      A492nm/min testA492nm/min blank) / (2.16X104M-1CM-1) (0.04)
      ΔA492nm/min Test= A492 (sample X) at saturation point - A492 (sample X) at 0 min
      ΔA492nm/min blank = A492nm (blank) at saturation point - A492nm (blank) at 0 min
      0.04 = dilution factor (10 μl/250 μl)
      To calculate specific activity, divide the value obtained in equation by the amount of protein present in the sample (converted to mg/ml).

Recipes

  1. Protein extraction working solution
    0.25 M sucrose
    50 mM HEPES
    3 mM EDTA
    1 mM DTT
    3.6 mM L-cysteine
    0.1 mM MgCl2
    0.6% PVP
    10 Tablets of Complete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets.
    Prepare the following stock solutions:
    1 M sucrose
    1 M HEPES (pH 7.2)
    0.25 M EDTA
    1 M DTT
    100 mM MgCl2
    In 80 ml of water add the following reagents:



    Add 10 Tablets of Complete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets.
    Mix well and adjust volume to 100 ml

Acknowledgments

This protocol has been adapted and modified to use in Arabidopsis from Jiang and Zhang (2002). This work was supported by the Samuel Roberts Noble Foundation.

References

  1. Able, A. J., Guest, D. I. and Sutherland, M. W. (1998). Use of a new tetrazolium-based assay to study the production of superoxide radicals by tobacco cell cultures challenged with avirulent zoospores of phytophthora parasitica var Nicotianae. Plant Physiol 117(2): 491-499.
  2. Jiang, M. and Zhang, J. (2002). Involvement of plasma-membrane NADPH oxidase in abscisic acid- and water stress-induced antioxidant defense in leaves of maize seedlings. Planta 215(6): 1022-1030.
  3. Rojas, C. M., Senthil-Kumar, M., Wang, K., Ryu, C. M., Kaundal, A. and Mysore, K. S. (2012). Glycolate oxidase modulates reactive oxygen species-mediated signal transduction during nonhost resistance in Nicotiana benthamiana and Arabidopsis. Plant Cell 24(1): 336-352.
  4. Sagi, M. and Fluhr, R. (2001). Superoxide production by plant homologues of the gp91(phox) NADPH oxidase. Modulation of activity by calcium and by Tobacco mosaic virus infection. Plant Physiol 126(3): 1281-1290.

简介

NADPH氧化酶是膜结合酶,其通过将电子从NADPH转移到分子氧O 2而产生(O 2 - )。 O 2被自发地分解成更稳定的形式H 2 O 2。 O 2 - 和H 2 O 2都是反应性氧物质(ROS)的形式,其参与许多细胞活性的调节,例如 转录,细胞内信号传导和宿主防御。 植物组织的总膜部分中NADPH氧化酶依赖性O 2 2-的产生已经通过四唑鎓盐XTT被O 2-还原而确定。 在O 2-的存在下,XTT产生可溶性黄色甲,其可以通过分光光度法定量。

材料和试剂

  1. 蔗糖
  2. HEPES
  3. EDTA
  4. DTT
  5. L-半胱氨酸
  6. MgCl 2
  7. PVP
  8. Complete,Mini,无EDTA的蛋白酶抑制剂混合物片剂(F.Hoffmann-La Roche,目录号:04693159001)
  9. BSA
  10. Bio-Rad蛋白测定(Bio-Rad Laboratories,目录号:500-0006)
  11. Tris-HCl
  12. 将3,3' - ( - [(苯基氨基)羰基] -3,4-四唑鎓) - 双(4-甲氧基-6-硝基)苯磺酸水合物(XTT)(Sigma-Aldrich,目录号:X4626)
  13. NADPH(Sigma-Aldrich,目录号:N1630)
  14. 蛋白质提取工作溶液(参见配方)

设备

  1. 微量滴定板读数器(Infinite M200 Pro,Tecan)
  2. 微量离心机(AqquSpin Micro R)(Thermo Fisher Scientific)
  3. 超速离心机(Optima TLX,Beckman)
  4. 微量滴定板(BD Biosciences,目录号:353075)

程序

  1. 蛋白质提取和膜组分与植物组织的分离
    1. 收获组织在液氮。 如果不立即使用,保持在-80℃直到处理
    2. 在液氮中研磨组织,并在已经在液氮中预冷并用于去皮的空Falcon管中称出0.5g的研磨组织。
    3. 加入6ml冰冷的蛋白质提取缓冲液在冰上研磨组织
    4. Vortex室温下彻底混合
    5. 通过四层奶酪布过滤均质组织,并在冰上将滤液(流通)转移到2ml微量离心管中。
    6. 在4℃下以10,000×g离心45分钟,并将上清液转移到超离心管中。
    7. 通过在4℃下以203,000×g超速离心60分钟分离总膜级分。
    8. 弃去上清液并在1ml冰冷的10mM Tris-HCl(pH7.4)中重悬沉淀

  2. 使用Bradford微量测定法(160μl)
    1. 制备从5μg至25μg/ml范围内的BSA标准品,如下所示:



      这些标准将用于生成标准曲线。
    2. 使用96孔微量滴定板制备反应混合物
    3. 准备空白,一次加入160μl水到一个孔,一式三份
    4. 通过加入2μl上清液(从第1部分)到158μl水制备测试样品
    5. 向BSA标准品,空白和测试样品中加入40μlBradford测定试剂
    6. 混合并在室温下孵育5分钟,并在平板读数器分光光度计上读取在595nm处的吸光度(A <595)。
      如果分光光度计不包括用于生成标准曲线以自动估计蛋白质含量的软件,可通过绘制已知蛋白质浓度)与吸光度(在Y轴)。通过绘制未知物的吸光度(在y轴上)作图并确定交叉点来估计给定未知样品的蛋白质浓度点与BSA标准曲线,然后找到与该特定点(在x轴)相关的浓度。如果使用excel,在绘制浓度与A 之后,获得趋势线并使用线的方程, sub> 595 来解析未知浓度。

  3. NADPH氧化酶活性测定
    1. 制备1mM XTT和1mM NADPH的新鲜溶液
    2. 准备两种不同的测定溶液A和B:


      因为膜部分可以自发地减少XTT,即使没有底物(NADPH),有必要制备两个空白, 更正此背景活动级别。
    3. 准备空白:
      空白1:向240μl测定溶液A中加入10μl水 空白2:向240μl测定溶液B中加入10μl膜级分
    4. 通过加入10μl膜部分(从部分1)到240μl测定溶液A制备样品。在0分钟时读取492nm处的吸光度(A 492),然后以20分钟的间隔读取1小时 或直到达到饱和点
    5. 为了获得最终的A 492 空白读数,减去A 492 Blank1和A <49>
    6. 通过使用消光系数2.16×10 4 cm -1计算O 2生成率(Jiang和Zhang 2002)。< br /> (ΔA 492nm/min测试/ΔA 492nm/min空白)/(2.16×10 4 M -1) 1 M-1 )(0.04)
      在饱和点-A A 492(样品X)处的ΔA 492nm /min测试 = A 492(样品X) 在0分钟
      在饱和点的ΔA 492nm /min空白 = A 492nm(空白) - 在0℃的A 492nm(空白) min
      0.04 =稀释因子(10μl/250μl)
      为了计算比活性,将公式中得到的值除以样品中存在的蛋白质量(换算成mg/ml)。

食谱

  1. 蛋白提取工作溶液
    0.25 M蔗糖 50 mM HEPES
    3 mM EDTA
    1 mM DTT
    3.6mM L-半胱氨酸 0.1mM MgCl 2·h/v 0.6%PVP
    10片,完整,微型,无EDTA的蛋白酶抑制剂鸡尾酒片 准备以下储备溶液:
    1 M蔗糖
    1 M HEPES(pH 7.2)
    0.25 M EDTA
    1 M DTT
    100mM MgCl 2/v/v 在80ml水中加入以下试剂:



    添加10片完整,微型,无EDTA的蛋白酶抑制剂鸡尾酒片 混合好,调整体积到100 ml

致谢

该协议已经被改编和修改以使用在拟南芥中,来自Jiang和Zhang(2002)。 这项工作得到了Samuel Roberts Noble基金会的支持。

参考文献

  1. Able,A.J.,Guest,D.I。和Sutherland,M.W。(1998)。 使用   的新的基于四氮唑的测定来研究超氧化物的生产 通过用无毒游动孢子攻击的烟草细胞培养物的自由基   phytophthora parasitica var Nicotianae 植物生理学 491-499。
  2. Jiang,M。和Zhang,J。(2002)。 参与  的血浆膜NADPH氧化酶在脱落酸和水中 胁迫诱导的玉米幼苗叶片中的抗氧化防御。 215(6):1022-1030。
  3. Rojas,C.M.,Senthil-Kumar,M.,Wang,K.,Ryu,C.M.,Kaundal,A.and Mysore,K.S.(2012)。 乙醇酸氧化酶调节本氏烟草中非宿主抵抗期间的反应性氧物质介导的信号转导,植物细胞 24(1):336-352。
  4. Sagi,M。和Fluhr,R。(2001)。 由植物同系物的gp91(phox)NADPH氧化酶产生的超氧化物。通过钙和烟草花叶病毒感染调节活性。 植物生理学 126(3):1281-1290。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kaundal, A., Rojas, C. M. and Mysore, K. S. (2012). Measurement of NADPH Oxidase Activity in Plants. Bio-protocol 2(20): e278. DOI: 10.21769/BioProtoc.278.
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a short summery of limitation of mtt and xtt for measuring cell viability
thanks
11/8/2012 2:10:55 PM Reply