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Measurement of ATP Hydrolytic Activity of Plasma Membrane H+-ATPase from Arabidopsis thaliana Leaves
拟南芥叶片质膜上H+-ATP酶的ATP水解活性测定   

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Abstract

Plant plasma membrane H+-ATPase, which is a P-type ATPase, couples ATP hydrolysis to H+ extrusion and thereby generates an electrochemical gradient across the plasma membrane. The proton gradient is necessary for secondary transport, cell elongation, and membrane potential maintenance. Here we describe a protocol for measurement of the ATP hydrolytic activity of the plasma membrane H+-ATPase from Arabidopsis thaliana leaves.

Keywords: Arabidopsis thaliana(拟南芥), ATP hydrolytic activity(ATP水解活性), Orthovanadate(原钒酸盐), P-type ATPase(P型ATP酶), Plasma membrane H+-ATPase(血浆膜H + -ATP酶)

Background

Determination of the plasma membrane H+-ATPase activity is important to elucidate its function and regulatory mechanism. However, it is sometimes difficult to determine the ATP hydrolytic activity of the plasma membrane H+-ATPase, because plant cells contain many ATP hydrolytic enzymes. This protocol is developed based on the publications by Uemura and Yoshida (1986) and Kinoshita et al. (1995). We used KNO3 as an inhibitor of V-type ATPases, ammonium molybdate as an inhibitor of acid phosphatases, oligomycin as an inhibitor of F-type ATPases, and NaF as an inhibitor of phosphatases (Shimazaki and Kondo,1987; Kinoshita et al.,1995). Orthovanadate inhibits the P-type ATPase and thus can be used to measure the activity of the plasma membrane H+-ATPase by assessing the vanadate-sensitive Pi release from ATP hydrolysis. The released Pi reacts with molybdate to form a blue complex which can then be quantified by measuring absorption at 750 nm.

Materials and Reagents

  1. Ultracentrifuge tube (Beckman Coulter, catalog number: 349623 )
  2. Cuvette (100 µl) (Beckman Coulter, catalog number: 523270 )
    Note: This product has been discontinued.
  3. Arabidopsis thaliana ecotype Col-0
  4. Dithiothreitol (DTT) (NACALAI TESQUE, catalog number: 14128-04 )
  5. Phenylmethylsulfonyl fluoride (PMSF) (NACALAI TESQUE, catalog number: 273-27 )
  6. Leupeptin (Wako Pure Chemical Industries, catalog number: 126-03754 )
  7. MOPS (NACALAI TESQUE, catalog number: 23415-54 )
  8. Oligomycin (Sigma-Aldrich, catalog number: 75351 )
  9. Sodium chloride (NaCl) (Wako Pure Chemical Industries, catalog number: 191-01665 )
  10. Ethylenediamine-N,N,N’,N’-tetraacetic acid (EDTA) (Dojindo Molecular Technologies, catalog number: N001-10 )
  11. Sodium fluoride (NaF) (NACALAI TESQUE, catalog number: 31420-82 )
  12. Tris (NACALAI TESQUE, catalog number: 35406-91 )
  13. 2-(N-morpholino)ethanesulfonic acid (MES) (NACALAI TESQUE, catalog number: 21623-26 )
  14. Magnesium sulfate (MgSO4) (Wako Pure Chemical Industries, catalog number: 131-00405 )
  15. Potassium chloride (KCl) (Wako Pure Chemical Industries, catalog number: 163-03545 )
  16. Potassium nitrate (KNO3) (Wako Pure Chemical Industries, catalog number: 160-04035 )
  17. Ammonium molybdate (Wako Pure Chemical Industries, catalog number: 016-06902 )
  18. Triton X-100 (Wako Pure Chemical Industries, catalog number: 169-21105 )
  19. ATP (NACALAI TESQUE, catalog number: 10406-61 )
  20. Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508 )
  21. SDS (NACALAI TESQUE, catalog number: 31607-65 )
  22. Sodium molybdate (Wako Pure Chemical Industries, catalog number: 196-02472 )
  23. Sulfuric acid (H2SO4) (Wako Pure Chemical Industries, catalog number: 192-04696 )
  24. 1-amino-2-naphthol-4-sulfonic acid (ANSA) (NACALAI TESQUE, catalog number: 02212-12 )
  25. Sodium bisulfite (NaHSO3) (Wako Pure Chemical Industries, catalog number: 190-01375 )
  26. Sodium sulfate (Na2SO4) (Wako Pure Chemical Industries, catalog number: 192-03415 )
  27. Potassium dihydrogen phosphate (KH2PO4) (Wako Pure Chemical Industries, catalog number: 169-04245 )
  28. DTT stock solution (see Recipes)
  29. Protease inhibitor solution (see Recipes)
  30. Oligomycin solution (see Recipes)
  31. Homogenization buffer (see Recipes)
  32. 2x ATPase buffer (see Recipes)
  33. ATPase reaction buffer (see Recipes)
  34. ATP solution (see Recipes)
  35. Vanadate solution (see Recipes)
  36. Stop solution (see Recipes)
  37. ANSA solution (see Recipes)
  38. Pi standard stock solution (see Recipes)

Equipment

  1. Mortar (90 mm diameter) and pestle
  2. Refrigerated centrifuge (TOMY DIGITAL BIOLOGY, model: MX-307 )
  3. Ultracentrifuge (Beckman Coulter, model: OptimaTM TLX )
  4. Vortex (Scientific Industry, model: SI-0286 )
  5. Heat block (TAITEC, model: e-ThermoBucket ETB )
  6. Spectrophotometer (Beckman Coulter, model: DU 730 )
    Note: This product has been discontinued.

Procedure

  1. Preparation of microsomal membranes
    1. Grow Arabidopsis thaliana in soil for 3 weeks at 23 °C under white light (50 µmol photons m-2 s-1) with a 16-h-light/8-h-dark cycle.
    2. Homogenize rosette leaves (about 100 mg) with a mortar and pestle in 2 ml ice-cold homogenization buffer and keep on ice.
    3. Centrifuge the homogenate at 13,000 x g for 10 min at 4 °C.
    4. Ultracentrifuge the supernatant at 100,000 x g for 1 h at 4 °C.
    5. Resuspend the pellet in 100 μl ice-cold homogenization buffer by pipetting up and down.
    6. Quantify the protein concentration by Bradford assay (Bradford, 1976).
    7. Protein concentration is adjusted to 0.45 µg/µl with homogenization buffer.
      Note: Keep microsomal membranes on ice until use.

  2. Measurement of vanadate-sensitive ATP hydrolytic activity
    1. Mix 100 μl of microsomal membranes with 100 μl ATPase reaction buffer, split the mixture in two tubes, 90 µl each (20 µg protein), and keep on ice.
    2. To determine vanadate-sensitive ATPase activity, add 2 μl vanadate solution to one tube and an equal volume of 1x ATPase buffer to the other tube.
    3. Add 10 μl ATP solution and gently vortex.
    4. Incubate at 30 °C for 30 min. Gently vortex once after 15 min of incubation.
    5. Add 1 ml stop solution.
    6. Add 50 μl ANSA solution and gently vortex.
    7. Incubate at 24 °C for 30 min. Gently vortex once after 15 min of incubation.
    8. Measure absorption at 750 nm by a spectrophotometer using a cuvette.
      Note: Do not centrifuge the samples.

  3. Preparation of Pi standard curve
    1. Prepare Pi dilution series as shown in Table 1.

      Table 1. Template for the preparation of the Pi standard curve


    2. Add 50 μl ATPase reaction buffer and 1 ml stop solution.
    3. Add 50 μl ANSA solution and incubate at 24 °C for 30 min. Gently vortex once after 15 min of incubation.
    4. Measure absorption at 750 nm using a cuvette and make a standard curve (Figure 1).

Data analysis

A typical Pi standard curve is shown in Figure 1. Calculate Pi content of samples using the standard curve. Vanadate-sensitive ATP hydrolytic activity is determined by subtracting Pi content in the presence of vanadate from that in the absence of vanadate, and is expressed as nmol Pi/h/mg of protein.

  1. Calculate a slope of Pi standard curve.
    A750 = 0.0038 (Pi content [nmol]) + 0.0088
  2. Determine Pi content of samples from the slope.
    (Pi content [nmol]) = (A750 - 0.0088)/0.0038
  3. Subtract Pi content in the presence of vanadate from that in the absence of vanadate.
  4. Divide by the amount of protein (mg), and reaction time (h). The following is an example of calculation.
    Pi content in the absence of vanadate = 10 nmol
    Pi content in the presence of vanadate = 5 nmol
    Reaction time = 0.5 h (30 min)
    Amount of protein = 0.02 mg (20 µg)
    Vanadate-sensitive ATP hydrolytic activity (nmol Pi/h/mg of protein)
    = ([Pi content in the absence of vanadate] - [Pi content in the presence of vanadate])/(reaction time)/(Amount of protein)
    = (10 - 5)/0.5/0.02 = 500 nmol Pi/h/mg of protein


    Figure 1. Standard curve generated by using known amounts of Pi. The absorption at 750 nm was measured.

Recipes

  1. DTT stock solution
    1 M DTT in sterile water
    Store at -20 °C in small aliquots
  2. Protease inhibitor solution
    200 mM PMSF and 4 mM leupeptin in DMSO
    Store at 4 °C in small aliquots
  3. Oligomycin solution
    5 mg/ml oligomycin in DMSO
    Store at -20 °C in small aliquots
  4. Homogenization buffer
    50 mM MOPS-KOH (pH 7.5)
    100 mM NaCl
    2.5 mM EDTA
    10 mM NaF
    5 mM DTT
    1 mM PMSF
    20 μM leupeptin
    Store at 4 °C. Add NaF, DTT, PMSF, and leupeptin just before use
  5. 2x ATPase buffer
    60 mM Tris-MES (pH 6.5)
    6 mM MgSO4
    100 mM KCl
    200 mM KNO3
    Store at 4 °C
  6. ATPase reaction buffer (freshly prepared)
    2x ATPase buffer supplemented with:
    1 mM ammonium molybdate
    10 μg/ml oligomycin
    0.1% (w/w) Triton X-100
    0.5 mM PMSF
    10 μM leupeptin
  7. ATP solution
    20 mM ATP in 1x ATPase buffer
    Solution should be aliquoted in small volumes to avoid freeze-thawing and can be stored at -80 °C for at least 6 months
  8. Vanadate solution (freshly prepared)
    10 mM sodium orthovanadate in 1x ATPase buffer
  9. Stop solution (freshly prepared)
    1.3% (w/v) SDS
    0.25% (w/v) sodium molybdate
    0.3 N H2SO4
  10. ANSA solution (freshly prepared)
    0.125% (w/v) 1-amino-2-naphthol-4-sulfonic acid (ANSA)
    15% (w/v) NaHSO3
    1% (w/v) Na2SO4
  11. Pi standard stock solution (freshly prepared)
    5 mM KH2PO4

Acknowledgments

This protocol is adapted from Okumura et al. (2016). This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (15H05956 and 15H04386 to T.K.) and by a Grant-in-Aid for JSPS fellows (253307 to M.O.).

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(1-2): 248-254.
  2. Kinoshita, T., Nishimura, M. and Shimazaki, K. (1995). Cytosolic concentration of Ca2+ regulates the plasma membrane H+-ATPase in guard cells of fava bean. Plant Cell 7(8): 1333-1342.
  3. Okumura, M., Inoue, S., Kuwata, K. and Kinoshita, T. (2016). Photosynthesis activates plasma membrane H+-ATPase via sugar accumulation. Plant Physiol 171(1): 580-589.
  4. Shimazaki, K. I. and Kondo, N. (1987). Plasma membrane H+-ATPase in guard-cell protoplasts from Vicia faba L. Plant Cell Physiol 28(5): 893-900.
  5. Uemura, M. and Yoshida, S. (1986). Studies on freezing injury in plant cells: II. Protein and lipid changes in the plasma membranes of Jerusalem artichoke tubers during a lethal freezing in vivo. Plant Physiol 80(1): 187-195. 

简介

作为P型ATP酶的植物质膜H sup + -ATPase将ATP水解耦合到H + +/- 挤出,从而在质膜上产生电化学梯度。质子梯度对于二次转运,细胞伸长和膜电位维持是必需的。这里我们描述用于测量来自拟南芥叶片的质膜H + -ATPase的ATP水解活性的方案。
关键词: strong> 拟南芥,ATP水解活性,原钒酸盐,P-型ATP酶,血浆膜H + -ATPase

质膜H + -ATPase活性的测定对于阐明其功能和调节机制是重要的。然而,有时难以确定质膜H sup + -ATP酶的ATP水解活性,因为植物细胞含有许多ATP水解酶。该协议是基于Uemura和Yoshida(1986)和Kinoshita等人的出版物开发的。 (1995)。我们使用KNO 3作为V型ATP酶的抑制剂,钼酸铵作为酸性磷酸酶的抑制剂,寡霉素作为F型ATP酶的抑制剂,NaF作为磷酸酶的抑制剂(Shimazaki和Kondo ,1987; Kinoshita等人,1995)。原钒酸盐抑制P型ATP酶,并且因此可以通过评估来自ATP水解的钒酸盐敏感性P 1释放来用于测量质膜H sup + -ATP酶的活性。释放的P 1与钼酸盐反应形成蓝色络合物,然后可以通过测量在750nm的吸收来量化。

关键字:拟南芥, ATP水解活性, 原钒酸盐, P型ATP酶, 血浆膜H + -ATP酶

材料和试剂

  1. 超声离心管(Beckman Coulter,目录号:349623)
  2. 比色杯(100μl)(Beckman Coulter,目录号:523270)
    注意:此产品已停产。
  3. 拟南芥生态型Col-0
  4. 二硫苏糖醇(DTT)(NACALAI TESQUE,目录号:14128-04)
  5. 苯基甲基磺酰氟(PMSF)(NACALAI TESQUE,目录号:273-27)
  6. 亮光素(Wako Pure Chemical Industries,目录号:126-03754)
  7. MOPS(NACALAI TESQUE,目录号:23415-54)
  8. 寡霉素(Sigma-Aldrich,目录号:75351)
  9. 氯化钠(NaCl)(Wako Pure Chemical Industries,目录号:191-01665)
  10. 乙二胺-N,N,N',N'-四乙酸(EDTA)(Dojindo Molecular Technologies,目录号:N001-10)
  11. 氟化钠(NaF)(NACALAI TESQUE,目录号:31420-82)
  12. Tris(NACALAI TESQUE,目录号:35406-91)
  13. 2-(N-吗啉代)乙磺酸(MES)(NACALAI TESQUE,目录号:21623-26)
  14. 硫酸镁(MgSO 4)(Wako Pure Chemical Industries,目录号:131-00405)
  15. 氯化钾(KCl)(Wako Pure Chemical Industries,目录号:163-03545)
  16. 硝酸钾(KNO 3)(Wako Pure Chemical Industries,目录号:160-04035)
  17. 钼酸铵(Wako Pure Chemical Industries,目录号:016-06902)
  18. Triton X-100(Wako Pure Chemical Industries,目录号:169-21105)
  19. ATP(NACALAI TESQUE,目录号:10406-61)
  20. 原钒酸钠(Sigma-Aldrich,目录号:S6508)
  21. SDS(​​NACALAI TESQUE,目录号:31607-65)
  22. 钼酸钠(Wako Pure Chemical Industries,目录号:196-02472)
  23. 硫酸(H 2 SO 4)(Wako Pure Chemical Industries,目录号:192-04696)
  24. 1-氨基-2-萘酚-4-磺酸(ANSA)(NACALAI TESQUE,目录号:02212-12)
  25. 亚硫酸氢钠(NaHSO 3)(Wako Pure Chemical Industries,目录号:190-01375)
  26. 硫酸钠(Na 2 SO 4)(Wako Pure Chemical Industries,目录号:192-03415)
  27. 磷酸二氢钾(KH 2 PO 4)(Wako Pure Chemical Industries,目录号:169-04245)
  28. DTT储备溶液(见配方)
  29. 蛋白酶抑制剂溶液(参见配方)
  30. 寡霉素溶液(见配方)
  31. 均质缓冲液(参见配方)
  32. 2x ATPase缓冲液(见配方)
  33. ATPase反应缓冲液(参见配方)
  34. ATP溶液(参见配方)
  35. 钒酸盐溶液(参见配方)
  36. 停止解决方案(参见配方)
  37. ANSA解决方案(参见配方)
  38. Pi标准储备溶液(见配方)

设备

  1. 砂浆(直径90mm)和研钵
  2. 冷冻离心机(TOMY DIGITAL BIOLOGY,型号:MX-307)
  3. 超速离心机(Beckman Coulter,型号:OptimaTM TLX)
  4. Vortex(科学工业,型号:SI-0286)
  5. 热块(TAITEC,型号:e-ThermoBucket ETB)
  6. 分光光度计(Beckman Coulter,型号:DU 730)
    注意:此产品已停产。

程序

  1. 微粒体膜的制备
    1. 在23℃下在白光(50μmol光子m -1 -2 s -1 s -1 -1 )下在土壤中生长拟南芥 3周, -h光/8小时黑暗周期。
    2. 用研钵和研杵在2ml冰冷的匀浆缓冲液中匀化玫瑰花叶(约100mg),并保持在冰上。
    3. 在4℃下将匀浆在13,000×g离心10分钟
    4. 在4℃下将上清液在100,000xg下超速离心1小时
    5. 通过上下吹吸,将沉淀重悬在100μl冰冷的匀浆缓冲液中
    6. 通过Bradford测定法(Bradford,1976)定量蛋白质浓度
    7. 用匀浆缓冲液将蛋白质浓度调整至0.45μg/μl 注意:将微粒体膜保存在冰上直到使用。

  2. 钒酸盐敏感性ATP水解活性的测量
    1. 混合100微升的微粒体膜与100微升ATP酶反应缓冲液,分开混合物在两个管,每个90微升(20微克蛋白质),并保持在冰上。
    2. 为了确定钒酸盐敏感性ATP酶活性,将2μl钒酸盐溶液加入一个试管中,并向另一试管加入等体积的1×ATP酶缓冲液。
    3. 加入10μlATP溶液,轻轻涡旋
    4. 在30℃孵育30分钟。孵育15分钟后轻轻涡旋一次。
    5. 加入1ml终止液。
    6. 加入50μlANSA溶液,轻轻涡旋
    7. 在24℃孵育30分钟。孵育15分钟后轻轻涡旋一次。
    8. 使用比色杯通过分光光度计测量750nm处的吸光度 注意:不要离心样品。

  3. 制备Pi标准曲线
    1. 准备Pi稀释系列,如表1所示。

      表1.用于准备Pi标准曲线的模板


    2. 加入50μlATP酶反应缓冲液和1ml终止液
    3. 加入50微升ANSA溶液,并在24°C孵育30分钟。孵育15分钟后轻轻涡旋一次。
    4. 使用比色杯测量750nm处的吸光度并制作标准曲线(图1)。

数据分析

典型的Pi标准曲线如图1所示。使用标准曲线计算样品的Pi含量。钒酸盐敏感性ATP水解活性通过从不存在钒酸盐的条件下减去钒酸盐存在下的P 1含量来确定,并表示为nmol Pi/h/mg蛋白质。

  1. 计算Pi标准曲线的斜率。
    A <750> = 0.0038(Pi含量[nmol])+ 0.0088
  2. 从斜率确定样品的Pi含量 (Pi含量[nmol])=(A 750〜0.0088)/0.0038
  3. 在没有钒酸盐的情况下,从钒酸盐的存在中减去Pi含量
  4. 除以蛋白质的量(mg)和反应时间(h)。以下是计算示例。
    在钒酸盐不存在下的Pi含量= 10nmol
    在钒酸盐存在下的Pi含量= 5nmol
    反应时间= 0.5小时(30分钟)
    蛋白质量= 0.02mg(20μg)
    钒酸盐敏感性ATP水解活性(nmol Pi/h/mg蛋白质)
    =([不存在钒酸盐时的Pi含量] - [钒酸盐存在时的Pi含量])/(反应时间)/(蛋白质量)
    =(10-5)/0.5/0.02=500nmol Pi/h/mg蛋白质


    图1.使用已知量的Pi产生的标准曲线。测量750nm处的吸收。

食谱

  1. DTT储备液
    1 M DTT在无菌水中
    以小量等分试样保存于-20°C
  2. 蛋白酶抑制剂溶液
    200mM PMSF和4mM亮抑酶肽的DMSO溶液 在4℃下以小等分保存
  3. 寡霉素溶液
    5mg/ml寡霉素的DMSO溶液 以小量等分试样保存于-20°C
  4. 均匀化缓冲液
    50mM MOPS-KOH(pH7.5) 100 mM NaCl
    2.5mM EDTA
    10mM NaF 5 mM DTT
    1mM PMSF
    20μM亮肽素 储存于4°C。在使用前加入NaF,DTT,PMSF和亮抑酶肽
  5. 2x ATPase缓冲液
    60mM Tris-MES(pH6.5) 6mM MgSO 4 100 mM KCl
    200mM KNO 3
    存储在4°C
  6. ATPase反应缓冲液(新鲜制备)
    2x ATP酶缓冲液,补充有:
    1mM钼酸铵 10μg/ml寡霉素 0.1%(w/w)Triton X-100 0.5 mM PMSF
    10μM亮肽素
  7. ATP溶液
    20mM ATP,在1x ATP酶缓冲液中 溶液应小份分装,以避免冻融,并可在-80℃下储存至少6个月。
  8. 钒酸盐溶液(新鲜制备)
    10mM原钒酸钠的1x ATP酶缓冲液中
  9. 停止溶液(新鲜制备)
    1.3%(w/v)SDS
    0.25%(w/v)钼酸钠 0.3 N H 2 SO 4子< br />
  10. ANSA解决方案(新鲜制备)
    0.125%(w/v)1-氨基-2-萘酚-4-磺酸(ANSA) 15%(w/v)NaHSO 3·
    1%(w/v)Na 2 SO 4 4 /
  11. Pi标准储备溶液(新鲜制备)
    5mM KH 2 PO 4 sub/

致谢

该协议改编自Okumura等人。 (2016年)。这项工作得到了日本教育,文化,体育,科学和技术部科学研究助理(15H05956和15H04386到TK)和JSPS研究员资助(253307到MO)。

参考文献

  1. Bradford,MM(1976)。  快速敏感的方法用于利用蛋白质 - 染料结合原理定量微克数量的蛋白质。 Anal Biochem 72(1-2):248-254。
  2. Kinoshita,T.,Nishimura,M.and Shimazaki,K.(1995)。  Ca 2+的胞质浓度调节蚕豆保卫细胞中的质膜H sup + -ATPase。 植物细胞 7(8):1333-1342。
  3. Okumura,M.,Inoue,S.,Kuwata,K.and Kinoshita,T.(2016)。  光合作用通过糖积累激活质膜H + -ATPase 植物生理学171(1):580- 589.
  4. Shimazaki,KI和Kondo,N。(1987)。  + -ATP酶。植物细胞生理学 28(5) :893-900。
  5. Uemura,M。和Yoshida,S。(1986)。  研究植物细胞中的冻伤:II。在体内致死冷冻期间,菊芋块茎的质膜中的蛋白质和脂质变化。 植物生理学80(1):187-195。
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Copyright: © 2016 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. Okumura, M. and Kinoshita, T. (2016). Measurement of ATP Hydrolytic Activity of Plasma Membrane H+-ATPase from Arabidopsis thaliana Leaves. Bio-protocol 6(23): e2044. DOI: 10.21769/BioProtoc.2044.
  2. Kinoshita, T., Nishimura, M. and Shimazaki, K. (1995). Cytosolic concentration of Ca2+ regulates the plasma membrane H+-ATPase in guard cells of fava bean. Plant Cell 7(8): 1333-1342.
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