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Extraction and Quantification of Poly P, Poly P Analysis by Urea-PAGE
提取和量化多磷酸盐以及使用尿素聚丙烯酰胺凝胶(Urea-PAGE)对其进行分析

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Abstract

Inorganic polyphosphate (poly P) molecules, linear chains containing hundreds of orthophosphate (Pi) residues linked by high-energy phosphoanhydride bonds are abundant in every cell in nature. These molecules are widely distributed among bacteria, including key pathogens, and eukaryotes, poly P is present in organelles, including nuclei, mitochondria, and vesicles.  

Remarkable properties of this molecule as a polyanion have been discovered and have made it suited for a crucial role in the emergence of cells on earth. Poly P is essential for bacterial responses to stresses and starvation, motility, quorum sensing, biofilm formation, and virulence and essential for survival. Polymers of different lengths are present in different locations and have different roles in the cell.

Keywords: Poly P(聚磷), Poly P Urea-PAGE Analysis(聚磷脲页分析), Poly P Quantification(聚磷定量)

Materials and Reagents

  1. [γ-32P] ATP, [32P] (Amersham Biosciences)
  2. Poly P (types 15 and 75) (apyrase, and common chemicals) (Sigma-Aldrich)
  3. Poly P35, Poly P50, Poly P300, and Poly P750
  4. Polyphosphatase (PPX) isolated from yeast
  5. Polyphosphate kinase (PPK) isolated from Escherichia coli (E. coli) (EcPPK)
  6. Tris-HCl (pH 8.0)
  7. Ammonium sulfate
  8. Toluidine blue
  9. 25% methanol
  10. 5% glycerol
  11. 5x sample buffer (see Recipes)
  12. THK buffer (see Recipes)
  13. Elution buffer (see Recipes)
  14. Aacrylamide solution (see Recipes)

Equipment

  1. DE81 disk
  2. Monolight 2010 (Analytical Luminescence Laboratory)
  3. Topcount (Packard Instruments)
  4. Microfuge (14,000 rpm or 16,000 x g)
  5. Phosphorimager (Molecular Dynamics) (conventional)
  6. Kodak X-Omat AR film

Procedure

Note: This protocol can be suitable for any kind of extract. The amount of sample for poly P quantification depends on the total amount of poly P in the sample and the right dilution should be determined experimentally.

  1. Extraction and quantification
    1. Acid-soluble and salt-soluble poly P fractions were extracted at 4 °C with 0.5 N HClO4 and a saturated solution of NaClO4 in 1 N HClO4, respectively.
    2. The remaining biomass was treated with 0.5 N HClO4 for 30 min at 90 °C, and the level of the acid-insoluble poly P fraction was measured as the amount of released Pi (Vagabov et al., 2000).
    3. Nucleotides were removed from the acid-soluble fraction by adsorption to Norit A charcoal (Ault-Riche et al., 1998).
    4. The levels of poly P in the acid-soluble and salt-soluble poly P fractions were calculated as a difference in the P amount before and after hydrolysis of samples in the presence of 1 N HCl for 10 min at 100 °C (labile phosphorus).
    5. The DE81 filter paper treatment was used in parallel to estimate the amount of poly P (Rao et al., 1998).
    6. An aliquot of the cell lysate (20 μl) was loaded onto a DE81 disk (1.3 cm diameter) and set on a paper towel for 2 to 5 min to dry.
    7. Each disk was washed three times by shaking in 10 ml of THK buffer.
    8. Partially air-dried disks were then placed in a 0.5-ml Eppendorf tube without the cap and with a tiny hole at the bottom; the tube with disk was then placed inside a 1.5-ml Eppendorf tube.
    9. Poly P was eluted with 50 μl of elution buffer by soaking the disk in the buffer for 10 min at room temperature and then centrifuging the tubes for 5 sec at 14,000 rpm in a microfuge; elution with 50 μl of buffer was repeated three times.
    10. To the combined 200 μl of eluate was added 2 μl of poly P, type P65 (20 μg), and 6 μl of a 15% Norit suspension in water.
    11. The suspension was mixed and centrifuged for 5 min at 14,000 rpm in a microfuge.
    12. The supernatant was transferred to a fresh Eppendorf tube and the pellet was washed twice with 400 μl of 10 mM Tris-HCl buffer (pH 8.0).
    13. The supernatants were pooled to yield a total volume of 1.0 ml, which constituted the purified poly P extract.
    14. Protein levels were determined using bovine serum albumin as a standard.
    15. To estimate total Poly P levels we used the method of Ault-Riché (Ault-Riche et al., 1998; Rao et al., 1998), in which the enzyme assay is performed with purified PPK from E. coli (Ec-PPK); in the presence of ADP and poly P, ATP is generated and measured using a luminescence assay as described in (Ault-Riche and Kornberg, 1999).

  2. Poly P assay
    1. 0.1 ml reaction mixture
      50 mM Tris-HCl (pH 7.4)
      40 mM ammonium sulfate
      4 mM MgCl2
      5 μM ADP
      24,000 U of Ec-PPK (pure enzyme)
      Incubated at 37 °C for 40 min and then at 90 °C for 2 min.
    2. The reaction mixture was diluted 1:100 in 100 mM Tris-HCl (pH 8.0) - 4 mM EDTA, of which 0.1 ml was added to 0.1 ml of luciferase reaction mixture.
    3. Luminescence was measured by using a luminometer (Monolight 2010 or Topcount).
    4. A standard curve for ATP (0, 0.55, 1.1, 1.65, and 3.3 pmol) in 100 mM Tris-HCl (pH 8.0) - 4 mM EDTA was used for comparison.
    5. Concentration of poly P is given in terms of Pi residues. [32P] poly P values are based on specific radioactivity and susceptibility to hydrolysis with PPX.

  3. Poly P analysis by urea-PAGE
    1. Urea-polyacrylamide gels were prepared by mixing:
      10.51 g of urea
      3.75 ml of acrylamide solution
      2.5 ml of 0.9 M Tris borate (pH 8.3)
      27 mM EDTA, to a final volume of 25 ml
      Ammonium persulfate (15 μg) and TEMED (25 μl)
    2. The gel was poured (14 x 23 x 0.75 mm) and allowed to polymerize.
    3. Gels of other strengths were made with appropriate volumes of the stock acrylamide solution. The gel was pre-electrophoresed at 300 V for 1 h.
    4. Poly P was mixed with 0.25 volume of 5x sample buffer and loaded on the gel.
    5. Electrophoresis was at 300 V until the dye was 7 to 8 cm from the top of the gel.
    6. Gels were stained with:
      0.05% toluidine blue in 25% methanol and 5% glycerol for 20 min followed by destaining in the same solvent without toluidine blue.
    7. Gels containing radioactivity were analyzed on a Phosphorimager and autoradiography on Kodak X-Omat AR film (Kumble and Kornberg, 1995).
    8. For determination of chain lengths, marker poly P of various sizes was electrophoresed along with the samples and the markers localized by toluidine blue staining (Figure 1).
    9. Centimeters migrated versus chain length was plotted, the mobility of the sample was measured, and its chain length was determined by extrapolating from the plot.


    Figure 1. Poly P products of DdPPK1. Poly P synthesis with 40 ng of DdPPK1 was carried out with [γ-32P] ATP under the optimal conditions for 30 min. Products were purified, treated with exopolyphosphatase (PPX) (as described in Materials and Reagents) and separated by 20% PAGE containing 7 M urea, and visualized by PhosphorImager. P750 is a purified product of EcPPK1, which has a chain length of ≈ 750 residues. P300, P50, and P30 indicate the position of poly P with different chain lengths. PPi, pyrophosphate. (A) Poly P products before (−) and after (+) PPX-treatment. (B) The region between poly P750 and PPi from gel A is magnified to highlight the chain lengths of poly P. (Zhang et al., 2007)

Notes

The poly P analysis in Synechococcus is just like extracting poly P from E. coli. Preparation of a cell lysate by sonication, Synechococcus has a lot of poly P, so, prepare 1/10 and 1/100 dilutions and follow step A4 above. This is true as well for any other prokaryotes with high poly P content.

Recipes

  1. 5x sample buffer
    50% sucrose
    0.125% bromophenol blue
    450 mM Tris borate at pH 8.3
    13.5 mM EDTA
  2. THK buffer
    10 mM Tris-HCl (pH 8.0)
    100 mM KCl
    5 mM KPO4 (pH 8.0)
  3. Elution buffer
    10 mM Tris-HCl (pH 8.0)
    500 mM KCl
  4. Aacrylamide solution
    38 g of acrylamide and 2 g of bisacrylamide dissolved in deionized water to a final volume of 100 ml

Acknowledgments

This protocol is adapted from Ault-Riche et al. (1998); Ault-Riche et al. (1999); Rao et al. (1998); and Zhang et al. (2007).

References

  1. Ault-Riche, D. and Kornberg, A. (1999). Definitive enzymatic assays in polyphosphate analysis. Prog Mol Subcell Biol 23: 241-252.
  2. Ault-Riche, D., Fraley, C. D., Tzeng, C. M. and Kornberg, A. (1998). Novel assay reveals multiple pathways regulating stress-induced accumulations of inorganic polyphosphate in Escherichia coli. J Bacteriol 180(7): 1841-1847.
  3. Kumble, K. D. and Kornberg, A. (1995). Inorganic polyphosphate in mammalian cells and tissues. J Biol Chem 270(11): 5818-5822.
  4. Rao, N. N., Liu, S. and Kornberg, A. (1998). Inorganic polyphosphate in Escherichia coli: the phosphate regulon and the stringent response. J Bacteriol 180(8): 2186-2193.
  5. Vagabov, V. M., Trilisenko, L. V. and Kulaev, I. S. (2000). Dependence of inorganic polyphosphate chain length on the orthophosphate content in the culture medium of the yeast Saccharomyces cerevisiae. Biochemistry (Mosc) 65(3): 349-354.
  6. Zhang, H., Gomez-Garcia, M. R., Shi, X., Rao, N. N. and Kornberg, A. (2007). Polyphosphate kinase 1, a conserved bacterial enzyme, in a eukaryote, Dictyostelium discoideum, with a role in cytokinesis. Proc Natl Acad Sci U S A 104(42): 16486-16491.

简介

无机多磷酸盐(poly P)分子,包含通过高能量磷酸氢键连接的数百个正磷酸(Pi)残基的线性链在自然界中的每个细胞中是丰富的。 这些分子广泛分布在细菌中,包括关键的病原体和真核生物,poly P存在于细胞器中,包括细胞核,线粒体和囊泡。

此分子作为聚阴离子的显着性质 发现并使其适合在地球上细胞出现中的关键作用。 Poly P对于应激和饥饿,运动,群体感应,生物膜形成和毒力的细菌应答和对于存活必不可少。 不同长度的聚合物存在于不同的位置,并在池中具有不同的作用

关键字:聚磷, 聚磷脲页分析, 聚磷定量

材料和试剂

  1. [γ-32 P] ATP,[32] P](Amersham Biosciences)
  2. Poly P(类型15和75)(腺苷三磷酸双磷酸酶和常见化学品)(Sigma-Aldrich)
  3. Poly P Sub 35,Poly P 50,Poly P 300和Poly P 750中的至少一种。
  4. 从酵母中分离的多磷酸酶(PPX)
  5. 从大肠杆菌(大肠杆菌)(EcPPK)分离的多磷酸激酶(PPK)
  6. Tris-HCl(pH 8.0)
  7. 硫酸铵
  8. 甲苯胺蓝
  9. 25%甲醇
  10. 5%甘油
  11. 5x样品缓冲液(见配方)
  12. THK缓冲区(参见配方)
  13. 洗脱缓冲液(见配方)
  14. 丙烯酰胺溶液(参见配方)

设备

  1. DE81磁盘
  2. Monolight 2010(分析发光实验室)
  3. Topcount(Packard Instruments)
  4. Microfuge(14,000 rpm或16,000 x g )
  5. 磷光体(分子动力学)(常规)
  6. 柯达X-Omat AR电影

程序

注意: 此协议可以适用于任何类型的提取。 用于聚P定量的样品量取决于样品中多聚P的总量,并且实验应确定正确的稀释度。

  1. 提取和定量
    1. 在4℃下提取酸溶性和盐溶性聚P级分 与0.5N HClO 4和在1N HClO 4中的NaClO 4的饱和溶液混合, 分别。
    2. 剩余的生物质在90℃下用0.5N HClO 4处理30分钟, 并测量酸不溶性聚P部分的水平 释放的量(Vagabov等人,2000)。
    3. 通过吸附到Norit A炭上从酸可溶性级分中去除核苷酸(Ault-Riche等人,1998)。
    4. 在酸溶性和盐溶性聚P中聚P的水平 分数计算为P值之前和之后的差异 在100℃下在1N HCl存在下水解样品10分钟   ℃(不稳定磷)。
    5. 并行使用DE81滤纸处理以估计聚P的量(Rao等人,1998)。
    6. 将细胞裂解物的等分试样(20μl)加载到DE81盘(1.3μm)上   cm直径)并置于纸巾上2至5分钟以干燥。
    7. 通过在10ml THK缓冲液中振荡将每个盘洗涤三次。
    8. 然后将部分空气干燥的盘置于0.5-ml Eppendorf管中 没有盖子和在底部有一个小孔; 管带盘 然后置于1.5-ml Eppendorf管中。
    9. 聚P 通过将盘浸泡在缓冲液中用50μl洗脱缓冲液洗脱 在室温下孵育10分钟,然后将管离心5秒 在微型离心机中以14,000rpm; 重复用50μl缓冲液洗脱 三次。
    10. 向合并的200μl洗脱液中加入2μl 的P型P 65型(20μg)和6μl的15%Norit悬浮液 水。
    11. 将悬浮液混合并在微量离心机中以14,000rpm离心5分钟
    12. 将上清液转移到新鲜的Eppendorf管中, 沉淀用400μl10mM Tris-HCl缓冲液(pH8.0)洗涤两次。
    13. 合并上清液以产生1.0ml的总体积,其构成纯化的聚P提取物。
    14. 使用牛血清白蛋白作为标准测定蛋白质水平。
    15. 为了估计总P值,我们使用了Ault-Riché的方法 (Ault-Riche等人,1998; Rao等人,1998),其中酶测定法 用来自E的纯化的PPK进行。 大肠杆菌(Ec-PPK); 在......的存在下   ADP和聚P,ATP,并使用发光测量 如(Ault-Riche和Kornberg,1999)所述。

  2. Poly P测定
    1. 0.1ml反应混合物
      50mM Tris-HCl(pH7.4) 40mM硫酸铵 4mM MgCl 2/
      5μMADP
      24,000U Ec-PPK(纯酶)
      在37℃孵育40分钟,然后在90℃孵育2分钟。
    2. 将反应混合物在100mM Tris-HCl(pH 8.0)-4中以1:100稀释   mM EDTA,其中0.1ml加入0.1ml荧光素酶反应中 混合物。
    3. 使用发光计(Monolight 2010或Topcount)测量发光。
    4. ATP(0,0.55,1.1,1.65和3.3pmol)在100mM中的标准曲线 Tris-HCl(pH8.0)-4mM EDTA用于比较。
    5. 聚P的浓度以P 1i残基的形式给出。 p P 值基于特定的放射性和易感性 用PPX水解。

  3. 通过尿素-PAGE的聚P分析
    1. 通过混合以下物质制备脲 - 聚丙烯酰胺凝胶:
      10.51克尿素 3.75ml丙烯酰胺溶液 2.5ml 0.9M Tris硼酸盐(pH8.3) 27mM EDTA,至终体积为25ml
      过硫酸铵(15μg)和TEMED(25μl)
    2. 将凝胶倒入(14×23×0.75mm)中并使其聚合。
    3. 其他强度的凝胶用适当体积的 股票丙烯酰胺溶液。 将凝胶在300V下预电泳1   H。
    4. Poly P与0.25体积的5x样品缓冲液混合,并装载在凝胶上
    5. 电泳在300V下进行,直到染料距离凝胶顶部7至8cm。
    6. 凝胶用:
      染色 0.05%   甲苯胺蓝在25%甲醇和5%甘油中的溶液处理20分钟,随后进行 在不含甲苯胺蓝的相同溶剂中脱色。
    7. 凝胶 在Phosphorimager上分析含荧光的放射性 在Kodak X-Omat AR膜上的放射自显影(Kumble和Kornberg,1995)。
    8. 为了测定链长,使用各种大小的标记聚P   连同样品和被定位的标记一起电泳 甲苯胺蓝染色(图1)。
    9. 绘制厘米迁移的对比链长度,样品的迁移率 测量,并且其链长度通过从的外推确定
    10. 凝胶用:
      染色 0.05%   甲苯胺蓝在25%甲醇和5%甘油中的溶液处理20分钟,随后进行 在不含甲苯胺蓝的相同溶剂中脱色。
    11. 凝胶 在Phosphorimager上分析含荧光的放射性 在Kodak X-Omat AR膜上的放射自显影(Kumble和Kornberg,1995)。
    12. 为了测定链长,使用各种大小的标记聚P   连同样品和被定位的标记一起电泳 甲苯胺蓝染色(图1)。
    13. 绘制厘米迁移的对比链长度,样品的迁移率 测量,并且其链长度通过从的外推确定... Recipes

      1. 5x sample buffer
        50% sucrose
        0.125% bromophenol blue
        450 mM Tris borate at pH 8.3
        13.5 mM EDTA
      2. THK buffer
        10 mM Tris-HCl (pH 8.0)
        100 mM KCl
        5 mM KPO4 (pH 8.0)
      3. Elution buffer
        10 mM Tris-HCl (pH 8.0)
        500 mM KCl
      4. Aacrylamide solution
        38 g of acrylamide and 2 g of bisacrylamide dissolved in deionized water to a final volume of 100 ml

      Acknowledgments

      This protocol is adapted from Ault-Riche et al. (1998); Ault-Riche et al. (1999); Rao et al. (1998); and Zhang et al. (2007).

      References

      1. Ault-Riche, D. and Kornberg, A. (1999). Definitive enzymatic assays in polyphosphate analysis. Prog Mol Subcell Biol 23: 241-252.
      2. Ault-Riche, D., Fraley, C. D., Tzeng, C. M. and Kornberg, A. (1998). Novel assay reveals multiple pathways regulating stress-induced accumulations of inorganic polyphosphate in Escherichia coli. J Bacteriol 180(7): 1841-1847.
      3. Kumble, K. D. and Kornberg, A. (1995). Inorganic polyphosphate in mammalian cells and tissues. J Biol Chem 270(11): 5818-5822.
      4. Rao, N. N., Liu, S. and Kornberg, A. (1998). Inorganic polyphosphate in Escherichia coli: the phosphate regulon and the stringent response. J Bacteriol 180(8): 2186-2193.
      5. Vagabov, V. M., Trilisenko, L. V. and Kulaev, I. S. (2000). Dependence of inorganic polyphosphate chain length on the orthophosphate content in the culture medium of the yeast Saccharomyces cerevisiae. Biochemistry (Mosc) 65(3): 349-354.
      6. Zhang, H., Gomez-Garcia, M. R., Shi, X., Rao, N. N. and Kornberg, A. (2007). Polyphosphate kinase 1, a conserved bacterial enzyme, in a eukaryote, Dictyostelium discoideum, with a role in cytokinesis. Proc Natl Acad Sci U S A 104(42): 16486-16491.
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引用:Garcia, M. R. (2014). Extraction and Quantification of Poly P, Poly P Analysis by Urea-PAGE. Bio-protocol 4(9): e1113. DOI: 10.21769/BioProtoc.1113.
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Ming-Yang Ho
BMMB, Penn State University
Hi Maria,

How much volume of 0.5 N HClO4 and and a saturated solution of NaClO4 in 1 N HClO4 would you use in the first step of extraction when using a certain amount of biomass?

Thanks,

Ming-Yang
6/10/2014 9:24:16 AM Reply
Rosario Gomez-Garcia
Department of Biochemistry, Stanford University School of Medicine, USA

Hello,
The answer depends on how much poly P do you have in your sample, probably I would start using the same volume HClO4 solution/sample.

Regards,

Maria

6/10/2014 11:41:35 PM