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Extraction and Quantification of Polyphosphate in the Budding Yeast Saccharomyces cerevisiae
出芽酿酒酵母中聚磷酸的提取和定量测定   

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Abstract

Inorganic polyphosphate (polyP) is a linear polymer present in both prokaryotic and eukaryotic organisms and made from three to hundreds of orthophosphate residues linked by phosphoanhydride bonds. The biological role of this molecule goes beyond serving as Pi store or energy source to replace ATP. For instance, in yeast polyP levels have been related to stress adaptation and this molecule has been shown to be the substrate for polyphosphorylation of proteins. Here we describe two different methods to purify polyP from the yeast Saccharomyces cerevisiae and the subsequent protocol to quantify polyP levels by spectrophotometrically measuring the Pi generated upon enzymatic hydrolysis of purified polyP. It must be noted that the purification protocol used greatly influences the polyP values obtained.


Figure 1. Enzymatic hydrolysis of polyP

Keywords: Yeast(酵母), Polyphosphate(多聚磷酸盐), Extraction methods(提取方法), Enzymatic phosphate release(酶促磷释放)

Materials and Reagents

  1. 1.5 ml screw cap tubes
  2. Silica-gel columns from QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28706 )
  3. Inoculation loop
  4. Magnetic stirrer
  5. 96-Well assay microplate non-treated clear polystyrene (Thermo Fisher Scientific, NuncTM MicroWellTM, catalog number: 269620 )
  6. Yeast Saccharomyces cerevisiae
  7. Yeast extract, peptone (YP) base medium (Conda, catalog number: 1511 )
  8. AE buffer
  9. Phenol solution (Sigma-Aldrich, catalog number: P4557 )
  10. Sodium dodecylsulfate (SDS) (Panreac AppliChem, catalog number: A7249 )
  11. Chloroform (CHCl3) (Merck Millipore, catalog number: 102445 )
  12. RNase A (100 mg/ml) (QIAGEN, catalog number: 19101 )
  13. DNase I (Roche Diagnostics, catalog number: 10104159001 )
  14. Sodium acetate trihydrate (NaC2H3O2·3H2O) (Merck Millipore, catalog number: 106267 )
  15. Ethanol absolute (CH3CH2OH) (Panreac Applichem, catalog number: 131086 )
  16. Milli-Q water
  17. 98% sulfuric acid (H2SO4) (Merck Millipore, catalog number: 112080 )
  18. Sodium hydroxide (NaOH) (Panreac Applichem, catalog number: 131687 )
  19. Tris (2-amino-2-hydroxymethyl-propane-1,3-diol) buffer (Panreac Applichem, catalog number: A1379 )
  20. Neutral red (Sigma-Aldrich, catalog number: N4638 )
  21. Sodium iodide (NaI) (Sigma-Aldrich, catalog number: 383112 )
  22. Acetic acid glacial (CH3COOH) (Panreac Applichem, catalog number: 131088 )
  23. Disodium ethylenediaminetetraacetate 2-hydrate (EDTA) (Panreac Applichem, catalog number: 131669 )
  24. Recombinant Ppx1 (rPpx1)
    Note: Ppx1 is a S. cerevisiae exopolyphosphatase that hydrolyses polyphosphate into Pi residues. It was obtained from E. coli containing a plasmid-borne, His-tagged version of the PPX1 gene, as described in (Ruiz et al., 2001), after one-step affinity purification with HisTrapTM HP columns (GE Healthcare Life Sciences ).
  25. Potassium dihydrogen phosphate (KH2PO4) (Merck Millipore, catalog number: 104877 )
  26. Sodium phosphate glass Type 45 (polyP45) (Sigma-Aldrich, catalog number: S4379 )
  27. Sodium tripolyphosphate (polyP3) (Sigma-Aldrich, catalog number: 238503 )
  28. Malachite green oxalate salt (Sigma-Aldrich, catalog number: M9015 )
  29. Polyvinyl alcohol (Sigma-Aldrich, catalog number: P1763 )
  30. D-Glucose monohydrate (Panreac Applichem, catalog number: A1349 )
  31. Ammonium acetate (NH4C2H3O2) (Panreac Applichem, catalog number: 131114 )
  32. Magnesium acetate tetrahydrate [Mg(CH3COO)2·4H2O] (Merck Millipore, catalog number: 105819 )
  33. Sodium chloride (NaCl) (Panreac Applichem, catalog number: 131659 )
  34. Ammonium heptamolybdate tetrahydrate [(NH4)6Mo7O24·4H2O] (Sigma-Aldrich, catalog number: 09878 )
  35. YPD (see Recipes)
  36. 3 M sodium acetate (see Recipes)
  37. 0.5 M EDTA (see Recipes)
  38. Buffer AE (see Recipes)
  39. 10% SDS (see Recipes)
  40. RNase A (10 mg/ml) (see Recipes)
  41. DNase I (10 mg/ml) (see Recipes)
  42. 70% ethanol (see Recipes)
  43. 0.1% neutral red solution (see Recipes)
  44. 1 M Tris-HCl (pH 7.5) supplemented to 6% (v/v) with 0.1% neutral red solution (w/v) (see Recipes)
  45. 2 M NaOH (see Recipes)
  46. 1 M sulfuric acid (see Recipes)
  47. 6 M NaI (see Recipes)
  48. 1 M ammonium acetate (see Recipes)
  49. 1 M magnesium acetate (see Recipes)
  50. 5 M NaCl (see Recipes)
  51. Wash buffer (see Recipes)
  52. Polyphosphate assay buffer (see Recipes)
  53. 50 mM potassium dihydrogen phosphate (see Recipes)
  54. Phosphate calibration curve (see Recipes)
  55. 222 µM polyP45 (10 mM Pi) (see Recipes)
  56. 3.3 mM polyP3 (10 mM Pi) (see Recipes)
  57. 6 µM polyP45 (see Recipes)
  58. 200 µM polyP3 (see Recipes)
  59. 28 mM ammonium heptamolybdate in 2.1 M H2SO4 (see Recipes)
  60. 0.76 mM malachite green in 0.35% polyvinyl alcohol (see Recipes)

Equipment

  1. Novaspec Plus spectrophotometer GE
  2. Thermo LabSystems Multiskan Ascents 354 microplate reader
  3. Eppendorf Thermomixer® compact
  4. Vortex mixer (Heidolph)
  5. Centrifuge (Eppendorf, MiniSpin®)

Procedure

  1. Polyphosphate purification by precipitation
    Protocol adapted from (Kumble and Kornberg, 1995)
    1. Inoculate a single colony of the yeast strain with a sterile inoculation loop from a fresh plate into 5 ml of liquid medium (YPD or appropriate selection medium) and incubate overnight on a rotary shaker at 200-220 rpm and 28 °C.
    2. After 16-24 h of growth, determine the OD600 of the yeast culture by using a spectrophotometer. Inoculate cells in 5 ml YPD (final OD600 0.2) and incubate in the shaker at 200-220 rpm and 28 °C until the OD600 reaches 0.6-0.8 (This will take 3 to 5 h.).
    3. Collect from 0.5 to 1 OD600 units of yeast exponential culture (1-2 x 107 cells) by centrifugation at 12,000 x g for 1 min at room temperature and discard the supernatant.
    4. Resuspend the cell pellet with 400 μl of AE buffer prechilled at 4 °C.
    5. Transfer the cell resuspension to previously prepared 1.5 ml screw cap tubes containing 300 µl phenol and 40 μl 10% SDS and securely fasten the lid.
    6. Mix by inversion 4 times and vortex 5 sec to homogenize.
    7. Incubate at 65 °C for 5 min and chill the tubes for 1 min on ice.
    8. Add 300 µl of chloroform, securely fasten the lid, mix by inversion 4 times, and vortex 5 sec to homogenize.
    9. Centrifuge at room temperature for 2 min at 13,000 x g to separate the aqueous phase containing the polyP from the organic phase.
    10. Carefully transfer the aqueous phase (top) to prepared 1.5 ml screw cap tubes containing 350 µl chloroform. Typically, the recovered volume will be approximately 450 µl. Be sure not to carry over any phenol during pipetting by avoid touching the bottom phase or the white protein containing interphase.
    11. Securely fasten the lid, mix by inversion 4 times, and vortex 5 sec to homogenize.
    12. Centrifuge at room temperature for 2 min at 13,000 x g.
    13. Recover the aqueous phase, approximately 400 µl, and transfer it to a new 1.5 ml microcentrifuge tube. As before, be sure not to carry over any phenol during pipetting by avoid touching the bottom phase.
    14. Add 2 µl of RNase A (10 mg/ml) and 2 µl of DNase I (10 mg/ml) to each tube and incubate 1 h at 37 °C. RNase A and DNase I are added before precipitation to degrade RNA and DNA, respectively, thus avoiding RNA and DNA precipitation with polyP.
    15. Transfer the aqueous phase to pre-cold at -20 °C, 1.5 ml microcentrifuge tube containing 1 ml of absolute ethanol and 40 µl of 3M sodium acetate (pH 5.3) and leave 3 h at -20 °C to precipitate polyphosphate.
    16. Centrifuge for 20 min at 13,000 x g at 4 °C.
    17. Discard the supernatant by decantation and add 500 µl of 70% ethanol.
    18. Centrifuge for 5 min at 13,000 x g at 4 °C.
    19. Discard the supernatant by decantation and centrifuge 1 min at 13,000 x g to remove traces of ethanol by pipetting.
    20. Open the tubes and dry the small translucent-white polyphosphate pellet that can be observed at the bottom of the tube at room temperature for 5 min or until the pellet is completely dry.
    21. Resuspend in 50 µl of Milli-Q water.
    22. The polyphosphate sample can be directly measured or stored at -20 °C.

  2. Polyphosphate purification with silica-gel columns
    Protocol adapted from (Werner et al., 2005)
    1. Inoculate a single colony of the yeast strain with a sterile inoculation loop from a fresh plate into 5 ml of liquid medium (YPD or appropriate selection medium) and incubate overnight on a rotary shaker at 200-220 rpm and 28 °C.
    2. After 16-24 h of growth, determine the OD600 of the yeast culture by using a spectrophotometer. Inoculate cells in 5 ml YPD (final OD600 0.2) and incubate in the shaking incubator at 200-220 rpm and 28 °C until the OD600 reaches 0.6-0.8 (This will take 3 to 5 h.).
    3. Collect from 0.5 to 1 OD600 units of yeast exponential culture (1-2 x 107 cells) by centrifugation at 12,000 x g for 1 min at room temperature and discard the supernatant.
    4. Add 50 µl of 1 M sulfuric acid over the pellet of cells, mix and leave 5 min at room temperature.
    5. Neutralize the suspension with 50 µl of 2 M NaOH and add 100 µl of 1 M Tris-HCl (pH 7.5) supplemented to 6% (v/v) with 0.1% neutral red solution (w/v).
    6. Remove the cell debris by centrifugation for 10 min at 800 x g at 4 °C.
    7. Recover 200 µl of supernatant, add 600 µl of 6 M NaI and mix well prior adjusting the pH of the sample. The pH should be around 7 (The sample colour becomes orange-red.). Samples too acidic (pink) or too basic (yellow) have to be corrected by addition of 2 M NaOH or 1 M sulfuric acid, respectively.
      Note: The correct adjustment of pH is a critical step for the reproducibility of polyP extraction.
    8. Apply the 800 µl of polyphosphate extract to the QIAquick purification column and centrifuge the column for 1 min at 13,000 x g at room temperature.
    9. Discard the flow-through and wash the column twice with 400 µl of wash buffer by centrifugation for 1 min at 13,000 x g at room temperature.
    10. After the last wash, discard the flow-through and dry the column by centrifugation for an additional 1 min at 13,000 x g at room temperature.
    11. Transfer the column to new 1.5 ml microcentrifuge tube and add 50 µl Milli-Q water. Let the column stand for 1 min and elute the polyphosphate by centrifugation for 1 min at 13,000 x g at room temperature. It is highly recommended to use Milli-Q water with pH > 7 for a better polyP elution.
    12. The eluted polyphosphate can be directly measured or stored at -20 °C.

  3. Colum regeneration
    If necessary, the columns can be reused after regeneration with the following protocol:
    1. Wash the column once with 750 µl of 0.2 M acetic acid and 50 mM EDTA (pH 8) by centrifugation 1 min at 13,000 x g at room temperature.
    2. Wash three times with 750 µl Milli-Q water by centrifugation 1 min at 13,000 x g each time at room temperature.
    3. Normally, a column can be used 3-5 times. New columns must be used if a decrease in the yield of polyP recovery is observed.

  4. Polyphosphate digestion and plate preparation
    1. Use 96-well plate for the reaction of polyphosphate degradation and the subsequent measure of the released phosphate. An excess of rPpx1 (500 ng of rPpx1 enzyme per reaction) is employed for enzymatic polyphosphate digestion.
    2. Add 90 µl of polyphosphate assay buffer to wells that will contain phosphate standard curve, sample background measurements, and blanks. Add 90 µl of the mix of Polyphosphate assay buffer and rPpx1 enzyme to the sample wells.
    3. Add 10 µl of the sample (duplicate), phosphate standard curve (0 to 500 µM KH2PO4), and positive control consisting in 200 µM polyP3 or 6 µM polyP45 to the 90 µl of Polyphosphate assay buffer. Mix by pipetting up and down several times.
    4. Incubate the plate at 37 °C for 60 min.


      Figure 2. Example plate of polyphosphate measurement. Row A contains the phosphate calibration curve. Rows B and E contain the polyphosphate sample without enzyme (italics) to measure the background and rows C, D, F, & G contain duplicate samples with rPpx1 enzyme. Similarly, the last row contains blanks and positive controls of polyP3 and polyP45 that are incubated with and without enzyme (italics and bold respectively).

  5. Phosphate quantification
    Protocol adapted from (Cogan et al., 1999). Quantification of phosphate is made directly on the PolyP digestion plate
    1. To quantify the released phosphate, add to each well 86 µl of 28 mM ammonium heptamolybdate in 2.1 M H2SO4 and 64 µl of 0.76 mM malachite green in 0.35% polyvinyl alcohol. Mix well by using the shaking option of the microplate reader or by pipetting up and down the samples to ensure a complete mixture of all reagents.
    2. After 20 min at room temperature measure the A595 in a multiplate reader. Do not let the reaction proceed more than 1 h as it can cause appearance of small precipitates in highly concentrated phosphate samples and polyP degradation in background samples, thus interfering with the correct phosphate measure.
    3. The amount of phosphate is obtained by comparing the absorbance value of each sample with the phosphate standard curve after subtracting the background value for each sample. The polyP3 and polyP45 are used as controls for the rPpx1 reaction over polyphosphates.


      Figure 3. Example plate of phosphate quantification

Notes

  1. The main advantage of silica column purification versus the precipitation protocol is that the former allows processing many samples at once. Even more, it is possible to use Qiagen 96 PCR purification plates instated of columns to process a high number of samples. However, the main drawback is that likely short chain polyPs are lost and the amount of PolyP is underestimated in comparison with the precipitation protocol. As a reference, when we used commercial polyP45, that is a mix of different polyP with enrichment in polyP chains with 45 phosphate residues, the polyP recovery using silica columns process was estimated to be 20-22%. Thus, it should be possible to approximately compare PolyP levels obtained by the column method with those generated by the precipitation method by multiplying by a factor of 4. As an example with S. cerevisiae samples, phenol-extracted material (that is, processed until step A14 of the "Polyphosphate purification by precipitation" protocol) was split in 2 halves. One half was processed following the same protocol and the other subjected to purification using the QIAquick columns. As shown in Figure 4, purification through the silica columns results in 3 to 4-fold decrease in the detected polyP levels. Consequently, if the objective of the extraction is to obtain high amounts of polyP of all sizes, the precipitation method is most suitable. A detailed method for assessing polyP size by gel electrophoresis can be found as a BioProtocol (Garcia, 2014).
    In contrast, the precipitation method is more economic, as it does not require purchasing purification columns.


    Figure 4. Comparison of ethanol precipitation and silica column purification methods. The figure shows the amount of Pi per unit of optical density of the culture. Data is presented as the mean ± SD from 5 experiments.

Recipes

  1. YPD
    Mix 30 g YP base medium and 20 g glucose in 500 ml dH2O
    Add dH2O to 1,000 ml and autoclave
  2. 3 M sodium acetate (pH 5.3)
    Mix 40.82 g sodium acetate trihydrate with 70 ml dH2O
    Adjust pH to 5.3 with acetic acid glacial
    Add dH2O to 100 ml and stored at room temperature
  3. 0.5 M EDTA (pH 8)
    Dissolve 18.61 g disodium ethylenediaminetetraacetate 2-hydrate in 80 ml dH2O
    Adjust pH to 8 by adding ~2 g NaOH pellets and stir vigorously on a magnetic stirrer
    Add dH2O to 100 ml and stored at room temperature
  4. Buffer AE [50 mM sodium acetate (pH 5.3), 10 mM EDTA]
    Mix 1.67 ml 3 M sodium acetate (pH 5.3) with 70 ml dH2O
    Add 2 ml 0.5 M EDTA
    Add dH2O to 100 ml and stored at room temperature
  5. 10% SDS
    Mix 10 g sodium dodecylsulfate (SDS) with 80 ml dH2O
    Add dH2O to 100 ml and stored at room temperature
  6. RNase A (10 mg/ml)
    Mix 100 µl RNase A (100 mg/ml) with 900 µl dH2O
    Stored at 4 °C
  7. DNase I (10 mg/ml)
    Mix 50 mg DNase I with 5 ml dH2O
    Stored at 4 °C
  8. 70% ethanol
    Mix 70 ml of ethanol absolute with 30 ml dH2O
    Stored at -20 °C
  9. 0.1% neutral red solution (w/v)
    Mix 0.1 g neutral red with 100 ml 70% ethanol
    Stored at room temperature
  10. 1 M Tris-HCl (pH 7.5) supplemented to 6% (v/v) with 0.1% neutral red solution (w/v)
    Mix 12.1 g Tris with 70 ml dH2O
    Add 6 ml 0.1% neutral red solution (w/v)
    Adjust pH to 7.5 with HCl
    Add dH2O to 100 ml and stored at room temperature
  11. 2 M NaOH
    Mix 8 g NaOH with 80 ml dH2O
    Add dH2O to 100 ml and stored at room temperature
  12. 1 M sulfuric acid (H2SO4)
    Mix 5.45 ml 98% sulfuric acid with 94.55 ml dH2O
    Stored at room temperature
  13. 6 M NaI
    Mix 13.5 g NaI with dH2O up to 15 ml
    Solution has to be freshly prepared each time and protected from light
  14. 1 M ammonium acetate
    Mix 7.71 g ammonium acetate with 80 ml dH2O
    Add dH2O to 100 ml and stored at room temperature
  15. 1 M magnesium acetate
    Mix 21.4 g magnesium acetate tetrahydrate with 70 ml dH2O
    Add dH2O to 100 ml and stored at room temperature
  16. 5 M NaCl
    Mix 29.22 g sodium chloride with 70 ml dH2O
    Add dH2O to 100 ml and stored at room temperature
  17. Wash buffer [10 mM Tris-HCl buffer (pH 7.5), 50% ethanol, 1 mM EDTA and 100 mM NaCl]
    Mix 500 µl 1M Tris-HCl (pH 7.5), 25 ml 100% ethanol, 100 µl 0.5 M EDTA, 1 ml 5 M NaCl, and 20 ml dH2O
    Adjust pH to 7.5 with HCl
    Add dH2O to 50 ml and stored at room temperature
    0.2 M acetic acid and 50 mM EDTA
    Mix 571 µl acetic acid glacial and 5 ml of 0.5 M EDTA
    Add dH2O to 50 ml and stored at room temperature
  18. Polyphosphate assay buffer [20 mM Tris-HCl (pH 7.5), 5 mM magnesium acetate, 100 mM ammonium acetate]
    Mix 2 ml 1 M Tris-HCl (pH 7.5), 500 µl 1 M magnesium acetate, and 10 ml 1 M ammonium acetate
    Adjust pH to 7.5 with HCl
    Add dH2O to 100 ml and stored at 4 °C
  19. 50 mM potassium dihydrogen phosphate
    Mix 340 mg potassium dihydrogen phosphate with 50 ml dH2O
    Stored room temperature
  20. Phosphate calibration curve (0 to 500 µM KH2PO4)
    Mix 100 µl 50 mM potassium dihydrogen phosphate with 9.9 ml dH2O
    From the 500 µM KH2PO4 make serial dilutions of 400, 300, 250, 200, 150, 100, 80, 60, 40, and 20 µM KH2PO4
    Stored room temperature or at 4 °C
  21. 222 µM polyP45 (10 mM Pi)
    Mix 10.4 mg sodium phosphate glass Type 45 (polyP45) with 10 ml dH2O
    Stored at 4 °C
  22. 3.3 mM polyP3 (10 mM Pi)
    Mix 12.3 mg sodium tripolyphosphate (polyP3) with 10 ml dH2O
    Stored at 4 °C
  23. 6 µM polyP45
    Mix 135.1 µl 222 µM polyP45 with 4.86 ml dH2O
    Stored at 4 °C
  24. 200 µM polyP3
    Mix 300 µl 3.3 mM polyP3 with 4.7 ml dH2O
    Stored at 4 °C
  25. 28 mM ammonium heptamolybdate in 2.1 M H2SO4
    Mix 3.46 g ammonium heptamolybdate tetrahydrate with 80 ml dH2O
    Add 11.2 ml 98% sulfuric acid
    Add dH2O to 100 ml and stored at room temperature
  26. 0.76 mM malachite green in 0.35% polyvinyl alcohol
    Mix 350 mg polyvinyl alcohol in 100 ml dH2O at 80 °C and stir vigorously on a magnetic stirrer until all polyvinyl alcohol dissolves completely
    Add 35 mg malachite green oxalate salt
    Add dH2O to 100 ml and stored at room temperature

Acknowledgments

We thank Dr. Roberto Docampo (University of Georgia, Athens, USA.) for the E. coli clone expressing rPpx1. JA is recipient of grants BFU2011-30197-C03-01 and BFU2014-54591-C2-1-P from the Ministerio de Economía y Competitividad (MINECO), Spain, and 2014SGR-4 from the Generalitat de Catalunya. JC is recipient of grant BFU 2013-44189-P from Ministerio de Economia y Competitividad (MINECO), Spain, and 2014 SGR-1014 from the Generalitat de Catalunya.

References

  1. Cogan, E. B., Birrell, G. B. and Griffith, O. H. (1999). A robotics-based automated assay for inorganic and organic phosphates. Anal Biochem 271(1): 29-35.
  2. Freimoser, F. M., Hurlimann, H. C., Jakob, C. A., Werner, T. P. and Amrhein, N. (2006). Systematic screening of polyphosphate (poly P) levels in yeast mutant cells reveals strong interdependence with primary metabolism. Genome Biol 7(11): R109.
  3. Garcia, M. R. (2014). Extraction and quantification of Poly P, Poly P analysis by Urea-PAGE. Bio-protocol 4(9): e1113.
  4. Kumble, K. D. and Kornberg, A. (1995). Inorganic polyphosphate in mammalian cells and tissues. J Biol Chem 270(11): 5818-5822.
  5. Rosenfeld, L., Reddi, A. R., Leung, E., Aranda, K., Jensen, L. T. and Culotta, V. C. (2010). The effect of phosphate accumulation on metal ion homeostasis in Saccharomyces cerevisiae. J Biol Inorg Chem 15(7): 1051-1062.
  6. Ruiz, F. A., Rodrigues, C. O. and Docampo, R. (2001). Rapid changes in polyphosphate content within acidocalcisomes in response to cell growth, differentiation, and environmental stress in Trypanosoma cruzi. J Biol Chem 276(28): 26114-26121.
  7. Werner, T. P., Amrhein, N. and Freimoser, F. M. (2005). Novel method for the quantification of inorganic polyphosphate (iPoP) in Saccharomyces cerevisiae shows dependence of iPoP content on the growth phase. Arch Microbiol 184(2): 129-136.

简介

无机多磷酸盐(polyP)是存在于原核和真核生物中的线性聚合物,由通过磷酸酐键连接的三至数百个正磷酸盐残基制成。 这种分子的生物学作用超越了作为P 1储存或能量源以代替ATP。 例如,在酵母中,polyP水平与应激适应相关,并且该分子已经显示为蛋白质多磷酸化的底物。 在这里,我们描述了从酵母酿酒酵母中纯化polyP的两种不同方法和随后的通过分光光度法测量在酶水解纯化的polyP时产生的Pi来定量polyP水平的方案。 必须注意的是,所使用的纯化方案极大地影响所获得的polyP值。

图1. polyP的酶水解

关键字:酵母, 多聚磷酸盐, 提取方法, 酶促磷释放

材料和试剂

  1. 1.5 ml螺旋盖管
  2. 来自QIAquick Gel Extraction Kit(QIAGEN,目录号:28706)的硅胶柱
  3. 接收回路
  4. 磁力搅拌器
  5. 96孔测定微孔板未处理的透明聚苯乙烯(Thermo Fisher Scientific,Nunc Microspec MicroWell TM ,目录号:269620)
  6. 酵母酿酒酵母
  7. 酵母提取物,蛋白胨(YP)基础培养基(Conda,目录号:1511)
  8. AE缓冲区
  9. 苯酚溶液(Sigma-Aldrich,目录号:P4557)
  10. 十二烷基硫酸钠(SDS)(Panreac AppliChem,目录号:A7249)
  11. 氯仿(CHCl 3)(Merck Millipore,目录号:102445)
  12. RNA酶A(100mg/ml)(QIAGEN,目录号:19101)
  13. DNase I(Roche Diagnostics,目录号:10104159001)
  14. 醋酸钠三水合物(NaC 2 H 3 O 3 H 2·3H 2 O)(Merck Millipore,目录号: 106267)
  15. 无水乙醇(CH 3 CH 2 OH)(Panreac Applichem,目录号:131086)
  16. Milli-Q水
  17. 98%硫酸(H 2 SO 4)(Merck Millipore,目录号:112080)
  18. 氢氧化钠(NaOH)(Panreac Applichem,目录号:131687)
  19. 三(2-氨基-2-羟甲基 - 丙烷-1,3-二醇)缓冲液(Panreac Applichem,目录号:A1379)
  20. 中性红(Sigma-Aldrich,目录号:N4638)
  21. 碘化钠(NaI)(Sigma-Aldrich,目录号:383112)
  22. 乙酸冰醋酸(CH 3 COOH)(Panreac Applichem,目录号:131088)
  23. 乙二胺四乙酸二钠2水合物(EDTA)(Panreac Applichem,目录号:131669)
  24. 重组Ppx1(rPpx1)
    注意:Ppx1是将多磷酸水解成Pi残基的酿酒酵母外多磷酸酶。在使用HisTrapTM HP柱(GE Healthcare Life Sciences)一步亲和纯化后,如(Ruiz等人,2001)所述,从含有质粒携带的His标签形式的PPX1基因的大肠杆菌。
  25. 磷酸二氢钾(KH 2 PO 4)(Merck Millipore,目录号:104877)
  26. 磷酸钠玻璃45型(polyP 45)(Sigma-Aldrich,目录号:S4379)
  27. 三聚磷酸钠(polyP 3)(Sigma-Aldrich,目录号:238503)
  28. 孔雀绿草酸盐(Sigma-Aldrich,目录号:M9015)
  29. 聚乙烯醇(Sigma-Aldrich,目录号:P1763)
  30. D-葡萄糖一水合物(Panreac Applichem,目录号:A1349)
  31. 乙酸铵(NH 4 Cl 2 H 3 3 O 2)(Panreac Applichem,目录号:131114) br />
  32. 乙酸镁四水合物[Mg(CH 3 COO)2·4H 2 O](Merck Millipore,目录号:105819)
  33. 氯化钠(NaCl)(Panreac Applichem,目录号:131659)
  34. 七钼酸铵四水合物[(NH 4)6 Mo 12 Mo 24 SO 4·4H 2 > O](Sigma-Aldrich,目录号:09878)
  35. YPD(见配方)
  36. 3 M乙酸钠(见配方)
  37. 0.5 M EDTA(见配方)
  38. 缓冲液AE(参见配方)
  39. 10%SDS(见配方)
  40. RNA酶A(10mg/ml)(参见配方)
  41. DNase I(10mg/ml)(参见配方)
  42. 70%乙醇(见配方)
  43. 0.1%中性红溶液(见配方)
  44. 补充至含0.1%中性红溶液(w/v)的6%(v/v)的1M Tris-HCl(pH7.5)(参见配方)
  45. 2 M NaOH(见配方)
  46. 1 M硫酸(参见配方)
  47. 6 M NaI(见配方)
  48. 1 M醋酸铵(见配方)
  49. 1 M醋酸镁(见配方)
  50. 5 M NaCl(见配方)
  51. 洗涤缓冲液(见配方)
  52. 多磷酸盐测定缓冲液(参见配方)
  53. 50 mM磷酸二氢钾(见配方)
  54. 磷酸盐校准曲线(参见配方)
  55. 222μMPolyP 45(10mM P sub)(参见配方)
  56. 3.3mM polyP 3 Sub(10mM P Sub)(参见配方)
  57. 6μMpolyP 45 (参见配方)
  58. 200μMpolyP 3 (参见配方)
  59. 28 mM的七钼酸铵在2.1M H 2 SO 4中(见配方)。
  60. 0.76mM孔雀绿,0.35%聚乙烯醇(参见配方)

设备

  1. Novaspec Plus分光光度计GE
  2. Thermo LabSystems Multiskan Ascents 354酶标仪
  3. Eppendorf Thermomixer ?紧凑型
  4. 涡流混合器(Heidolph)
  5. 离心机(Eppendorf,MiniSpin )

程序

  1. 多磷酸盐沉淀纯化
    协议改编自(Kumble and Kornberg,1995)
    1. 用无菌接种环将来自新鲜平板的酵母菌株的单个菌落接种到5ml液体培养基(YPD或合适的选择培养基)中,并在200-220rpm和28℃下在旋转振荡器上孵育过夜。 >
    2. 生长16-24小时后,通过使用分光光度计测定酵母培养物的OD 600。在5ml YPD(最终OD 600)中接种细胞并在振荡器中在200-220rpm和28℃孵育直到OD 600达到0.6-0.8(这个将需要3到5小时。)。
    3. 通过在12,000×g离心1分钟,收集0.5至1OD 600单位的酵母指数培养物(1-2×10 7个细胞)并弃去上清液。
    4. 用400μl预先在4°C预冷的AE缓冲液重悬细胞沉淀
    5. 将细胞重悬浮至预先准备的含有300μl苯酚和40μl10%SDS的1.5 ml螺旋盖管中,并牢固固定盖子。
    6. 通过颠倒混合4次并涡旋5秒以匀化
    7. 在65℃孵育5分钟,并在冰上冷却管子1分钟
    8. 加入300μl氯仿,牢固固定盖子,颠倒混合4次,涡旋5秒以匀化
    9. 在室温下以13,000×g离心2分钟,以从有机相中分离含有polyP的水相。
    10. 小心地将水相(顶部)转移到含有350μl氯仿的制备的1.5ml螺旋盖管中。通常,回收的体积将为约450μl。确保在移液期间不要携带任何苯酚,避免接触底部相或含白色蛋白质的相间
    11. 牢固固定盖子,颠倒混合4次,涡旋5秒以均质化
    12. 在室温下以13,000×g离心2分钟。
    13. 回收水相,约400微升,并将其转移到一个新的1.5毫升微量离心管。和以前一样,在移液期间,不要接触底部相,以免不要携带任何苯酚
    14. 加入2微升的核糖核酸酶A(10毫克/毫升)和2微升的DNA酶I(10毫克/毫升)到每个管,并在37℃孵育1小时。在沉淀之前分别加入RNA酶A和DNA酶I以分别降解RNA和DNA,从而避免polyP的RNA和DNA沉淀。
    15. 将水相转移至-20℃预冷,含有1ml无水乙醇和40μl3M乙酸钠(pH 5.3)的1.5ml微量离心管中,在-20℃下保持3小时以沉淀多磷酸盐。
    16. 在4℃下以13,000×g离心20分钟
    17. 通过倾析弃去上清液,加入500μl70%乙醇
    18. 在4℃下以13,000×g离心5分钟
    19. 通过倾析弃去上清液,并在13,000×g离心1分钟,通过移液除去痕量的乙醇。
    20. 打开管并干燥小的半透明的白色多磷酸盐沉淀,其可以在管的底部在室温下观察5分钟或直到沉淀完全干燥。
    21. 重悬于50μlMilli-Q水中。
    22. 多聚磷酸盐样品可直接测量或储存在-20℃下
  2. 用硅胶柱纯化聚磷酸盐
    协议改编自(Werner等人,2005)
    1. 用无菌接种环将来自新鲜平板的酵母菌株的单个菌落接种到5ml液体培养基(YPD或合适的选择培养基)中,并在200-220rpm和28℃下在旋转振荡器上孵育过夜。 >
    2. 生长16-24小时后,通过使用分光光度计测定酵母培养物的OD 600。在5ml YPD(最终OD 600)中接种细胞,并在振荡培养箱中在200-220rpm和28℃孵育,直到OD 600达到0.6-0.8这将需要3到5小时。)。
    3. 通过在12,000×g离心1分钟,收集0.5至1OD 600单位的酵母指数培养物(1-2×10 7个细胞)并弃去上清液。
    4. 在细胞团块上加入50μl1M硫酸,混合并在室温下离开5分钟
    5. 用50μl2M NaOH中和悬浮液,并加入100μl添加有6%(v/v)和0.1%中性红溶液(w/v)的1M Tris-HCl(pH7.5)。
    6. 通过在4℃以800×g离心10分钟除去细胞碎片
    7. 回收200μl上清液,加入600μl6M NaI,并在调整样品的pH之前充分混合。 pH应该在7左右(样品颜色变成橙红色)。样品过酸性(粉红色)或太碱性(黄色)必须分别通过加入2M NaOH或1M硫酸来校正。
      注意:正确的pH调节是polyP提取的重现性的关键步骤。
    8. 将800μl多聚磷酸盐提取物应用于QIAquick纯化柱,并在室温下以13,000×g离心该柱1分钟。
    9. 弃去流出液并用400μl洗涤缓冲液通过在室温下以13,000×g离心1分钟洗涤柱两次。
    10. 在最后一次洗涤后,弃去流出液并通过在室温下以13,000×g离心另外1分钟来干燥柱。
    11. 将柱转移到新的1.5 ml微量离心管中,加入50μlMilli-Q水。使柱静置1分钟,并通过在室温下以13,000×g离心1分钟来洗脱多磷酸盐。强烈推荐使用pH> 10的Milli-Q水。 7用于更好的polyP洗脱。
    12. 洗脱的多磷酸盐可以直接测量或储存在-20℃
  3. COLUM再生
    如果需要,可以在再生后使用以下方案重新使用色谱柱:
    1. 通过在室温下以13,000×g离心1分钟,用750μl的0.2M乙酸和50mM EDTA(pH 8)洗涤柱一次。
    2. 用750μlMilli-Q水洗涤三次,每次在室温下以13,000×g离心1分钟。
    3. 通常,一列可以使用3-5次。如果观察到polyP回收率下降,必须使用新的色谱柱。

  4. 多磷酸盐消化和板制备
    1. 使用96孔板用于多磷酸盐降解的反应和随后测量释放的磷酸盐。使用过量的rPpx1(每个反应500ng的rPpx1酶)用于酶促多磷酸盐消化。
    2. 向含有磷酸盐标准曲线,样品背景测量和空白的孔中加入90μl多磷酸盐测定缓冲液。向样品孔中加入90μl多磷酸盐测定缓冲液和rPpx1酶的混合物
    3. 加入10μl样品(一式两份),磷酸盐标准曲线(0至500μMKH 2 PO 4)和由200μMpolyP 3组成的阳性对照/或6μMpolyP 45加到90μl多磷酸测定缓冲液中。通过上下吹打数次混合。
    4. 孵育板在37℃下60分钟。


      图2.聚磷酸盐测量示例板。 A行包含磷酸盐校准曲线。行B和E含有不含酶的多磷酸盐样品( )以测量背景和行C,D,F, G含有具有rPpx1酶的重复样品。类似地,最后一行包含在有和没有酶(分别为斜体和阴影)的情况下孵育的polyP 3 3和polyP 45的空白和阳性对照( 。

  5. 磷酸盐定量
    改编的方案(Cogan等人,1999)。磷酸盐的定量直接在PolyP消化板
    上进行
    1. 为了定量释放的磷酸盐,向每个孔中加入86μl在2.1MH 2 SO 4 SO 4中的28mM七钼酸铵和64μl在0.35%聚乙烯醇中的0.76mM孔雀绿。通过使用酶标仪的摇动选项或通过上下移动样品以确保所有试剂的完全混合来充分混合。
    2. 在室温下20分钟后,在多层读数器中测量A 595。不要让反应进行超过1小时,因为它可能导致在高度浓缩的磷酸盐样品中出现小的沉淀,并且背景样品中的polyP降解,从而干扰正确的磷酸盐测量。
    3. 磷酸盐的量通过将每个样品的吸光度值与减去每个样品的背景值之后的磷酸盐标准曲线进行比较而获得。 polyP sub 3和subP 45用作对聚磷酸盐的rPpx1反应的控制。


      图3.磷酸盐定量示例板

笔记

  1. 二氧化硅柱纯化与沉淀方案的主要优点是前者允许一次处理许多样品。甚至更多,可以使用Qiagen 96 PCR纯化板来处理大量样品。然而,主要缺点是可能的短链多肽丢失,并且与沉淀方案相比PolyP的量被低估。作为参考,当我们使用商业polyP 45时,其是具有45个磷酸根残基的polyP链中富集的不同polyP的混合物,使用二氧化硅柱方法的polyP回收率估计为20-22% 。因此,应该可以近似地比较通过柱方法获得的PolyP水平与通过乘以因子4的沉淀方法产生的PolyP水平。作为使用S的例子。啤酒酵母样品,苯酚提取的材料(即,直到"通过沉淀的多磷酸盐纯化"方案的步骤A14处理)分成两半。一半按照相同的方案进行处理,另一半使用QIAquick柱进行纯化。如图4所示,通过二氧化硅柱的纯化导致检测到的polyP水平降低3至4倍。因此,如果提取的目的是获得所有尺寸的大量polyP,则沉淀方法是最合适的。通过凝胶电泳评估polyP大小的详细方法可以作为BioProtocol(Garcia,2014)找到 相比之下,沉淀法更经济,因为它不需要购买纯化柱

    图4.乙醇沉淀和二氧化硅柱纯化方法的比较。该图显示培养物的每单位光密度的Pi量。数据表示为5次实验的平均值±SD

食谱

  1. YPD
    在500ml dH 2 O中混合30g YP基础培养基和20g葡萄糖 将dH 2 O加入到1000ml和高压釜中
  2. 3 M乙酸钠(pH 5.3)
    将40.82g乙酸钠三水合物与70ml dH 2 O混合 用冰醋酸
    调节pH至5.3 将dH 2 O加到100ml中并在室温下贮存
  3. 0.5 M EDTA(pH 8)
    将18.61g乙二胺四乙酸二钠2-水合物溶解在80ml dH 2 O中
    通过加入?2g NaOH颗粒将pH调节至8,并在磁力搅拌器上剧烈搅拌
    将dH 2 O加到100ml中并在室温下贮存
  4. 缓冲液AE [50mM乙酸钠(pH5.3),10mM EDTA] 将1.67ml 3M乙酸钠(pH5.3)与70ml dH 2 O混合 加入2ml 0.5M EDTA
    将dH 2 O加到100ml中并在室温下贮存
  5. 10%SDS
    将10g的十二烷基硫酸钠(SDS)与80ml dH 2 O混合 将dH 2 O加到100ml中并在室温下贮存
  6. 核糖核酸酶A(10mg/ml) 混合100μlRNase A(100mg/ml)与900μldH 2 O
    储存在4°C
  7. DNase I(10mg/ml)
    将50mg DNA酶I与5ml dH 2 O混合 储存在4°C
  8. 70%乙醇
    将70ml无水乙醇与30ml dH 2 O混合 储存于-20°C
  9. 0.1%中性红溶液(w/v) 将0.1g中性红与100ml 70%乙醇混合 在室温下贮存
  10. 添加有0.1%中性红溶液(w/v)的6%(v/v)的1M Tris-HCl(pH7.5) 将12.1g Tris与70ml dH 2 O混合 加入6ml 0.1%中性红溶液(w/v) 用HCl
    调节pH至7.5 将dH 2 O加到100ml中并在室温下贮存
  11. 2 M NaOH
    将8g NaOH与80ml dH 2 O混合 将dH 2 O加到100ml中并在室温下贮存
  12. 1M硫酸(H 2 SO 4)
    将5.45ml 98%硫酸与94.55ml dH 2 O混合 在室温下贮存
  13. 6 M NaI
    将13.5g NaI与dH 2 O混合直到15ml
    解决方案必须每次新鲜准备,避免光线
  14. 1M乙酸铵 将7.71g乙酸铵与80ml dH 2 O混合 将dH 2 O加到100ml中并在室温下贮存
  15. 1M乙酸镁 将21.4g乙酸镁四水合物与70ml dH 2 O混合 将dH 2 O加到100ml中并在室温下贮存
  16. 5 M NaCl
    将29.22g氯化钠与70ml dH 2 O混合 将dH 2 O加到100ml中并在室温下贮存
  17. 洗涤缓冲液[10mM Tris-HCl缓冲液(pH7.5),50%乙醇,1mM EDTA和100??mM NaCl] 将500μl1M Tris-HCl(pH7.5),25ml 100%乙醇,100μl0.5M EDTA,1ml 5M NaCl和20ml dH 2 O混合。 用HCl
    调节pH至7.5 将dH 2 O加至50ml并在室温下贮存
    0.2M乙酸和50mM EDTA 混合571μl冰醋酸和5ml 0.5M EDTA
    将dH 2 O加到50ml中并在室温下贮存
  18. 多磷酸盐测定缓冲液[20mM Tris-HCl(pH7.5),5mM乙酸镁,100mM乙酸铵] 混合2ml 1M Tris-HCl(pH7.5),500μl1M乙酸镁和10ml 1M乙酸铵。 用HCl
    调节pH至7.5 将dH 2 O加到100ml中并在4℃下贮存
  19. 50mM磷酸二氢钾 将340mg磷酸二氢钾与50ml dH 2 O混合 储存室温度
  20. 磷酸盐校准曲线(0?500μMKH 2 PO 4)
    将100μl50mM磷酸二氢钾与9.9ml dH 2 O混合 从500μMKH 2 PO 4 4制备400,300,250,200,150,100,80,60,40和20μMKH 2 PO 4
    存储的室温或4°C
  21. 222μM聚P 45(10mM P sub)
    将10.4mg的45型磷酸钠玻璃(polyP 45)与10ml dH 2 O混合。
    储存在4°C
  22. 3.3mM polyP 3 3(10mM P sub)
    将12.3mg三聚磷酸钠(polyP 3)与10ml dH 2 O混合。
    储存在4°C
  23. 6μMpolyP 45
    将135.1μl222μM聚P 45与4.86ml dH 2 O混合。
    储存在4°C
  24. 200μMpolyP sub 3
    将300μl3.3mM polyP 3与4.7ml dH 2 O混合。
    储存在4°C
  25. 在2.1M H 2 SO 4中的28mM七钼酸铵
    将3.46g七钼酸铵四水合物与80ml dH 2 O混合 加入11.2ml 98%硫酸
    将dH 2 O加到100ml中并在室温下贮存
  26. 0.76mM孔雀绿,0.35%聚乙烯醇 在80℃下将350mg聚乙烯醇在100ml dH 2 O中混合,并在磁力搅拌器上剧烈搅拌,直到所有聚乙烯醇完全溶解为止。
    加入35mg孔雀石绿草酸盐
    将dH 2 O加到100ml中并在室温下贮存

致谢

我们感谢Roberto Docampo博士(格鲁吉亚大学,雅典,美国)。表达rPpx1的大肠杆菌克隆。 JA是西班牙经济部长会议(MINECO)的赠款BFU2011-30197-C03-01和BFU2014-54591-C2-1-P的接受者,以及来自加泰罗尼亚地方政府的2014SGR-4。 JC是西班牙经济部长奖(BFEC)2013-44189-P和2014年加泰罗尼亚通用汽车公司(SGR-1014)的获得者。

参考文献

  1. Cogan,EB,Birrell,GB和Griffith,OH(1999)。  基于机器人的自动测定无机和有机磷酸盐。 Anal Biochem 271(1):29-35。
  2. Freimoser,FM,Hurlimann,HC,Jakob,CA,Werner,TP和Amrhein,N。(2006)。  酵母突变体细胞中多磷酸(poly P)水平的系统性筛选揭示了与初级代谢的强相互依赖性。基因组生物学7(11): R109。
  3. Garcia,MR(2014)。  Poly P,Poly P的提取和定量通过Urea-PAGE分析。 生物协议 4(9):e1113。
  4. Kumble,KD和Kornberg,A。(1995)。  Inorganic polyphosphate in mammalian cells and tissues。 J Biol Chem <270(11):5818-5822。
  5. Rosenfeld,L.,Reddi,AR,Leung,E.,Aranda,K.,Jensen,LT和Culotta,VC(2010)。< a class ="ke-insertfile"href ="http:磷酸盐积累对酿酒酵母中金属离子稳态的影响 /em> 15(7):1051-1062。
  6. Ruiz,FA,Rodrigues,CO和Docampo,R。(2001)。  在响应于细胞生长,分化和环境胁迫的酸聚合体内的多磷酸盐含量在克氏锥虫中的快速变化。

    J Biol Chem ):26114-26121。
  7. Werner,TP,Amrhein,N.and Freimoser,FM(2005)。  用于酿酒酵母中无机多磷酸盐(iPoP)的定量的新方法显示iPoP含量对生长期的依赖性。 Arch Microbiol 184 2):129-136。

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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Canadell, D., Bru, S., Clotet, J. and Ariño, J. (2016). Extraction and Quantification of Polyphosphate in the Budding Yeast Saccharomyces cerevisiae. Bio-protocol 6(14): e1874. DOI: 10.21769/BioProtoc.1874.
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