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
发布: 2016年07月20日第6卷第14期 DOI: 10.21769/BioProtoc.1874 浏览次数: 12564
评审: Fanglian HeYanjie LiRosario Gomez-Garcia
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The authors used this protocol in:
Feb 2015
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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
Polyphosphate (多聚磷酸盐)
Extraction methods (提取方法)
Enzymatic phosphate release (酶促磷释放)
Materials and Reagents
- 1.5 ml screw cap tubes
- Silica-gel columns from QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28706 )
- Inoculation loop
- Magnetic stirrer
- 96-Well assay microplate non-treated clear polystyrene (Thermo Fisher Scientific, NuncTM MicroWellTM, catalog number: 269620 )
- Yeast Saccharomyces cerevisiae
- Yeast extract, peptone (YP) base medium (Conda, catalog number: 1511 )
- AE buffer
- Phenol solution (Sigma-Aldrich, catalog number: P4557 )
- Sodium dodecylsulfate (SDS) (Panreac AppliChem, catalog number: A7249 )
- Chloroform (CHCl3) (Merck Millipore, catalog number: 102445 )
- RNase A (100 mg/ml) (QIAGEN, catalog number: 19101 )
- DNase I (Roche Diagnostics, catalog number: 10104159001 )
- Sodium acetate trihydrate (NaC2H3O2·3H2O) (Merck Millipore, catalog number: 106267 )
- Ethanol absolute (CH3CH2OH) (Panreac Applichem, catalog number: 131086 )
- Milli-Q water
- 98% sulfuric acid (H2SO4) (Merck Millipore, catalog number: 112080 )
- Sodium hydroxide (NaOH) (Panreac Applichem, catalog number: 131687 )
- Tris (2-amino-2-hydroxymethyl-propane-1,3-diol) buffer (Panreac Applichem, catalog number: A1379 )
- Neutral red (Sigma-Aldrich, catalog number: N4638 )
- Sodium iodide (NaI) (Sigma-Aldrich, catalog number: 383112 )
- Acetic acid glacial (CH3COOH) (Panreac Applichem, catalog number: 131088 )
- Disodium ethylenediaminetetraacetate 2-hydrate (EDTA) (Panreac Applichem, catalog number: 131669 )
- 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 ). - Potassium dihydrogen phosphate (KH2PO4) (Merck Millipore, catalog number: 104877 )
- Sodium phosphate glass Type 45 (polyP45) (Sigma-Aldrich, catalog number: S4379 )
- Sodium tripolyphosphate (polyP3) (Sigma-Aldrich, catalog number: 238503 )
- Malachite green oxalate salt (Sigma-Aldrich, catalog number: M9015 )
- Polyvinyl alcohol (Sigma-Aldrich, catalog number: P1763 )
- D-Glucose monohydrate (Panreac Applichem, catalog number: A1349 )
- Ammonium acetate (NH4C2H3O2) (Panreac Applichem, catalog number: 131114 )
- Magnesium acetate tetrahydrate [Mg(CH3COO)2·4H2O] (Merck Millipore, catalog number: 105819 )
- Sodium chloride (NaCl) (Panreac Applichem, catalog number: 131659 )
- Ammonium heptamolybdate tetrahydrate [(NH4)6Mo7O24·4H2O] (Sigma-Aldrich, catalog number: 09878 )
- YPD (see Recipes)
- 3 M sodium acetate (see Recipes)
- 0.5 M EDTA (see Recipes)
- Buffer AE (see Recipes)
- 10% SDS (see Recipes)
- RNase A (10 mg/ml) (see Recipes)
- DNase I (10 mg/ml) (see Recipes)
- 70% ethanol (see Recipes)
- 0.1% neutral red solution (see Recipes)
- 1 M Tris-HCl (pH 7.5) supplemented to 6% (v/v) with 0.1% neutral red solution (w/v) (see Recipes)
- 2 M NaOH (see Recipes)
- 1 M sulfuric acid (see Recipes)
- 6 M NaI (see Recipes)
- 1 M ammonium acetate (see Recipes)
- 1 M magnesium acetate (see Recipes)
- 5 M NaCl (see Recipes)
- Wash buffer (see Recipes)
- Polyphosphate assay buffer (see Recipes)
- 50 mM potassium dihydrogen phosphate (see Recipes)
- Phosphate calibration curve (see Recipes)
- 222 µM polyP45 (10 mM Pi) (see Recipes)
- 3.3 mM polyP3 (10 mM Pi) (see Recipes)
- 6 µM polyP45 (see Recipes)
- 200 µM polyP3 (see Recipes)
- 28 mM ammonium heptamolybdate in 2.1 M H2SO4 (see Recipes)
- 0.76 mM malachite green in 0.35% polyvinyl alcohol (see Recipes)
Equipment
- Novaspec Plus spectrophotometer GE
- Thermo LabSystems Multiskan Ascents 354 microplate reader
- Eppendorf Thermomixer® compact
- Vortex mixer (Heidolph)
- Centrifuge (Eppendorf, MiniSpin®)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
微生物学 > 微生物生物化学 > 其它化合物
微生物学 > 微生物生物化学 > 蛋白质 > 修饰
生物化学 > 其它化合物 > 离子 > 磷酸根
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