Assaying Glycogen and Trehalose in Yeast
酵母中糖原和海藻糖的测定   

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Molecular Cell
May 2016

Abstract

Organisms store carbohydrates in several forms. In yeast, carbohydrates are stored in glycogen (a multi-branched polysaccharide) and in trehalose (a disaccharide). As in other organisms, the amount of stored carbohydrate varies dramatically with physiological state, and accordingly, an assay of stored carbohydrate can help reveal physiological state. Here, we describe relatively easy and streamlined assays for glycogen and trehalose in yeast that can be applied either to a few samples, or in a moderately high-throughput fashion (dozens to hundreds of samples).

Keywords: Glycogen (糖原), Trehalose (海藻糖), Yeast (酵母), Storage carbohydrate (贮藏碳水化合物), Cell cycle (细胞周期)

Background

Glycogen and trehalose are the two storage carbohydrates of yeast and many other organisms. In yeast, both these storage carbohydrates accumulate when the medium starts to be depleted and the rate of cell growth decreases. Methods for assaying storage carbohydrates in yeast date back at least to 1956 (Trevelyan and Harrison, 1956a and 1956b), and have been updated many times since (e.g., [Becker, 1978; Quain, 1981; Schulze et al., 1995; Parrou and Francois, 1997; Plata et al., 2013], among others). There are three basic steps in assaying these two storage carbohydrates: first, lysing or permeabilizing the cells; second, freeing glucose from the glycogen or trehalose; and third, assaying the resulting glucose.

Cells can be lysed mechanically (Schulze et al., 1995), but this is inevitably somewhat tedious and time-consuming, and tends to require larger numbers of cells. Cells can be permeabilized by alkali, but glycogen forms large, multi-branched granules, and can be difficult to extract, and so some protocols use both an alkali and an acid extraction (Trevelyan and Harrison, 1956a and 1956b; Quain, 1981). However, alkali treatment alone extracts the vast majority of the glycogen (and probably all of the trehalose) (Becker, 1978; Quain, 1981; Parrou and Francois, 1997); and it may allow enzymes such as amyloglucosidase access to the interior of the permeabilized cell, where it can liberate glucose from any residual glycogen, and alkali extraction alone is much easier than a dual alkali/acid extraction. Therefore, like Becker, and Parrou and Francois, we use only an alkali extraction. However, it is possible that this may fail to assay a relatively small amount of acid-extractable glycogen (Quain, 1981).

In older assays (e.g., [Trevelyan and Harrison, 1956a and 1956b]), glucose was released and/or assayed by purely chemical methods. However, these were relatively non-specific, and also assayed glucose present in other molecules, such as cell wall glucans. Therefore more modern methods use enzymes to liberate glucose from specific polysaccharides; e.g., amyloglucosidases are used to liberate glucose from glycogen (Becker, 1978), and trehalases are used to liberate glucose from trehalose (Parrou and Francois, 1997). A challenge to these methods is that some enzymes are contaminated with other activities. For instance, Parrou and Francois found that some amyloglucosidases were contaminated with trehalases. Therefore either purer enzymes need to be used, or less pure enzymes need to be used under conditions that inhibit the unwanted activities. Here, like Parrou and Francois, we use Aspergillus niger α-amyloglucosidase, which may also contain a trehalase activity (Parrou and Francois, 1997), depending on the specific preparation of enzyme, but we use it at high temperature (55 °C to 57 °C), approximately the optimum temperature for this enzyme, where the trehalase is inactive (Parrou and Francois, 1997).

Finally, the enzymatically-released glucose must be assayed. There are many well-developed assays for glucose. We use the glucose oxidase/peroxidase/o-dianisidine reagent of the Sigma-Aldrich glucose oxidase kit, which produces oxidized o-dianisidine, which has a pink/purple color, easily assayed by absorbance at 540 nm.

Our procedure is adapted from that of Parrou and Francois (1997). However, at most steps, we use smaller volumes of reagents, which make the assay easier in some respects. The small volumes allow us to adapt the procedure to 96-well microtitre dishes, which allows the assay to become moderately high-throughput. We give two procedures, one for 2 ml screw-capped tubes, and one for 96-well microtitre dishes.

Materials and Reagents

  1. Protective eye wear/safety glasses/face shield
  2. Pipette tips
  3. 2 ml screw cap tubes with o-ring (e.g., SARSTEDT, catalog number: 72.694.406 or 72.694.217 )
  4. Microplate sealing tape (e.g., Corning aluminum tape, Corning, catalog number: 6570 )
  5. QuickSeal Foil PCR Self Adhesive Seal (Biosero)
    Or 4titude PCR Foil Seal (4titude, catalog number: 4ti-0550 )
    Or Peelable heat-sealing foil seals and a heat sealer
  6. For 96 well microtitre plate assay
    a. Polypropylene, round-bottom 96-well plates, 360 microlitre capacity (e.g., Corning, catalog number: 3359 )
    b. Polystyrene, flat-bottom 96-well plates (for plate reader) (e.g., Corning, catalog numbers: 3370 and 3915 )
  7. Yeast cells
    Note: This protocol has been developed for S. cerevisiae. It has not been tried with other species of yeast, but should work.
  8. Milli-Q or double-distilled water
  9. Glucose assay kit (Sigma-Aldrich, catalog number: GAGO-20 )
  10. Sulphuric acid
  11. Glacial acetic acid
  12. NaAcetate trihydrate*
  13. Aspergillus niger α-amyloglucosidase (Biochemika, ~70 U/mg) (Sigma-Aldrich, catalog number: 10115 )
    Alternatively: 120 U/mg, may be higher purity (Sigma-Aldrich, catalog number: 10113 ).
  14. Porcine trehalase (about 2.3 U/ml) (Sigma-Aldrich, catalog number: T8778 )
  15. Concentrated H2SO4 (sulfuric acid)*
  16. Sodium carbonate anhydrous (Na2CO3)*
  17. 1 M acetic acid (see Recipes)*
  18. 0.2 M NaAcetate, pH 5.2 (see Recipes)
  19. 0.2 M NaAcetate, ~pH 8 (see Recipes)
  20. For 96 well microtitre plate assay
    1. Concentrated amyloglucosidase buffer (see Recipes)
    2. Concentrated trehalase buffer (see Recipes)
    3. Trehalase dilution buffer (0.1 M NaAcetate, pH 5.7) (see Recipes)
  21. 9 N H2SO4 (see Recipes)
  22. 0.25 M Na2CO3 (see Recipes)

    Note: *Reagents from any qualified company are suitable for this experiment.

Equipment

  1. Roller or shaker for growing yeast
  2. Adjustable micropipettes, volumes from 2 to 500 μl
  3. Spectrophotometer and cuvettes
  4. Centrifuge (room temperature or chilled) for volumes of 5 to 15 ml
  5. Microcentrifuge for 1.5 and 2 ml tubes
  6. Vortex mixer
  7. pH meter
  8. Water bath (95 °C)
  9. Water bath or air incubator, 57 °C and 37 °C
  10. Glass pipet
  11. Fume hood
  12. For 96-well plate assay
    1. Centrifuge and adaptors for microtitre plates
    2. Multichannel pipettes
    3. Plate reader

      Note: All those items can be ordered from any qualified company.

Procedure

  1. Glycogen and trehalose measurements in 2 ml screw-capped tubes
    Note: Methods are based on Becker, 1978; Parrou and Francois, 1997.
    1. Cell concentration is measured carefully (e.g., using a Coulter Counter or light scattering in a spectrophotometer) and noted. Transfer samples (of necessarily different, carefully-measured volumes) containing either 5.0 x 107 cells, or, if available, 1.0 x 108 cells, to a 2 ml screw-capped tube and centrifuge at about 20,000 x g for 10 sec. (e.g., 10 sec at maximum speed [~16,000 rpm] at room temperature in an Eppendorf 5415 D).
    2. Remove the supernatant, wash the cell pellet once in 1 ml ice-cold water to remove residual glucose from the medium. Resuspend cell pellets in 125 μl of 0.25 M Na2CO3 solution with initial vigorous/violent vortexing to thoroughly disperse all cells, and incubate at 95 °C for about 3 h with occasional vortexing (~once per hour, 5 sec), with care to maintain temperature at the top of the tube to avoid excessive condensation.
      Note: This can be done by incubating the tubes in an enclosed water bath, so that the air above the tubes is ~95 °C. Somewhat longer or shorter incubations have no apparent effect on the result; Becker (1978) and Quain (1981) incubated for only 90 min. At later times, the alkali-treated cells tend to clump together and are difficult to disperse, but failure to disperse the cells at these late times has no apparent effect on the result.
    3. After incubation at 95 °C, adjust the pH to 5.5, and the volume to 0.5 ml, by addition of 75 μl 1 M acetic acid and 300 μl 0.2 M NaAcetate, pH 5.2 (an appropriate mixture of acetic acid and NaAcetate was made first, and then 375 μl of the mixture was added to the 125 μl of sample).
    4. Vortex the sample vigorously to resuspend and disperse cell debris, then immediately divide it into two 250 μl in fresh tubes, for glycogen and trehalose measurements respectively.
      Note: It may be important to divide the cell debris equally, because the permeabilized cells may still contain glycogen granules, from which glucose will be liberated by amyloglucosidase in the glycogen assay.
    5. For glycogen measurement, make a 20 mg/ml solution of Aspergillus niger α-amyloglucosidase (~70 U/mg) freshly in 0.2 M NaAcetate, pH 5.2. (The pH optimum of the enzyme is ~pH 5, but with high activity between pH 3.0 and pH 6.5 [Pazur and Ando, 1959]). Add 10 μl of this solution to the 250 μl sample. Incubate the mixture overnight at 57 °C, close to the temperature optimum, with occasional vortexing as convenient.
      Note: The exact length of the incubation has little apparent effect on the result. However, incubation at temperatures below 55 °C may allow contaminating trehalase activities in the amyloglucosidase to become active, and to release glucose from trehalose, resulting in an over-estimation of the amount of glycogen (Parrou and Francois, 1997).
    6. For trehalose measurement, adjust the pH slightly upwards by addition of 15 μl of 0.2 M NaAcetate (as made by dissolving NaAcetate trihydrate in water, ~pH 8) to the 250 μl sample. The pH optimum for porcine trehalase is about pH 5.8. Add 3 μl of porcine trehalase (2.27 U/ml) (i.e., about 0.007 U of trehalase is added) and mix it well. Incubate the mixture overnight at 37 °C.
    7. Glucose liberated in the above procedures is quantified using a glucose assay kit.
      a. Briefly, prepare the glucose oxidase/peroxidase/o-dianisidine reagent as described by the manufacturer.
      b. Centrifuge cell samples for 5 min to pellet cell debris.
      c. Mix 50 μl of supernatant from the cell samples with 100 μl of assay reagent, and incubate the mixture in a pre-warmed rack at 37 °C for 30 min.
      d. Then add 100 μl of 9 N H2SO4 carefully to each reaction to stop the reaction and develop color (see Recipe 4).
    8. Prepare 50 μl samples of glucose standards (0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5 μg glucose) in 0.2 M NaAcetate buffer pH 5.2 and assayed in parallel. Typically prepare four or more ‘blank’ (0 glucose) standards. Finally, measure absorbance at 540 nanometers.
    9. Samples are diluted and re-developed if A540 readings are > 0.5, above which the assay is highly non-linear. A540 readings are converted to absolute amounts of glucose using the standard curve, and normalized for the number of cells used in the sample.

  2. Glycogen and trehalose measurements in 96-well microtitre dishes
    1. For large numbers of assays, the above protocol has been adapted to 96-well microtitre dishes. Cell concentration is measured carefully (e.g., using a Coulter Counter or light scattering in a spectrophotometer) and noted. Transfer samples (of necessarily different, carefully-measured volumes) containing either 5.0 x 107 cells, or, if available, 1.0 x 108 cells, in less than 250 μl of liquid into the wells (~360 μl) of a polypropylene 96-well plate. Centrifuge the plates and aspirate the supernatant. Wash the cell pellets once in 250 μl ice-cold water to remove residual glucose. After centrifugation, remove the supernatant by aspiration.
    2. Resuspend the cell pellets in 125 μl of 0.25 M Na2CO3 solution. Seal the plates are tightly with a peelable PCR foil microtitre dish sealing film (e.g., QuickSeal Foil PCR Self Adhesive Seal [Biosero] or 4titude PCR Foil Seal) (see Notes). Shake the sealed plates violently by hand, then press to a vortex mixer, to thoroughly disperse all cells.
    3. Incubate the plates at 95 °C for about 3 h with occasional vortexing, with care to maintain temperature at the top surface of the plate to avoid excessive condensation. This can be done by incubating the plate in an enclosed water bath, so that the air above the plate is ~95 °C.
      Note: The polystyrene plates should not be used at this temperature. At later times, the alkali-treated cells tend to clump together and are difficult to disperse, but failure to disperse the cells at these late times has no apparent effect on the result.
    4. After incubation at 95 °C, plates are cooled to room temperature. Immediately before the contents are dispensed into new 96-well plates for treatment with amyloglucosidase or trehalase, shake the plates violently and vortex to disperse cell debris, then tap on the benchtop to bring liquid to the bottom of the wells, then unseal.
    5. For the glycogen assay, add 188 μl of concentrated amyloglucosidase buffer to each well of a 96-well plate (see Recipes). Pipet 62 μl (half) of the heat-and-alkali treated, freshly-shaken cell suspension into each well. It may be important to divide the cell debris equally, because the permeabilized cells may still contain some glycogen granules, from which glucose will be liberated by amyloglucosidase in the glycogen assay.
    6. Make a 20 mg/ml solution of Aspergillus niger α-amyloglucosidase (~70 U/mg) freshly in 0.2 M NaAcetate buffer, pH 5.2. Add 10 μl of Aspergillus niger α-amyloglucosidase (~70 U/mg) solution to the 250 μl already in each well (step B3). Seal the plates using ordinary foil seals (e.g., Corning aluminum tape) (see Notes) and shake violently and vortex to mix. Incubate the plates overnight at 57 °C, with occasional vortexing as convenient, with care to maintain high temperature at the top of the plate to prevent condensation (e.g., by placing the entire plate inside a water bath or air incubator.
      Note: The exact length of the incubation has little apparent effect on the result. However, incubation at temperatures below 55 °C may allow contaminating trehalase activities in the amyloglucosidase to become active, and to release glucose from trehalose, resulting in an over-estimation of the amount of glycogen (Parrou and Francois, 1997).
    7. For the trehalose assay, add 188 μl of concentrated trehalase buffer (see Recipes) to each well of a 96-well plate. Pipet 62 μl (half) of the heat-and-alkali treated, freshly-shaken cell suspension into each well. Porcine trehalase (2.27 U/ml) is diluted ~3 fold in trehalase dilution buffer (see Materials and Reagents) to 0.7 U/ml, and 10 μl of the diluted enzyme (i.e., about 0.007 U of trehalase) is added to each well. Seal the plate with ordinary foil seals (e.g., Corning aluminum tape) (see Notes), then shake violently and vortex. Incubate the plate overnight at 37 °C.
    8. Glucose liberated in the above procedures is quantified using a glucose assay kit. Prepare the glucose oxidase/peroxidase/o-dianisidine reagent as described by the manufacturer.
      1. Add 100 μl of this reagent to each well of a fresh 96-flat-bottom-well, polystyrene plate (i.e., a plate suitable for a plate reader, see Materials and Reagents).
      2. Centrifuge the 96-well plates containing cell samples for 5 min to pellet cell debris. Pipette 50 μl of supernatant from the cell samples into the 100 μl of assay reagent in the assay plate. Seal the plates and mix by violent shaking and vortexing.
      3. Incubate the mixture in a water bath at 37 °C for 30 min. Unseal the plates and then add 100 μl of 9 N H2SO4 carefully to each well to stop the reaction and develop color.
        Note: Mixing can be accomplished by pipetting up and down; alternatively the plate could be resealed and shaken/vortexed, but with care (e.g., after wrapping in paper towels) because of the sulphuric acid in the wells.
      4. Measure absorbance at 540 nanometers using a plate reader. (see Recipe 4)
    9. Prepare 50 μl samples of glucose standards (0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5 μg glucose) in 0.2 M NaAcetate pH 5.2 and assayed in parallel. Typically four or more ‘blank’ (0 glucose) standards are prepared and assayed (Zhao et al., 2016).
    10. Samples are diluted and re-developed if A540 readings are > 0.5, above which the assay is non-linear. A540 readings are converted to absolute amounts of glucose using the standard curve, and normalized for the number of cells used in the sample.

Data analysis

Linear regression is used to fit a line to the linear part of the standard curve; points in the non-linear range (the higher glucose amounts) are not used in this analysis. The linear regression line is used to convert readings from samples into absolute amounts of glucose. From knowledge of the number of cells in the original sample (here, either 5 x 107 or 1 x 108), and knowledge of the fraction of the sample used in the final color assay (for the 2 ml tube glycogen assay, 50/260 = 0.192), the amount of carbohydrate per cell can be calculated in ng or pg of glucose-equivalents. Two key points for the analysis are accurate knowledge of the number of cells in the starting sample; and use of measurements within the linear range.

Notes

  1. These assays are highly robust in the sense that the results are not significantly changed by minor or even moderate variation in the incubation times or temperatures, or even the exact volumes of the reagents added. However, at least with some preparations of amyloglucosidase, the incubation temperature must be above 55 °C to avoid contaminating trehalase activity. An indication of amounts of contaminating trehalase activity can be found by using particular preparations of amyloglucosidase trehalose, and then assaying how much glucose is produced.
  2. We have not asked whether the cell debris remaining after alkali treatment still contains glycogen. This could be done by centrifuging the cell suspension after alkali treatment, and assaying the supernatant and pellet separately for glycogen. If there is little or no glycogen in the pellet fraction, then the protocol could be improved by centrifugation after the alkali step, and use of only the supernatant.
  3. Maintaining the seal on a 96-well dish for a 3 h incubation at 95 °C can be challenging. A heat sealer with a heat-sealing foil tape provides a robust seal, but heat sealers are expensive and not always available. An adhesive PCR foil tape (e.g., QuickSeal Foil PCR Self Adhesive Seal [Biosero] or 4titude PCR Foil Seal) will suffice if applied carefully. Ordinary foil sealing tape (e.g., Corning) will work if a second, round-bottom 96-well dish with the outer, rectangular flange removed is positioned on top of the foil seal of the sample plate, and the 96 empty wells of the top plate are used to press down, and maintain pressure on, the foil seal of the sample plate. Pressure can be maintained throughout the 3 h incubation with C-clamps, or with a weight. Any particular combination of foil seal, and microtitre dish, should be tested at 95 °C before committing to an experiment.
  4. A pipetting step could be saved by mixing the enzyme (amyloglucosidase or trehalase) with the buffer before mixing the buffer with the sample. However, we have not tested whether these enzymes are stable at the relatively low pH of these buffers.
  5. Strains that cannot synthesize glycogen, or that cannot synthesize trehalose (Zhao et al., 2016) can serve as negative controls for the assays. Wild-type cells grown to stationary phase can serve as positive controls.

Recipes

  1. 1 M acetic acid
    Add 1 ml glacial acetic acid (17.4 M) to 16.4 ml ddH2O
  2. 0.2 M NaAcetate buffer, pH 5.2
    Dissolve 2.05 g anhydrous NaAcetate in 75 ml of ddH2O in a beaker. While stirring (magnetic stirrer), adjust pH to 5.2 using 1 M acetic acid and a pH meter. Adjust final volume to 100 ml
  3. 0.2 M NaAcetate (~pH 8)
    Dissolve 2.05 g anhydrous NaAcetate in 75 ml of ddH2O in a beaker. Adjust final volume to 100 ml
  4. Concentrated amyloglucosidase buffer
    Per 96-well plate, mix 3.7 ml of 1 M acetic acid with 15.1 ml of 0.2 M NaAcetate, pH 5.2
  5. Concentrated trehalase buffer
    Per 96-well plate, mix 1.5 ml of 0.2 M NaAcetate (pH ~8), 3.7 ml of 1 M acetic acid, and 15.1 ml of 0.2 M NaAcetate, pH 5.2
  6. Trehalase dilution buffer (0.1 M NaAcetate, pH 5.7)
    Dissolve 1.025 g anhydrous NaAcetate in 75 ml of ddH2O in a beaker. While stirring (magnetic stirrer), adjust pH to 5.7 using 1 M acetic acid and a pH meter. Adjust final volume to 100 ml
  7. 9 N H2SO4
    1 vol concentrated H2SO4 diluted to 4 vols final
    Note: The preparation of 9 N H2SO4 is highly exothermic and requires care and protective eye and other equipment. It should be done in a fume hood. Add small amounts of the acid, slowly, with stirring, to the water. Have the vessel where the acid and water are mixed pointing away from the experimenter. Wear eye protection. Do not use a polystyrene pipet to add the acid, as the heat generated can be sufficient to melt/deform a polystyrene pipet.
  8. 0.25 M Na2CO3
    Dissolve 2.85 g of anhydrous Na2CO3 in 90 ml ddH2O. Make volume up to 100 ml

Acknowledgments

This work was funded by NIH RO1 GM 119175.

References

  1. Becker, J. U. (1978). A method for glycogen determination in whole yeast cells. Anal Biochem 86(1): 56-64.
  2. Pazur, J. H. and Ando, T. (1959). The action of an amyloglucosidase of Aspergillus niger on starch and malto-oligosaccharides. J Biol Chem 234(8): 1966-1970.
  3. Parrou, J. L. and Francois, J. (1997). A simplified procedure for a rapid and reliable assay of both glycogen and trehalose in whole yeast cells. Anal Biochem 248(1): 186-188.
  4. Plata, M. R., Koch, C., Wechselberger, P., Herwig, C. and Lendl, B. (2013). Determination of carbohydrates present in Saccharomyces cerevisiae using mid-infrared spectroscopy and partial least squares regression. Anal Bioanal Chem 405(25): 8241-8250.
  5. Quain, D. E. (1981). The determination of glycogen in yeasts. J I Brewing 87: 289-291.
  6. Schulze, U., Larsen, M. E. and Villadsen, J. (1995). Determination of intracellular trehalose and glycogen in Saccharomyces cerevisiae. Anal Biochem 228(1): 143-149.
  7. Trevelyan, W. E. and Harrison, J. S. (1956a). Studies on yeast metabolism. 5. The trehalose content of baker's yeast during anaerobic fermentation. Biochem J 62(2): 177-183.
  8. Trevelyan, W. E. and Harrison, J. S. (1956b). Studies on yeast metabolism. 7. Yeast carbohydrate fractions. Separation from nucleic acid, analysis, and behaviour during anaerobic fermentation. Biochem J 63(1): 23-33.
  9. Zhao, G., Chen, Y., Carey, L. and Futcher, B. (2016). Cyclin-dependent kinase co-ordinates carbohydrate metabolism and cell cycle in S. cerevisiae. Mol Cell 62(4): 546-557.

简介

生物体以多种形式储存碳水化合物。 在酵母中,碳水化合物储存在糖原(多支链多糖)和海藻糖(二糖)中。 如在其他生物体中,存储的碳水化合物的量与生理状态显着变化,因此,存储的碳水化合物的测定可以帮助揭示生理状态。 在这里,我们描述了酵母中糖原和海藻糖的相对容易和流线型的测定法,其可以应用于少量样品,或以适度高通量的方式(几十至几百个样品)。
【背景】糖原和海藻糖是酵母和许多其他生物的两种储存碳水化合物。在酵母中,当培养基开始耗尽并且细胞生长速率降低时,这两种储存碳水化合物都会积累。测定酵母中存储碳水化合物的方法可追溯到至少1956年(Trevelyan和Harrison,1956a和1956b),并且自(例如,Becker,1978; Quain,1981; Schulze ,1995; Parrou和Francois,1997; Plata等人,2013)等)。测定这两种储存碳水化合物有三个基本步骤:首先,裂解或渗透细胞;第二,从糖原或海藻糖释放葡萄糖;第三,测定所得葡萄糖。
细胞可以机械裂解(Schulze等人,1995),但这不可避免地有点乏味和耗时,并且往往需要更多的细胞。细胞可以被碱渗透,但是糖原形成大的多支化颗粒,并且难以提取,因此一些方案既使用碱和酸提取(Trevelyan和Harrison,1956a和1956b; Quain,1981)。然而,单独的碱处理提取绝大多数糖原(并且可能全部是海藻糖)(Becker,1978; Quain,1981; Parrou和Francois,1997);并且它可以允许诸如淀粉葡糖苷酶之类的酶进入透化细胞的内部,其中可以从任何残留的糖原释放葡萄糖,并且单独的碱提取比双重碱/酸提取容易得多。因此,像Becker,Parrou和Francois一样,我们只使用碱提取。然而,这可能无法测定相对少量的酸可萃取糖原(Quain,1981)。
 在较老的测定中(例如,[Trevelyan和Harrison,1956a和1956b]),通过纯化学方法释放和/或测定葡萄糖。然而,这些是相对非特异性的,并且还测定存在于其它分子中的葡萄糖,例如细胞壁葡聚糖。因此,更现代的方法使用酶从特定的多糖中释放葡萄糖;例如,淀粉葡糖苷酶用于从糖原释放葡萄糖(Becker,1978),海藻糖酶用于从海藻糖释放葡萄糖(Parrou和Francois,1997)。对这些方法的挑战是一些酶被其他活性污染。例如,Parrou和Francois发现一些淀粉葡萄糖苷酶被海藻糖酶污染。因此,需要使用更纯的酶,或者在抑制不需要的活性的条件下需要使用较不纯的酶。在这里,像Parrou和Francois一样,我们使用黑曲霉α-淀粉葡萄糖苷酶,其还可以含有海藻糖酶活性(Parrou和Francois,1997),这取决于酶的具体制备,但是我们使用它高温(55°C至57°C),约为该酶的最适温度,其中海藻糖酶无活性(Parrou和Francois,1997)。
最后,必须测定酶释放的葡萄糖。有许多发展良好的葡萄糖测定。我们使用Sigma-Aldrich葡萄糖氧化酶试剂盒的葡萄糖氧化酶/过氧化物酶/邻联茴香胺试剂,其产生具有粉红色/紫色的氧化的邻联茴香胺,容易通过540nm处的吸光度测定。
 我们的程序改编自Parrou和Francois(1997)的程序。然而,在大多数步骤中,我们使用较小体积的试剂,这使得测定在某些方面更容易。小容量使我们能够使程序适应96孔微量滴定皿,这使得测定成为适度高通量。我们给出两个程序,一个用于2ml螺旋盖管,一个用于96孔微量滴定皿。

关键字:糖原, 海藻糖, 酵母, 贮藏碳水化合物, 细胞周期

材料和试剂

  1. 防护眼戴/安全眼镜/面罩
  2. 移液器提示
  3. 带有O形环的2ml螺旋盖管(例如,SARSTEDT,目录号:72.694.406或72.694.217)
  4. 微孔板密封带(例如,Corning铝带,Corning,目录号:6570)
  5. QuickSeal Foil PCR自粘密封(Biosero)
    或4t PCR聚酯箔密封(4份,目录号:4ti-0550)
    或可剥离热封箔密封件和热封机
  6. 对于96孔微量滴定板测定
    一个。聚丙烯,圆底96孔板,360微升容量(例如Corning,目录号:3359)
    湾聚苯乙烯,平底96孔板(用于读板机)(例如,Corning,目录号:3370和3915)
  7. 酵母细胞
    注意:本协议已针对酿酒酵母开发。它没有被其他种类的酵母尝试,但应该工作。
  8. Milli-Q或双蒸水
  9. 葡萄糖测定试剂盒(Sigma-Aldrich,目录号:GAGO-20)
  10. 硫酸
  11. 冰醋酸
  12. 醋酸钠三水合物*
  13. 黑曲霉α-淀粉葡萄糖苷酶(Biochemika,〜70U / mg)(Sigma-Aldrich,目录号:10115)
    或者:120U / mg,可以是更高的纯度(Sigma-Aldrich,目录号:10113)。
  14. 猪海藻糖酶(约2.3U / ml)(Sigma-Aldrich,目录号:T8778)
  15. 浓H 2 SO 4(硫酸)*
  16. 无水碳酸钠(Na 2 CO 3)*
  17. 1M乙酸(参见食谱)*
  18. 0.2 M醋酸钠,pH 5.2(参见食谱)
  19. 0.2 M醋酸钠,〜pH 8(见配方)
  20. 96孔微量滴定板测定
    1. 浓缩淀粉葡糖苷酶缓冲液(参见食谱)
    2. 浓缩海藻糖酶缓冲液(参见食谱)
    3. 海藻糖酶稀释缓冲液(0.1M乙酸钠,pH 5.7)(参见食谱)
  21. 9 N H 2 SO 4(见配方)
  22. 0.25 M Na 2 CO 3(参见食谱)

    注意:*任何合格公司的试剂都适合本实验。

设备

  1. 用于生长酵母的滚筒或摇床
  2. 可调节的微量移液器,体积从2到500μl
  3. 分光光度计和比色皿
  4. 离心机(室温或冷藏)体积为5至15 ml
  5. 微量离心机用于1.5和2ml管子
  6. 涡街搅拌机
  7. pH计
  8. 水浴(95°C)
  9. 水浴或空气培养箱,57°C和37°C
  10. 玻璃移液器
  11. 通风柜
  12. 96孔平板测定
    1. 微量滴定板离心机和适配器
    2. 多通道移液器
    3. 读板器

      注意:所有这些项目都可以从任何合格的公司订购。

程序

  1. 糖精和海藻糖测量在2毫升螺旋盖管中 注意:方法基于Becker,1978; Parrou和Francois,1997.
    1. 仔细测量细胞浓度(例如,使用库尔特计数器或分光光度计中的光散射),并注意到。将含有5.0×10 7个细胞的样品(必需不同的仔细测量的体积)或如果有的话,将1.0×10 8个细胞转移到2ml螺杆并以约20,000×g离心10秒钟。 (例如,在Eppendorf 5415 D)中,在室温下以最大速度[〜16,000rpm] 10秒钟)。
    2. 取出上清液,用1ml冰冷的水清洗细胞沉淀,以从培养基中除去残留的葡萄糖。将细胞沉淀重新悬浮在125μl0.25M Na 2 CO 3溶液中,初步剧烈/剧烈涡旋以使所有细胞充分分散,并在95℃下孵育约3小时偶尔涡旋(〜每小时一次,5秒),小心保持管顶部的温度,以避免过度冷凝。
      注意:这可以通过在封闭的水浴中孵育管来完成,使得管上方的空气为〜95℃。有些更长或更短的孵化对结果没有明显的影响; Becker(1978)和Quain(1981)仅孵育90分钟。在稍后的时间,碱处理的细胞倾向于聚集在一起并且难以分散,但是在这些晚期不能分散细胞对结果没有明显的影响。
    3. 在95℃下温育后,通过加入75μl1M乙酸和300μl0.2M乙酸钠(pH5.2)(醋酸和乙酸钠的合适混合物)先调节pH至5.5,体积至0.5ml ,然后将375μl混合物加入到125μl样品中)。
    4. 将样品剧烈旋转以重悬和分散细胞碎片,然后立即将其分成新鲜管中的两个250μl,分别用于糖原和海藻糖测量。
      注意:细胞碎片平均分配可能很重要,因为透化细胞可能仍然含有糖原颗粒,葡萄糖在糖原测定中由淀粉葡糖苷酶释放出来。
    5. 对于糖原测量,在0.2M的乙酸钠(pH 5.2)中新鲜制备20mg / ml黑曲霉α-淀粉葡萄糖苷酶(〜70U / mg)的溶液。 (酶的pH最佳值为〜pH 5,但pH 3.0和pH 6.5之间具有较高的活性[Pazur and Ando,1959])。加入10μl该溶液至250μl样品。将混合物在57℃下孵育过夜,接近温度最佳,偶尔涡旋方便。
      注意:孵化的确切长度对结果几乎没有明显的影响。然而,在低于55℃的温度下孵育可能允许淀粉葡糖苷酶中的污染海藻糖酶活性变得活跃,并从海藻糖释放葡萄糖,导致对糖原量的过度估计(Parrou和Francois,1997)。 >
    6. 对于海藻糖测量,通过加入15μl0.2M的乙酸钠(通过将NaAcetate三水合物溶解在水中,pH8)制成)向250μl样品中调节pH稍微升高。猪海藻糖酶的pH最适pH约为5.8。加入3μl猪海藻糖酶(2.27U / ml)(即,加入约0.007U海藻糖酶)并充分混合。将混合物在37℃下孵育过夜。
    7. 使用葡萄糖测定试剂盒对上述程序释放的葡萄糖进行定量。
      一个。简言之,如制造商所述制备葡萄糖氧化酶/过氧化物酶/邻联茴香胺试剂。
      湾离心细胞样品5分钟以沉淀细胞碎片。
      C。将100μl测定试剂从细胞样品中混合50μl上清液,并将混合物在37℃的预热架中孵育30分钟。
      天。然后向每个反应中小心地加入100μl的9 N H 2 O 3 SO 4,以停止反应并显色(参见方案4)。
    8. 在0.2M乙酸钠缓冲液pH 5.2中制备50μl葡萄糖标准品(0,0.5,1,1.5,2,2.5,3,4,5μg葡萄糖)样品,并平行测定。通常准备四个或更多的“空白”(0葡萄糖)标准品。最后,测量540纳米的吸光度
    9. 如果A540读数为> 100,样品将被稀释并重新开发。 0.5以上,高分子量非常非线性。使用标准曲线将A540读数转换为绝对量的葡萄糖,并对样品中使用的细胞数进行标准化。

  2. 在96孔微量滴定皿中测定糖原和海藻糖
    1. 对于大量的测定,上述方案已经适应于96孔微量滴定皿。仔细测量细胞浓度(例如,使用库尔特计数器或分光光度计中的光散射),并注意到。转移含有5.0×10 7个细胞的样品(必需不同的仔细测量的体积),或者如果有的话,将其转移到小于250μl的1.0×10 8个细胞中的液体进入聚丙烯96孔板的孔(〜360μl)中。离心板并吸出上清液。在250μl冰冷的水中洗涤细胞沉淀物一次以除去残留的葡萄糖。离心后,通过抽吸去除上清液
    2. 将细胞沉淀重悬于125μl的0.25M Na 2 CO 3溶液中。用可剥离的PCR箔微量滴定盘密封膜(例如,QuickSeal Foil PCR Self Adhesive Seal [Biosero]或4t PCR聚合物箔密封)密封板(参见注释)。用手摇动密封的板,然后按下涡旋混合器,彻底分散所有的细胞
    3. 将板在95℃孵育约3小时,偶尔涡旋,注意保持板顶部的温度,以避免过度冷凝。这可以通过在封闭的水浴中孵育板来实现,使得板上方的空气为〜95℃。
      注意:聚苯乙烯板不应在此温度下使用。在稍后的时间,碱处理的细胞倾向于聚集在一起并且难以分散,但是在这些晚期不能分散细胞对结果没有明显的影响。
    4. 在95℃温育后,将板冷却至室温。在将内容物分配到新的96孔板中以便用淀粉葡糖苷酶或海藻糖酶处理之前,立即将板猛烈摇动并涡旋以分散细胞碎片,然后点击台面将液体带到孔的底部,然后开封。
    5. 对于糖原测定,向96孔板的每个孔中加入188μl浓缩的淀粉葡糖苷酶缓冲液(参见Recipes)。将62μl(一半)热和碱处理的新鲜摇动的细胞悬浮液吸入每个孔中。将细胞碎片平均分配可能是重要的,因为透化细胞可能仍然含有一些糖原颗粒,糖原测定中葡萄糖将由淀粉葡糖苷酶释放出来。
    6. 新鲜将20mg / ml的黑曲霉α-淀粉葡萄糖苷酶(〜70U / mg)溶液在0.2M的乙酸钠缓冲液(pH 5.2)中制备。向已经在每个孔中的250μl添加10μl黑曲霉α-淀粉葡萄糖苷酶(〜70U / mg)溶液(步骤B3)。使用普通铝箔密封(例如,Corning铝胶带)密封板(参见注释),并猛烈摇动并涡旋混合。在57℃孵育板过夜,偶尔涡旋方便,小心地在板的顶部保持高温以防止冷凝(例如,通过将整个板放置在水浴中或空气培养箱。
      注意:孵化的确切长度对结果几乎没有明显的影响。然而,在低于55℃的温度下孵育可能允许淀粉葡糖苷酶中的污染海藻糖酶活性变得活跃,并从海藻糖释放葡萄糖,导致对糖原量的过度估计(Parrou和Francois,1997)。 >
    7. 对于海藻糖测定,向96孔板的每个孔中加入188μl浓缩的海藻糖酶缓冲液(参见食谱)。将62μl(一半)热和碱处理的新鲜摇动的细胞悬浮液吸入每个孔中。在海藻糖酶稀释缓冲液(见材料和试剂)中将猪海藻糖酶(2.27U / ml)稀释至约0.7U / ml,稀释的酶(即约0.007U海藻糖酶)加入到每个孔中。用普通箔片密封板(例如,Corning铝胶带)(见注释),然后猛烈摇动并旋转。在37℃孵育板过夜。
    8. 使用葡萄糖测定试剂盒对上述程序释放的葡萄糖进行定量。如制造商所述,制备葡萄糖氧化酶/过氧化物酶/邻联茴香胺试剂。
      1. 向新鲜的96平底井聚苯乙烯板(即,适用于平板读数器的板,参见材料和试剂)的每个孔中加入100μl该试剂。
      2. 将含有细胞样品的96孔板离心5分钟以沉淀细胞碎片。将细胞样品中的50μl上清液吸取到测定板中的100μl测定试剂中。密封板,并通过剧烈摇晃和涡旋混合。
      3. 将混合物在37℃的水浴中孵育30分钟。开封板,然后小心地向每个孔中加入100μl9N H 2 O 3 SO 4,以停止反应并显色。
        注意:混合可以通过上下移动来完成;或者,由于孔中的硫酸,板可以被再密封并摇动/涡旋,但要小心(例如,在包裹在纸巾中之后)。
      4. 使用读板器测量540纳米的吸光度。 (见配方4)
    9. 在0.2M醋酸钠pH 5.2中制备50μl葡萄糖标准品(0,0.5,1,1.5,2,2.5,3,4,5μg葡萄糖)样品,并并行测定。通常制备和测定四种或更多的“空白”(0葡萄糖)标准(Zhao等人,2016)。
    10. 如果A540读数为> 100,样品将被稀释并重新开发。 0.5以上,测定是非线性的。使用标准曲线将A540读数转换为绝对量的葡萄糖,并对样品中使用的细胞数进行标准化。

数据分析

线性回归用于将线拟合到标准曲线的线性部分;在该分析中不使用非线性范围(较高葡萄糖量)的点。线性回归线用于将样本的读数转换为葡萄糖的绝对量。从原始样品中的细胞数量(这里为5×10 7 或1×10 8 )的知识,以及使用的样品的分数的知识最终颜色测定(对于2ml管糖原测定,50/260 = 0.192),每个细胞的碳水化合物的量可以以ng或pg的葡萄糖当量计算。分析的两个关键点是准确了解起始样品中细胞数量;并在线性范围内使用测量。

笔记

  1. 这些测定是非常稳定的,因为结果并没有显着改变,因为培养时间或温度的微小或甚至中度变化,甚至添加的试剂的确切体积。但是,至少有一些淀粉葡萄糖苷酶的制备,培养温度必须高于55℃,以避免污染海藻糖酶的活性。通过使用淀粉葡糖苷酶海藻糖的特定制剂,可以发现污染海藻糖酶活性的量的指示,然后测定产生多少葡萄糖。
  2. 我们还没有问过碱处理后残留的细胞残留物是否还含有糖原。这可以通过在碱处理后离心细胞悬浮液,并分别测定上清液和沉淀糖原来进行。如果颗粒级分中糖原少,则可以通过碱步骤离心来提高方案,只使用上清液。
  3. 将密封保持在96孔培养皿中,在95℃孵育3小时可能是具有挑战性的。具有热封箔带的热封机提供了坚固的密封,但是热封件是昂贵的并且并不总是可用的。如果仔细应用,粘合剂PCR箔片胶带(例如,QuickSeal Foil PCR Self Adhesive Seal [Biosero]或4t PCR聚合物箔密封)就足够了。如果拆除外部矩形凸缘的第二个圆底96孔盘定位在样品板的箔密封件的顶部,则常规箔密封胶带(例如,Corning)将起作用,以及使用顶板的96个空孔将样品板的箔片密封压下并保持压力。压力可以用C型夹具或重量保持3小时。在进行实验之前,应在95℃下测试箔密封和微量滴定皿的任何特定组合。
  4. 在将缓冲液与样品混合之前,通过将酶(淀粉葡糖苷酶或海藻糖酶)与缓冲液混合可以节省移液步骤。然而,我们还没有测试这些酶在这些缓冲液的相对低的pH下是否稳定
  5. 不能合成糖原或不能合成海藻糖的菌株(Zhao等人,2016)可用作测定的阴性对照。生长至固定相的野生型细胞可作为阳性对照

食谱

  1. 1M乙酸
    加入1ml冰醋酸(17.4M)至16.4ml ddH 2 O→/
  2. 0.2 M醋酸钠缓冲液,pH 5.2
    将2.05g无水乙酸钠溶于75ml的ddH 2 O烧杯中。搅拌(磁力搅拌器)时,使用1M乙酸和pH计将pH调节至5.2。将最终体积调整为100 ml
  3. 0.2 M醋酸钠(〜pH 8)
    将2.05g无水乙酸钠溶于75ml的ddH 2 O烧杯中。将最终体积调整为100 ml
  4. 浓缩淀粉葡糖苷酶缓冲液
    每96孔板中混合3.7ml 1M乙酸与15.1ml 0.2M乙酸钠,pH5.2
  5. 浓缩海藻糖酶缓冲液
    每96孔板中混合1.5ml 0.2M乙酸钠(pH〜8),3.7ml 1M乙酸和15.1ml 0.2M乙酸钠,pH5.2
  6. 海藻糖酶稀释缓冲液(0.1M乙酸钠,pH 5.7) 将1.025g无水乙酸钠溶于75ml ddH 2 O的烧杯中。搅拌(磁力搅拌器)时,使用1M乙酸和pH计将pH调节至5.7。将最终体积调整为100 ml
  7. 9 N H 2 SO 4
    1体积浓缩的H 2 O 3 SO 4稀释至4体积的终浓度
    注意:9 NH 2 SO 4 em高度放热,需要护理和保护眼睛和其他设备。它应该在通风橱里完成。在搅拌下将少量酸缓慢加入水中。将容器中的酸和水混合指向远离实验者。戴眼睛保护不要使用聚苯乙烯移液管来加酸,因为产生的热量足以使聚苯乙烯移液管熔化/变形。
  8. 0.25M Na 2 CO 3
    将2.85g无水Na 2 CO 3溶于90ml ddH 2 O中。使体积达到100 ml

致谢

这项工作由NIH RO1 GM 119175资助。

参考

  1. Becker,JU(1978)。  糖原测定方法在整个酵母细胞中。 Anal Biochem 86(1):56-64。
  2. Pazur,JH和Ando,T.(1959)。  淀粉和麦芽低聚糖上黑曲霉的淀粉葡糖苷酶的作用.J Biol Chem 234(8):1966-1970。
  3. Parrou,JL和Francois,J.(1997)。  用于在全酵母细胞中糖原和海藻糖的快速和可靠测定的简化程序。 248(1):186-188。
  4. Plata,MR,Koch,C.,Wechselberger,P.,Herwig,C.and Lendl,B。(2013)。  使用中红外光谱和偏最小二乘回归确定酿酒酵母中存在的碳水化合物。 Anal Bioanal Chem 405(25):8241-8250。
  5. Quain,DE(1981)。< a class =“ke-insertfile”href =“http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1981.tb04038.x/abstract”target =“ _blank“>确定酵母中的糖原。 JI酿造 87:289-291。
  6. Schulze,U.,Larsen,ME和Villadsen,J。(1995)。酿酒酵母中细胞内海藻糖和糖原的测定。 Anal Biochem 228(1):143-149。
  7. Trevelyan,WE和Harrison,JS(1956a)。研究对酵母代谢。 5.厌氧发酵过程中面包酵母的海藻糖含量。生物化学J 62(2):177-183。
  8. Trevelyan,WE和Harrison,JS(1956b)。  研究对酵母代谢。酵母碳水化合物。在厌氧发酵过程中与核酸,分析和行为分离。生物化学J 63(1):23-33。
  9. Zhao,G.,Chen,Y.,Carey,L.and Futcher,B。(2016)。  细胞周期蛋白依赖性激酶协调酿酒酵母中的碳水化合物代谢和细胞周期。分子细胞 62(4):546-557。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, Y. and Futcher, B. (2017). Assaying Glycogen and Trehalose in Yeast. Bio-protocol 7(13): e2371. DOI: 10.21769/BioProtoc.2371.
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