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Extraction and Assays of ADP-glucose Pyrophosphorylase, Soluble Starch Synthase and Granule Bound Starch Synthase from Wheat (Triticum aestivum L.) Grains
从小麦谷粒提取和检测ADP-葡萄糖焦磷酸化酶、可溶性淀粉合成酶和淀粉粒束缚淀粉合成酶   

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Abstract

Starch biosynthesis in plants involves a network of enzymes of which adenosine-5’-diphosphoglucose (ADP-glucose) pyrophosphorylase (AGPase, E.C. 2.7.7.27), and soluble and granule bound starch synthases (SSS and GBSS, E.C. 2.4.1.21) play central roles. Here, we outline the protocol for extraction and assay of these enzymes in developing grains of wheat (Triticum aestivum L.). The principle of the assays outlined is based on a coupling enzymatic reactions where the product of the initial reaction is used as a substrate for subsequent reactions in order to generate NADPH, which can be measured easily by spectrophotometer. This protocol does not need expensive labelled chemicals and can be carried out using equipment commonly found in a biochemical laboratory. We applied this protocol to study the dynamics of AGPase, SSS and GBSS activity in developing wheat grains at different time points after anthesis.

Keywords: Enzyme(酶), Grain(粮食), Starch synthesis(淀粉的合成), Wheat(小麦)

Background

Starch is a carbohydrate polymer made up of amylose, a linear glucan polymer composed of α-1,4-linked glucose molecules, and amylopectin, another glucan polymer composed of α-1,4-linked glucose molecules branched by α-1,6-glycosidic bonds. The enzyme adenosine-5’-diphosphoglucose (ADP-glucose) pyrophosphorylase (AGPase, E.C. 2.7.7.27) catalyzes the first committed step of starch synthesis, converting glucose-1-phosphate and ATP to ADP-glucose and inorganic pyrophosphate (PPi). ADP-glucose is subsequently used by soluble starch synthases (SSS) and granule bound starch synthases (GBSS) (E.C. 2.4.1.21), and starch branching enzymes to elongate and branch the glucan chains of the starch granule.
   Initially, AGPase and starch synthase assays were carried out using 14C- and 32P-labelled ADP-glucose (Ghosh and Price, 1966; Vos-Scheperkeuter et al., 1986), which requires the use of expensive chemicals as well as specialized equipment to work with labelled compounds. Here, we outline the method adopted and applied for extracting and assaying AGPase, SSS and GBSS activity in developing wheat (Triticum aestivum L.) grains (Mukherjee et al., 2015). Our protocol is based on the methods reported previously by Nakamura et al. (1989) for measuring AGPase and SSS activities in the endosperm of developing rice grains, and by Schaffer and Petreikov (1997) for measuring the activities of SSS and GBSS in tomato fruits. The method is based on coupling enzymatic reactions where the product of the initial reaction is used as a substrate for subsequent reactions in order to generate NADPH, which can be easily measured by spectrophotometers. The protocol can be carried out using the equipment commonly found in biochemical laboratories and does not require the use of labeled compounds.
   Because this protocol is an adoption of the methods developed for different tissues of other plant species (endosperm of rice grain and tomato fruit), the reaction mixture composition for all enzymes was optimized for reaction buffer pH and substrate concentration so that enzyme activity was within the linear phase with respect to incubation time and protein concentration. The amounts of enzyme preparation added (PGM, pyruvate kinase, hexokinase, G6PDH) have been adjusted to achieve completion of coupling reaction in expected time frame. This protocol can be used for studying the activities of AGPase, SSS and GBSS in other tissues of wheat as well as in different tissues of other plant species; however, optimization of reaction buffer pH and substrate concentrations is required.
   The principle of AGPase assay is presented in Figure 1. AGPase present in plant tissue extract catalyzes the conversion of ADP-glucose and PPi into glucose-1-phosphate and ATP. After the first reaction is stopped by boiling, phosphoglucomutase (PGM) is added to the reaction mixture; PGM converts quantitatively glucose-1-phosphate into glucose-6-phosphate. Subsequently, glucose-6-phosphate dehydrogenase (G6PDH) is added; G6PDH converts glucose-6-phosphate in the presence of NADP into 6-phosphogluconic acid. At the same time, NADP is converted into NADPH and the quantity of NADPH formed is equivalent to the quantity of glucose-6-phosphate oxidized. By measuring NADPH concentration spectrophotometrically at 340 nm, we can quantify the amount of ADP-glucose degraded by AGPase activity.


Figure 1. Principle for ADP-glucose pyrophosphorylase (AGPase) assay

   The principle of SSS/GBSS assay is presented in Figure 2. Starch synthases present in plant tissue extract (SSS) or in starch granule suspension (GBSS) catalyze the conversion of ADP-glucose into ADP coupled with the elongation of amylopectin primer for one glucose residue. Pyruvate kinase added to the reaction mixture after the first reaction is stopped by boiling converts quantitatively ADP into ATP in the presence of PEP. Subsequently, addition of glucose together with hexokinase leads to the conversion of ATP into glucose-6-phosphate. In the next step of assay, G6PDH converts glucose-6-phosphate in the presence of NADP into 6-phosphogluconic acid; NADP is converted into NADPH at the same time and the quantity of NADPH formed is equivalent to the quantity of glucose-6-phosphate oxidized. By measuring NADPH concentration spectrophotometrically at 340 nm, we can quantify the amount of ADP-glucose (at the first step of the analysis) degraded by SSS/GBSS activity.


Figure 2. Principle for soluble and granule bound starch synthases (SSS and GBSS) assay

Materials and Reagents

  1. Chemicals
    1. Common chemicals, buffers and kits
      1. Protein assay kit (Bio-Rad Laboratories, catalog number: 5000002 )
      2. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP310 )
      3. Sodium hydroxide (NaOH) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP359 )
      4. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670 )
      5. Ethylenediaminetetraacetic acid (EDTA), disodium salt dihydrate (Na2EDTA·2H2O) (Sigma-Aldrich, catalog number: E1644 )
      6. DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
      7. Polyvinylpyrrolidone (average molecular weight 10,000) (Sigma-Aldrich, catalog number: PVP10 )
      8. Potassium chloride (KCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP366 )
      9. Tetrasodium pyrophosphate (PPi) decahydrate (Na4P2O7·10H2O) (Sigma-Aldrich, catalog number: S6422 )
    2. Substrates and cofactors
      1. Adenosine-5’-diphosphoglucose (ADP-glucose), disodium salt (Sigma-Aldrich, catalog number: A0627 )
      2. Amylopectin from maize (Sigma-Aldrich, catalog number: 10120 )
      3. β-Nicotinamide adenine dinucleotide phosphate (NADP) sodium salt hydrate (Sigma-Aldrich, catalog number: N0505 )
      4. Phospho(enol)pyruvic acid (PEP) monosodium salt hydrate (Sigma-Aldrich, catalog number: P0564 )
    3. Enzymes
      1. Hexokinase (E.C. 2.7.1.1) from Saccharomyces cerevisiae (Sigma-Aldrich, catalog number: H5000 )
      2. Glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) from Saccharomyces cerevisiae (Sigma-Aldrich, catalog number: G7877 )
      3. Phosphoglucomutase (PGM, E.C. 5.4.2.2) from rabbit muscle (Sigma-Aldrich, catalog number: P3397 )
      4. Pyruvate kinase (E. C. 2.7.1.40) from rabbit muscle (Sigma-Aldrich, catalog number: P1506 )
    4. Stock solutions (see Recipes)
      1. 20 mM ADP-glucose
      2. 10 mM NADP
      3. 20 mM PEP

  2. Enzymatic activities reaction mixtures
    1. Extraction buffer (see Recipes, section 1)
    2. AGPase activity reaction mixture (see Recipes, section 3a)
    3. Starch synthase activity reaction mixture (see Recipes, section 3b)
    4. Solution 1 for starch synthase activity assay (Pyruvate kinase reaction mixture) (see Recipes, section 3c)
    5. Solution 2 for starch synthase activity assay (Glucose-6-phosphate dehydrogenase reaction mixture) (see Recipes, section 3d)

  3. Plant material
    1. Grains of common wheat (Triticum aestivum L.)

  4. Other materials
    1. Eppendorf tubes (1.5 and 2.0 ml)
    2. Falcon tubes (15 ml)
    3. Liquid nitrogen
    4. Microcentrifuge tube locker (for example, Sigma-Aldrich, catalog number: Z708372 , or Sorenson Bioscience MCT LidLockTM, catalog number: 11870 )
      Note: Product Z708372 has been discontinued.
    5. Miracloth (Merck Millipore, catalog number: 475855 )

Equipment

  1. Porcelain mortar and pestle
  2. Analytical balance (capacity - 100 g or higher, resolution - 0.001 g or better)
  3. Digital heat block or water bath suitable for 30 °C and digital heat block or water bath suitable for 100 °C
    Note: For some steps of the analysis immediate transfer from 30 °C to 100 °C is necessary. This is why two heating devices are necessary simultaneously for the protocol.
  4. Refrigerating ultracentrifuge with appropriate rotor suitable for spinning of 15 ml Falcon tube and 1.5-2 ml Eppendorf tubes at up to 12,000 x g
  5. Ultraviolet (UV)/Visible spectrophotometer equipped with heated cell holder suitable for maintaining cuvette temperature at 30 °C. We used GE Healthcare/Amersham Bioscience Ultrospec 3100 Pro UV/Visible spectrophotometer (Biochrom Ltd, Cambridge, UK).

Procedure

Summary of the enzyme extraction procedure is shown in a flowchart (Figure 3).


Figure 3. Flowchart for extraction of AGPase, SSS and GBSS from developing wheat seeds. Please, refer to the main text of the protocol for specific conditions and buffers/media composition required for each step.

  1. Wheat grain harvesting and storage before analysis
    Developing grains of wheat were harvested from primary and secondary tillers at different time points after anthesis (Figure 4), in liquid nitrogen and stored at -80 °C until further use. Care has to be taken not to thaw the frozen grains till enzyme assay as thawing the grains would seriously affect the result of the analysis.


    Figure 4. Developing seeds of wheat cv. Yecora Rojo at 4, 8, 16 and 25 days after anthesis

  2. Extraction
    1. Wheat grains were ground to a fine powder using liquid nitrogen in a pre-cooled mortar and pestle and ground tissue was transferred into empty pre-weighed Falcon tube pre-cooled with liquid nitrogen. The weight of the ground tissue was determined. This was performed by first weighing an empty Falcon tube, and then weighing it again filled with the ground tissue. The difference of the two readings equals to the net weight of the ground tissue. This value was recorded and used for further calculations (m, see Calculations, Equations 1, 2, 4, 5, 7).
    2. The extraction buffer was prepared and kept on ice (see Recipes, section 1).
    3. 1 ml of ice-cold extraction buffer was added to each 100 mg of the ground tissue and then homogenized.
    4. The homogenate was filtered through two layers of Miracloth moistened with extraction buffer and centrifuged at 12,000 x g for 10 min at 4 °C, and the supernatant containing the crude enzyme was transferred to a fresh tube and stored on ice.
    5. The yellow layer of storage protein that forms over the pellet of starch granules was scraped off and discarded (Figure 5).


      Figure 5. Fractioning of plant tissue homogenate. After centrifugation, the pellet consists of two layers; the upper layer formed by storage proteins and the lower layer by starch granules. The supernatant is collected into a separate tube and is used for AGPase and SSS assay. The upper layer of the pellet (storage proteins) is removed by spatula and discarded. The lower layer (starch granules) is used for GBSS assay after washing.

    6. The pellet of starch granules was washed with 2-4 ml* of extraction buffer twice by centrifugation to remove all traces of SSS. Supernatant obtained after first washing was combined with initial one (step B4) and the pooled supernatant was used for the AGPase and SSS activity assays. The volume of the pooled supernatant was recorded for further calculations (V1, see Calculations, Equations 1, 2, 4, 5). Supernatant after second washing was discarded**.
      *Note: The volume of extraction buffer used for washing should be sufficient to wash the starch granule pellet, but not in excess to avoid dilution of the extract. Use of extraction buffer volume that is equal to 20-25% of the volume used in step B3 is a good practice.
      **Note: The aim of starch granule pellet washing is to eliminate SSS contamination of the GBSS fraction. The amount of SSS in the first wash may significantly affect the final result of the SSS activity assay. This is why the supernatant from the first wash is combined with the main extract. The second wash may contain some SSS activity, but this amount does not significantly affect the final result of the assay and it can be discarded to avoid extra dilution of the main extract.
    7. The pellet of starch granules was re-suspended in 0.5-2.0 ml* of extraction buffer and stored on ice until used for the GBSS activity assay. The volume of suspension was recorded and used for further calculations of GBSS activity (V6, see Calculations, Equation 7).
      *Note: The volume of buffer used to re-suspend starch granules depends on the amount of granules collected. For larger quantities of granules, use larger volume of re-suspending buffer, but do not exceed the volume used in step B6.

  3. Enzyme activity assay
    1. AGPase activity assay
      Summary of the procedures for AGPase activity assay is shown in a flowchart (Figure 6).


      Figure 6. Flowchart of AGPase assay. Please, refer to the main text of the protocol for specific conditions and buffers/media composition needed for each step of the assay.

      1. Two tubes with AGPase reaction mixture, each containing 50 mM HEPES-NaOH (pH 7.5), 1.2 mM ADP-glucose*, 5 mM PPi, 6 mM MgCl2, 3 mM DTT and 50 µl of enzyme extract (V2, see Calculations, Equations 1, 3) in a total volume of 0.45 ml were prepared.
        *Note: The reaction mixture is prepared first by mixing all components other than ADP-glucose in a volume of 423 µl as shown in Table 1 (see Recipes, section 3a).
      2. The tubes were pre-incubated at 30 °C for 2 min prior to starting the reaction by adding 27 µl of 20 mM ADP-glucose stock solution.
      3. The reaction in the first tube (reference tube) was stopped immediately after the addition of ADP-glucose by incubating the tube in water bath at 100 °C for 1 min, whereas the second tube (experimental tube) was incubated at 30 °C for 15 min (t, reaction time, see Calculations, Equations 1, 3) followed by incubation of the tube at 100 °C for 1 min*. Subsequently (steps C1d-f), the reactions in the experimental and reference tubes were processed simultaneously in a similar manner.
        *Note: Use special microcentrifuge tube lockers (see Materials and Reagents, section D4) to prevent tube opening during boiling. Tube opening at this stage may lead to partial evaporation of water and uncontrolled decrease of reaction mixture volume that may seriously affect the accuracy of the measurement.
      4. The reaction mixtures were allowed to cool down to room temperature followed by centrifuging at 10,000 x g at room temperature for 10 min.
      5. The supernatant (0.45 ml) was transferred to a new tube, and 0.1 ml of 6 mM NADP and 0.3 ml of 50 mM HEPES-NaOH (pH 7.5) were added to it. The total volume of the final reaction mix (0.85 ml, V3, see Calculations, Equations 1, 3) was recorded and used for further calculations.
      6. 0.08 U of PGM and 0.07 U of G6PDH were added in a volume of 1 μl each and the tubes were incubated at 30 °C for 10 min.
      7. The reaction mix of experimental tube was monitored in a spectrophotometer at 340 nm using the reaction mix of reference tube as a reference and the increase in readings noted every minute until constant. The final value of absorbance (A340, see Calculations, Equations 1, 3) was recorded and used for further calculations.
    2. Soluble starch synthase (SSS) activity assay
      Summary of the procedures for SSS and GBSS activity assay is shown in a flowchart (Figure 7). 


      Figure 7. Flowchart of SSS and GBSS assay. Please, refer to the main text of the protocol for specific conditions and buffers/media composition needed for each step of the assay.

      1. For SSS activity assay, two tubes with reaction mixture, each containing 50 mM HEPES-NaOH (pH 7.5), 1.6 mM ADP-glucose*, 1.4 mg amylopectin, 15 mM DTT, and 0.2 ml of enzyme extract in a total volume of 0.45 ml were prepared. The volume of enzyme extract added to starch synthase reaction mixture, as well as total volume of reaction mixture (V2 and V4, correspondingly; see Calculations, Equations 4, 6, 7) were recorded and used for further calculations.
        Note*: The reaction mixture is prepared first by mixing all the components other than ADP-glucose in a volume of 423 µl as shown in Table 2 (see Recipes, section 3b).
      2. The tubes were pre-incubated in a water bath at 30 °C for 2 min prior to starting the reaction by adding 27 µl of 20 mM ADP-glucose stock solution.
      3. The reaction in the first tube (reference tube) was stopped immediately after the addition of ADP-glucose by incubating the tube in a water bath at 100 °C for 1 min*, whereas the second tube (experimental tube) was incubated at 30 °C for 20 min (t, reaction time, see Calculations, Equations 4, 6, 7) followed by incubating the tube at 100 °C for 1 min*. Subsequently (steps C2d-g), the reactions in the experimental and reference tubes were processed simultaneously in a similar manner.
        *Note: Use special microcentrifuge tube lockers (see Materials and Reagents, section D4) to prevent tube opening during boiling. Tube opening at this stage leads to partial evaporation of water and uncontrolled decrease of reaction mixture volume that may seriously affect the accuracy of the measurement.
      4. The reaction mixtures were allowed to cool down to room temperature followed by the centrifuging at 10,000 x g at room temperature for 10 min, 0.3-0.4 ml aliquot of supernatant was collected for further analysis. The volume of the aliquot was recorded and used for further calculations (V5, see Calculations, Equations 4, 6, 7).
      5. 0.2 ml of Solution 1 (see Recipes, section 3c) was added to the aliquot of supernatant from step C2d. along with 1.2 U of pyruvate kinase in a volume of 1-2 μl and incubated at 30 °C for 20 min followed by heating the tube at 100 °C for 1 min.
      6. The reaction mixture was allowed to cool down to room temperature followed by the centrifuging at 10,000 x g at room temperature for 10 min, supernatant was transferred to a fresh tube containing 0.4 ml of Solution 2 (see Recipes, section 3d). The final volume of reaction mixture was recorded and used for further calculations (V3, see Calculations, Equations 4, 6, 7).
      7. 1.4 U hexokinase and 0.35 U G6PDH were added to the final mixture in a volume of 1-2 μl each and the tubes were incubated at 30 °C for 10 min.
      8. The reaction mix of experimental tube was monitored in a spectrophotometer at 340 nm using the reaction mix of reference tube as a reference and the increase in readings noted every minute until constant. The final value of absorbance (A340, see Calculations, Equations 4, 6, 7) was recorded and used for further calculations.
    3. Granule bound starch synthase (GBSS) activity assay
      1. Insoluble starch synthase was assayed as described above for SSS (step C2), but the reaction mixture described in section C2a was modified (see Recipes, section 3b). Amylopectin and enzyme extract were omitted, while 0.1 ml aliquot of the starch granules pellet suspension was added as a source of both enzyme and reaction substrate. The suspension was vigorously mixed before being added to the reaction mixture. Settling of granules during the assay was prevented by slow and continuous shaking of the reaction tubes.
      2. The volume of starch granules pellet suspension added was recorded and used for further calculations (V7, see Calculations, Equation 7).
      3. All further steps of GBSS assay were similar to that described for SSS (see steps C2b-h).
    4. Determination of protein content
      1. Protein content was determined in grain extracts by Bradford assay (Bradford, 1976) using Bio-Rad Protein Assay Kit and BSA as a standard.
      2. To perform the analysis, follow the instructions in the manual supplied with the kit available in your laboratory.
      3. Protein content in the enzyme extract (CPR, expressed in mg·ml-1) was used for calculation of enzymatic activity on the basis of mg of protein according to Equations 2, 3, 5, 6 (see Calculations, sections D1c and D2c).

  4. Calculations
    1. AGPase
      1. Enzyme activity of AGPase was expressed in enzyme units. In terms of this protocol, one enzyme unit (U) of AGPase is determined as the amount of enzymatic activity that converts 1 micromole of ADP-glucose into glucose-1-phosphate per 1 min at pH 7.5 and at 30 °C.
      2. Enzyme activity of AGPase on g of tissue fresh weight may be calculated according to equation (1).


        Where,
        6.22      -     Micromolar extinction coefficient for NAD(P)H at 340 nm, cm2·μmol-1,
        Abs340     -     Absorbance of reaction mixture of experimental tube measured at 340 nm against the reaction mixture of reference tube,
        AFW           -     Enzyme activity of AGPase calculated on fresh weight basis, U·g-1,
        L           -     Length of optical path, cm. This parameter is 1.0 cm in most cases,
        m          -     Fresh weight of the sample, g,
        t            -     Reaction time, min,
        V1         -     The volume of the extract, ml,
        V2         -     The volume of the extract added to the reaction mixture, ml,
        V3         -     The volume of final reaction mixture, ml.
      3. Enzyme activity of AGPase on mg of protein basis may be calculated according to equations (2) and (3)

        Where,
        Apr         -     Enzyme activity of AGPase calculated on protein basis, U·mg-1,
        Cpr             -     Protein concentration in the extract, mg·ml-1,
        and other parameters are the same as for equation (1).
    2. Starch synthase
      1. Enzyme activity of starch synthase (both SSS and GBSS) was expressed in enzyme units. In terms of this protocol, one enzyme unit (U) of starch synthase (both SSS and GBSS) is determined as the amount of enzymatic activity that converts 1 micromole of ADP-glucose into starch and 1 micromole of ADP per 1 min at pH 7.5 and at 30 °C.
      2. Enzyme activity of SSS on g of tissue fresh weight may be calculated according to equation (4).

        Where,
        AFW         -     Enzyme activity of SSS calculated on fresh weight basis, U·g-1,
        6.22      -     Micromolar extinction coefficient for NAD(P)H at 340 nm, cm2·μmol-1,
        Abs340    -     Absorbance of reaction mixture of experimental tube measured at 340 nm against the reaction mixture of reference tube,
        L           -     Length of optical path, cm. This parameter is 1.0 cm in most cases,
        m          -     Fresh weight of the sample, g,
        t            -     Reaction time, min,
        V1         -     The volume of the extract, ml,
        V2         -     The volume of the extract added to the reaction mixture, ml,
        V3         -     The volume of final reaction mixture, ml,
        V4         -     The volume of SSS reaction mixture, ml,
        V5         -     The volume of an aliquot of SSS reaction mixture taken after stopping the reaction by boiling, ml.
      3. Enzyme activity of SSS on mg of protein basis may be calculated according to equations (5) and (6)

        Where,
        Apr           -     Enzyme activity of SSS calculated on protein basis, U·mg-1,
        Cpr        -     Protein concentration in the extract, mg·ml-1,
        and other parameters are the same as for equation (4).
      4. Enzyme activity of GBSS on g of tissue fresh weight may be calculated according to equation (7)

        Where,
        AFW          -     Enzyme activity calculated on fresh weight basis,
        6.22       -     Micromolar extinction coefficient for NAD(P)H at 340 nm, cm2·μmol-1,
        Abs340    -     Absorbance of reaction mixture of experimental tube measured at 340 nm against the reaction mixture of reference tube,
        L            -     Length of optical path, cm. This parameter is 1.0 cm in most cases,
        m           -     Weight of sample, g,
        t             -     Reaction time, min,
        V3          -     The final volume of the reaction mixture, ml,
        V4          -     The volume of GBSS reaction mixture, ml,
        V5          -     The volume of an aliquot of GBSS reaction mixture taken after stopping the reaction by boiling, ml,
        V6          -     The volume of starch granules suspension, ml,
        V7          -     The volume of the starch granules suspension aliquot added to the reaction mixture, ml.
      5. In this protocol, we did not measure protein content in starch granule suspension and this is why GBSS activity in the frame of this protocol can be calculated on g of fresh weight basis only.

Recipes

  1. Extraction buffer
    100 mM HEPES-NaOH (pH 7.5)
    8 mM MgCl2
    2 mM EDTA
    1 mM DTT
    12.5% (v/v) glycerol
    5% (w/v) polyvinylpyrrolidone (PVP-10)
  2. Stock solutions
    1. ADP-glucose, disodium salt (MW = 633.31), 20 mM
      ADP-glucose stock solution was prepared in the stock container. 0.79 ml of sterile water was added directly to a bottle containing 10 mg of the powder. Solution was aliquoted in small amounts and stored at -20 °C to avoid repetitive thawing.
    2. NADP, sodium salt hydrate (MW = 765.39, anhydrous basis), 10 mM
      7.8 mg of NADP was dissolved in 1 ml sterile water. The solution was aliquoted in small amounts and stored at -20 °C to avoid repetitive thawing. Being frozen, NADP solution is stable for about a year.
    3. PEP monosodium salt hydrate (MW = 190.02, anhydrous basis), 20 mM
      3.9 mg of PEP were dissolved in 1 ml sterile water. The solution was aliquoted in small amounts and stored at -80°C to avoid repetitive thawing.
  3. Enzymatic activities reaction mixtures
    1. AGPase activity reaction mixture (Table 1)
      Note: If AGPase activity is low (Abs340 is below 0.05), the volume of enzyme extract can be increased at the expense of corresponding decrease of dH2O volume added.

      Table 1. Composition of AGPase activity reaction mixture


    2. Starch synthase activity reaction mixture (Table 2)
      Note: If SSS/GBSS activity is low (Abs340 is below 0.05), the volume of enzyme extract/starch granule suspension can be increased at the expense of corresponding decrease of dH2O volume added. In case of SSS, more concentrated DDT can be used. For example, in case of 250 mM stock, 27 μl instead of 135 μl of DTT stock has to be added and 308 μl instead of 200 μl of enzyme extract can be added to the reaction mixture.

      Table 2. Composition of starch synthase activity reaction mixture


    3. Solution 1 for starch synthase activity assay (Pyruvate kinase reaction mixture) (Table 3)
      Note: Solution 1 should be prepared fresh every time while performing the assay.

      Table 3. Composition of Solution 1 (Pyruvate kinase reaction mixture)


    4. Solution 2 for starch synthase activity assay (Glucose-6-phosphate dehydrogenase reaction mixture) (Table 4)
      Note: Solution 2 should be prepared fresh every time while performing the assay.

      Table 4. Composition of Solution 2 (G6PDH reaction mixture)

Acknowledgments

This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to BTA. The authors would like to acknowledge Ms. Nina Kulichikhina for her technical assistance. This protocol is adapted from Nakamura et al. (1989) and Schaffer and Petreikov (1997).

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: 248-254.
  2. Ghosh, H. P. and Preiss, J. (1966). Adenosine diphosphate glucose pyrophosphorylase. A regulatory enzyme in the biosynthesis of starch in spinach leaf chloroplasts. J Biol Chem 241(19): 4491-4504.
  3. Mukherjee, S., Liu, A., Deol, K. K., Kulichikhin, K., Stasolla, C., Brule-Babel, A. and Ayele, B. T. (2015). Transcriptional coordination and abscisic acid mediated regulation of sucrose transport and sucrose-to-starch metabolism related genes during grain filling in wheat (Triticum aestivum L.). Plant Sci 240: 143-160.
  4. Nakamura, Y., Yuki, K., Park, S. Y. and Ohya, T. (1989). Carbohydrate metabolism in the developing endosperm of rice grains. Plant and Cell Physiology 30(6): 833-839.
  5. Schaffer, A. A. and Petreikov, M. (1997). Sucrose-to-starch metabolism in tomato fruit undergoing transient starch accumulation. Plant Physiology 113(3): 739-746.
  6. Vos-Scheperkeuter, G. H., de Boer, W., Visser, R. G., Feenstra, W. J. and Witholt, B. (1986). Identification of granule-bound starch synthase in potato-tubers. Plant Physiology 82(2): 411-416.

简介

植物中的淀粉生物合成涉及酶的网络,其中腺苷-5'-二磷酸葡萄糖(ADP-葡萄糖)焦磷酸化酶(AGPase,EC 2.7.7.27)和可溶性和颗粒结合的淀粉合酶(SSS和GBSS,EC 2.4.1.21)中心角色。在这里,我们概述了用于提取和测定这些酶在开发小麦谷粒( Triticum aestivum L)中的方案。概述的测定法的原理基于偶联酶反应,其中初始反应的产物用作随后反应的底物,以便产生NADPH,其可以通过分光光度计容易地测量。该方案不需要昂贵的标记的化学品,并且可以使用通常在生化实验室中发现的设备进行。我们应用这个协议来研究开花后不同时间点小麦籽粒中AGPase,SSS和GBSS活性的动力学。

[背景] 淀粉是一种碳水化合物聚合物由α-1,4-连接的葡萄糖分子组成的线性葡聚糖聚合物和支链淀粉(由α-1,6-糖苷键支化的α-1,4-连接的葡萄糖分子组成的另一种葡聚糖聚合物)。腺苷-5'-二磷酸葡萄糖(ADP-葡萄糖)焦磷酸化酶(AGPase,E.C.2.7.7.27)催化淀粉合成的第一承诺步骤,将葡萄糖-1-磷酸和ATP转化为ADP-葡萄糖和无机焦磷酸(PPi)。 ADP-葡萄糖随后被可溶性淀粉合成酶(SSS)和颗粒结合的淀粉合酶(GBSS)(EC 2.4.1.21)和淀粉分支酶使用以延长和分支淀粉颗粒的葡聚糖链。  最初,使用14 P-和32 P-标记的ADP-葡萄糖进行AGP酶和淀粉合酶测定(Ghosh和Price,1966; Vos-Scheperkeuter et al。 al。,1986),其需要使用昂贵的化学品以及使用标记的化合物的专门设备。在这里,我们概述了采用和应用于在开发小麦(小麦(Triticum aestivum))谷物中提取和测定AGPase,SSS和GBSS活性的方法(Mukherjee等人, )。我们的方案是基于先前由Nakamura等人报道的方法。 (1989)用于测量发育中的谷粒胚乳中的AGP酶和SSS活性,以及Schaffer和Petreikov(1997)用于测量番茄果实中SSS和GBSS的活性。该方法基于偶联酶反应,其中初始反应的产物用作随后反应的底物以产生NADPH,其可以容易地通过分光光度计测量。该方案可以使用生化实验室中常见的设备进行,不需要使用标记的化合物。
 因为该方案是采用针对其他植物物种(米粒和番茄果实的胚乳)的不同组织开发的方法,所以针对反应缓冲液pH和底物浓度优化所有酶的反应混合物组成,使得酶活性在相对于孵育时间和蛋白质浓度的线性相。已经调节加入的酶制剂(PGM,丙酮酸激酶,己糖激酶,G6PDH)的量以在预期的时间框架内实现偶联反应的完成。该方案可用于研究AGPase,SSS和GBSS在小麦的其它组织以及其他植物物种的不同组织中的活性;然而,需要优化反应缓冲液的pH和底物浓度。
   AGP酶测定的原理示于图1中。存在于植物组织提取物中的AGP酶催化ADP-葡萄糖和PPi向葡萄糖-1-磷酸和ATP的转化。在通过煮沸停止第一反应后,将磷酸葡萄糖变位酶(PGM)加入到反应混合物中; PGM定量地将葡萄糖-1-磷酸转化为葡萄糖-6-磷酸。随后,加入葡萄糖-6-磷酸脱氢酶(G6PDH) G6PDH在NADP存在下将葡萄糖-6-磷酸转化为6-磷酸葡萄糖酸。同时,NADP转化为NADPH,形成的NADPH的量等于氧化的葡萄糖-6-磷酸的量。通过在340nm下分光光度测量NADPH浓度,我们可以量化由AGP酶活性降解的ADP-葡萄糖的量。


图1. ADP-葡萄糖焦磷酸化酶(AGPase)测定的原理

  SSS/GBSS测定的原理示于图2中。存在于植物组织提取物(SSS)或淀粉颗粒悬浮液(GBSS)中的淀粉合酶催化ADP-葡萄糖转化为ADP,与一种葡萄糖的支链淀粉引物延长残基。丙酮酸激酶在第一次r后加入到反应混合物中

关键字:酶, 粮食, 淀粉的合成, 小麦

材料和试剂

  1. 化学品
    1. 常见化学品,缓冲液和试剂盒
      1. 蛋白质测定试剂盒(Bio-Rad Laboratories,目录号:5000002)
      2. 4-(2-羟乙基)-1-哌嗪乙磺酸(HEPES)(Thermo Fisher Scientific,Fisher Scientific,目录号:BP310)
      3. 氢氧化钠(NaOH)(Thermo Fisher Scientific,Fisher Scientific,目录号:BP359)
      4. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M2670)
      5. 乙二胺四乙酸(EDTA),二钠盐二水合物(Na 2 EDTA·2H 2 O)(Sigma-Aldrich,目录号:E1644)
      6. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
      7. 聚乙烯吡咯烷酮(平均分子量10,000)(Sigma-Aldrich,目录号:PVP10)
      8. 氯化钾(KCl)(Thermo Fisher Scientific,Fisher Scientific,目录号:BP366)
      9. 焦磷酸四钠(PPi)十水合物(Na 4 P 2 O 7 O 10·10H 2 O)(Sigma-Aldrich ,目录号:S6422)
    2. 底物和辅因子
      1. 腺苷-5'-二磷酸葡萄糖(ADP-葡萄糖),二钠盐(Sigma-Aldrich,目录号:A0627)
      2. 来自玉米的支链淀粉(Sigma-Aldrich,目录号:10120)
      3. β-烟酰胺腺嘌呤二核苷酸磷酸(NADP)钠盐水合物(Sigma-Aldrich,目录号:N0505)
      4. 磷酸(烯醇)丙酮酸(PEP)单钠盐水合物(Sigma-Aldrich,目录号:P0564)
      1. 来自酿酒酵母的己糖激酶(E.C.2.7.1.1)(Sigma-Aldrich,目录号:H5000)
      2. 来自酿酒酵母的葡萄糖-6-磷酸脱氢酶(G6PDH,E.C.1.1.1.49)(Sigma-Aldrich,目录号:G7877)
      3. 来自兔肌肉(Sigma-Aldrich,目录号:P3397)的磷酸葡萄糖变位酶(PGM,E.C.5.4.2.2)
      4. 来自兔肌肉的丙酮酸激酶(E.C.2.7.1.40)(Sigma-Aldrich,目录号:P1506)
    3. 库存解决方案(参见配方)
      1. 20mM ADP-葡萄糖
      2. 10 mM NADP
      3. 20mM PEP

  2. 酶活性反应混合物
    1. 提取缓冲液(参见配方,第1部分)
    2. AGPase活性反应混合物(参见食谱,第3a部分)
    3. 淀粉合酶活性反应混合物(参见食谱,第3b部分)
    4. 用于淀粉合酶活性测定的溶液1(丙酮酸激酶反应混合物)(参见Recipes,section 3c)
    5. 用于淀粉合酶活性测定的溶液2(葡萄糖-6-磷酸脱氢酶反应混合物)(参见Recipes,第3d部分)

  3. 植物材料
    1. 普通小麦(<普通小麦 L.)颗粒

  4. 其他材料
    1. Eppendorf管(1.5和2.0ml)
    2. Falcon管(15ml)
    3. 液氮
    4. 微量离心管锁盒(例如,Sigma-Aldrich,目录号:Z708372或Sorenson Bioscience MCT LidLock TM ,目录号:11870) 注意:产品Z708372已停产。
    5. Miracloth(Merck Millipore,目录号:475855)

设备

  1. 瓷砂浆和杵
  2. 分析天平(容量 - 100g或更高,分辨率 - 0.001g或更好)
  3. 适用于30°C的数字加热块或水浴和适用于100°C的数字加热块或水浴
    注意:对于某些分析步骤,需要立即从30°C转移至100°C。这就是为什么需要同时使用两个加热装置的协议。
  4. 带适当转子的冷冻超速离心机适用于以高达12,000×g 旋转15ml Falcon管和1.5-2ml Eppendorf管。
  5. 紫外(UV)/可见分光光度计配备有加热的细胞保持器,适于保持比色皿温度在30℃。我们使用GE Healthcare/Amersham Bioscience Ultrospec 3100 Pro UV/Visible分光光度计(Biochrom Ltd,Cambridge,UK)。

程序

酶提取程序的总结如流程图(图3)所示。


图3.从发育中的小麦种子中提取AGPase,SSS和GBSS的流程图请参阅协议正文,了解每个步骤所需的特定条件和缓冲液/培养基组成。

  1. 分析前的小麦籽粒收获和储存
    在开花后不同时间点(图4),在液氮中从初级和次级分蘖收获小麦的发育谷物,并储存在-80℃直至进一步使用。必须注意不要解冻冷冻的谷物直到酶测定,因为解冻谷物将严重影响分析的结果。


    图4.开发小麦cv的种子。开花后4,8,16和25天的Yecora Rojo

  2. 萃取
    1. 在预冷却的研钵中使用液氮将小麦籽粒研磨成细粉末,并将研杵和研磨的组织转移到用液氮预冷却的空的预先称重的Falcon管中。测定磨碎的组织的重量。这通过首先称重空的Falcon管,然后称重,再次填充研磨的组织来进行。两个读数的差等于地面组织的净重。记录该值并用于进一步的计算( m ,参见计算,等式1,2,4,5,7)。
    2. 制备提取缓冲液并保存在冰上(见Recipes,第1部分)
    3. 向每100mg的研磨组织中加入1ml冰冷的提取缓冲液,然后匀化
    4. 将匀浆通过用提取缓冲液润湿的两层Miracloth过滤,并在4℃以12,000×g离心10分钟,将含有粗酶的上清液转移到新管中,并储存在冰上。
    5. 将淀粉颗粒的颗粒上形成的黄色储存蛋白层刮掉并丢弃(图5)

      图5.植物组织匀浆的分级。离心后,沉淀由两层组成;上层由储存蛋白形成,下层由淀粉颗粒形成。将上清液收集到单独的管中并用于AGP酶和SSS测定。通过刮刀除去沉淀的上层(储存蛋白质)并丢弃。下层(淀粉颗粒)在洗涤后用于GBSS测定
    6. 通过离心除去所有痕量的SSS,用2-4ml *的提取缓冲液洗涤淀粉颗粒的颗粒两次。将第一次洗涤后获得的上清液与起始的(步骤B4)组合,并将合并的上清液用于AGP酶和SSS活性测定。记录合并的上清液的体积用于进一步的计算(参见计算,方程式1,2,4和5)。弃去第二次洗涤后的上清液** 注意:用于洗涤的提取缓冲液的体积应足以洗涤淀粉颗粒沉淀,但不能过量以避免提取物的稀释。使用提取缓冲液体积等于步骤B3中使用的体积的20-25%是一种好习惯。
      注意:淀粉颗粒丸洗涤的目的是消除GBSS级分的SSS污染。第一次洗涤中SSS的量可以显着影响SSS活性测定的最终结果。这就是为什么第一次洗涤的上清液与主要提取物混合的原因。第二次洗涤可能含有一些SSS活性,但该量不会显着影响测定的最终结果,并且可以丢弃以避免主提取物的额外稀释。
    7. 将淀粉颗粒的颗粒重悬于0.5-2.0ml *的提取缓冲液中,并保存在冰上直至用于GBSS活性测定。记录悬浮液的体积并用于进一步计算GBSS活性(参见计算,等式7)。
      注意:用于重新悬浮淀粉颗粒的缓冲液的体积取决于收集的颗粒的量。对于大量的颗粒,使用较大体积的重悬缓冲液,但不要超过步骤B6中使用的体积。

  3. 酶活性测定
    1. AGPase活性测定
      AGPase活性测定程序的总结显示在流程图中(图6)。


      图6. AGPase测定的流程图。请参阅方案的正文,了解测定每个步骤所需的特定条件和缓冲液/培养基组成。

      1. 两个具有AGP酶反应混合物的管,每个含有50mM HEPES-NaOH(pH 7.5),1.2mM ADP-葡萄糖*,5mM PPi,6mM MgCl 2,3mM DTT和50μl酶提取(总计体积为0.45ml)的溶液(见计算,方程式1,3)。
        注意:如表1所示,首先通过混合除了ADP-葡萄糖之外的所有组分(体积为423μl)来制备反应混合物(参见Recipes,第3a节)。
      2. 将管在30℃下预温育2分钟,然后通过加入27μl的20mM ADP-葡萄糖储备溶液开始反应。
      3. 通过在100℃的水浴中孵育1分钟,在加入ADP-葡萄糖后立即停止第一管(参考管)中的反应,而将第二管(实验管)在30℃下孵育15分钟(反应时间,见计算,方程式1,3),然后在100℃下孵育1分钟*。随后(步骤C1d-f),以类似的方式同时处理实验管和参考管中的反应 *注意:使用特殊的微量离心管储物柜(见材料和试剂,第D4节),以防止沸腾期间管子打开。在这个阶段的管开口可以导致水的部分蒸发和反应混合物体积的不受控制的减少,这可能严重影响测量的精度。
      4. 使反应混合物冷却至室温,随后在室温下以10,000×g离心10分钟。
      5. 将上清液(0.45ml)转移至新管中,并向其中加入0.1ml的6mM NADP和0.3ml的50mM HEPES-NaOH(pH7.5)。记录最终反应混合物的总体积(0.85ml,<3> ,参见计算,方程式1,3),并用于进一步计算。
      6. 以每个1μl的体积加入0.08U PGM和0.07U G6PDH,将管在30℃下孵育10分钟。
      7. 在分光光度计中在340nm下使用参考管的反应混合物作为参照并且每分钟记录的读数的增加直到恒定来监测实验管的反应混合物。记录吸光度的最终值( ,见计算,方程式1,3),并用于进一步计算。
    2. 可溶性淀粉合成酶(SSS)活性测定
      SSS和GBSS活性测定程序的总结如流程图(图7)所示。


      图7. SSS和GBSS测定的流程图。请参阅方案的正文,了解测定每个步骤所需的特定条件和缓冲液/培养基组成。

      1. 对于SSS活性测定,将具有反应混合物的两个管,每个含有总体积为0.45μL的50mM HEPES-NaOH(pH 7.5),1.6mM ADP-葡萄糖*,1.4mg支链淀粉,15mM DTT和0.2ml酶提取物ml。加入到淀粉合酶反应混合物中的酶提取物的体积,以及反应混合物的总体积(V 1/2和V 4) ;参见计算,方程式4,6,7),并用于进一步计算。
        注:*如表2所示,首先通过混合除了ADP-葡萄糖之外的所有组分(体积为423μl)来制备反应混合物(参见配方,第3b节)。
      2. 将管在30℃的水浴中预温育2分钟,然后通过加入27μl20mM ADP-葡萄糖储备溶液开始反应。
      3. 通过在100℃的水浴中孵育1分钟*,在加入ADP-葡萄糖后立即停止第一管(参比管)中的反应,而将第二管(实验管)在30℃反应时间,参见计算,方程式4,6,7),然后将管在100℃下孵育1分钟*。随后(步骤C2d-g),以类似的方式同时处理实验管和参考管中的反应 *注意:使用特殊的微量离心管储物柜(见材料和试剂,第D4节),以防止沸腾期间管子打开。在这个阶段的管开口导致水的部分蒸发和反应混合物的不受控制的减少体积,这可能严重影响测量的准确性。
      4. 将反应混合物冷却至室温,随后在室温下以10,000×g离心10分钟,收集0.3-0.4ml等分的上清液用于进一步分析。记录等分试样的体积并用于进一步的计算(参见计算,方程4,6,7)。
      5. 将0.2ml溶液1(参见Recipes,section 3c)加入到来自步骤C2d的上清液的等分试样中。连同1.2U体积为1-2μl的丙酮酸激酶,并在30℃下温育20分钟,然后在100℃下加热管子1分钟。
      6. 将反应混合物冷却至室温,随后在室温下以10,000×g离心10分钟,将上清液转移至含有0.4ml溶液2的新管中(参见Recipes,section 3d)。记录反应混合物的最终体积并用于进一步的计算(参见计算,方程式4,6,7)。
      7. 将1.4U己糖激酶和0.35U G6PDH加入到最终混合物中,每个体积为1-2μl,将管在30℃温育10分钟。
      8. 在分光光度计中在340nm下使用参考管的反应混合物作为参照并且每分钟记录的读数的增加直到恒定来监测实验管的反应混合物。记录吸光度的最终值( ,见计算,方程式4,6,7),并用于进一步计算。
    3. 颗粒结合淀粉合酶(GBSS)活性测定
      1. 如上所述对SSS(步骤C2)测定不溶性淀粉合酶,但修饰C2a部分中描述的反应混合物(参见Recipes,第3b节)。省略支链淀粉和酶提取物,同时加入0.1ml等分的淀粉颗粒悬浮液作为酶和反应底物的来源。将悬浮液剧烈混合,然后加入到反应混合物中。通过缓慢和连续振荡反应管来防止测定期间颗粒的沉降
      2. 记录添加的淀粉颗粒丸粒悬浮液的体积并用于进一步的计算(参见计算,方程式7)。
      3. GBSS测定的所有其它步骤与针对SSS所描述的相似(参见步骤C2b-h)。
    4. 蛋白质含量的测定
      1. 通过Bradford测定(Bradford,1976),使用Bio-Rad蛋白测定试剂盒和BSA作为标准,在谷物提取物中测定蛋白质含量。
      2. 要执行分析,请按照实验室中提供的套件提供的手册中的说明进行操作
      3. 酶提取物中的蛋白质含量( C PR 以 mg·ml 1 )用于根据等式2,3,5,6(基于计算,D1c和D2c部分)基于mg蛋白的酶活性计算, 。

  4. 计算
    1. AGPase
      1. AGP酶的酶活性以酶单位表示。 就该方案而言,将AGP酶的一个酶单位(μg)确定为在pH7.5下每1分钟将1微摩尔ADP-葡萄糖转化为葡萄糖-1-磷酸酯的酶活性量, 在30℃
      2. AGP酶的酶活性对组织鲜重的影响可以根据公式(1)计算。


        在哪里,
        6.22      -        NAD(P)H在340nm处的微摩尔吸光系数, cm 2 ·μmol< em> -1 ,
        Abs 340      -       Absorbance在340nm处测量的实验管对参比管的反应混合物的反应混合物,
        FW          -       AGPase的酶活性以鲜重计算,U·g -1 em>,
        L               -   -      光程长度,/em>。在大多数情况下,此参数为1.0 cm,
        m         -     样品的鲜重, g ,
        t                 ,
        V 1             -      提取量, ml ,
        V 2            -      添加到反应混合物中的提取液体积, ml ,

        在哪里,
        AGP酶的酶活性以蛋白质计算,U·mg -1
        C pr              -      提取物中的蛋白质浓度, mg·ml -1
        和其他参数与等式(1)相同。
    2. 淀粉合酶
      1. 淀粉合酶(SSS和GBSS)的酶活性以酶单位表示。 就该方案而言,将淀粉合酶(SSS和GBSS)的一个酶单位(U e)确定为将1微摩尔ADP-葡萄糖转化为淀粉的酶活性量和1微摩尔 ADP每1分钟,pH7.5和30℃
      2. SSS的酶活性对组织鲜重的影响可以根据等式(4)计算。

        在哪里,
        ; SSS的酶活性基于鲜重计算,U·g -1
        6.22       - 在340nm处的NAD(P)H的微摩尔吸光系数,cm 2 ·μmol -1
        Abs 340nm,相对于参比管的反应混合物,
                     在大多数情况下,此参数为1.0 cm,
        m             -       样品的鲜重,/em>,
        t            -      反应时间,最小,
         提取量, ml ,
        V  添加到反应混合物中的提取物体积, ml ,
         最终反应混合物的体积, ml ,
          SSS反应混合物的体积, ml ,
         在通过煮沸停止反应后取得的SSS反应混合物的等分试样的体积,em 。
      3. 可以根据等式(5)和(6)计算SSS对蛋白质的酶活性。

        在哪里,
        ;以蛋白质计算的SSS的酶活性,U·mg -1
        C 提取物中的蛋白质浓度, mg·ml -1
        和其他参数与等式(4)相同。
      4. GBSS的酶活性对组织鲜重的影响可以根据等式(7)计算:

        在哪里,
        FW           -      酶活性以鲜重计算,
        6.22        - 在340nm处的NAD(P)H的微摩尔吸光系数,cm 2 ·μmol -1
        Abs 在340nm下相对于参比管的反应混合物,
                          > cm 。在大多数情况下,此参数为1.0 cm,
        m            -       /em>,
        t > min ,
        V 3            -      反应混合物的最终体积,
        V 4           -        GBSS反应混合物的体积, ml ,
        V 5         -      通过煮沸停止反应后取得的GBSS反应混合物的等分试样的体积, ml ,
        V 6          -      淀粉颗粒悬浮液的体积, ml ,
        V 7          -       将添加到反应混合物中的淀粉颗粒悬浮液等分试样的体积, ml
      5. 在该方案中,我们没有测量淀粉颗粒悬浮液中的蛋白质含量,这就是为什么本协议框架中的GBSS活性只能基于鲜重计算。

食谱

  1. 提取缓冲区
    100mM HEPES-NaOH(pH7.5) 8mM MgCl 2/v/v 2mM EDTA 1 mM DTT
    12.5%(v/v)甘油 5%(w/v)聚乙烯吡咯烷酮(PVP-10)
  2. 库存解决方案
    1. ADP-葡萄糖,二钠盐(MW = 633.31),20mM
      在储备容器中制备ADP-葡萄糖储备溶液。将0.79ml无菌水直接加入含有10mg粉末的瓶中。将溶液少量分装,并储存在-20℃以避免重复解冻
    2. NADP,钠盐水合物(MW = 765.39,无水基),10mM 将7.8mg NADP溶解于1ml无菌水中。将溶液少量等分并保存在-20℃以避免重复解冻。被冷冻后,NADP溶液稳定约一年。
    3. PEP单钠盐水合物(MW = 190.02,无水基),20mM/dm 将3.9mg PEP溶解于1ml无菌水中。将溶液少量分装并储存在-80℃下以避免重复解冻。
  3. 酶活性反应混合物
    1. AGP酶活性反应混合物(表1) 注意:如果AGP酶活性低(Abs <340>低于0.05),则可以以相应降低dH 2 O O为代价增加酶提取物的体积 已添加卷。

      表1. AGPase活性反应混合物的组成


    2. 淀粉合酶活性反应混合物(表2)
      注意:如果SSS/GBSS活性低(Abs 340低于0.05),则酶提取物/淀粉颗粒悬浮液的体积可以以dH的相应降低为代价而增加, 2 O体积。 在SSS的情况下,可以使用更浓缩的DDT。 例如,在250mM储备液的情况下,必须加入27μl而不是135μl的DTT储液,并且可以向反应混合物中加入308μl而不是200μl的酶提取物。

      表2.淀粉合酶活性反应混合物的组成


    3. 用于淀粉合酶活性测定的溶液1(丙酮酸激酶反应混合物)(表3)
      注意:在进行测定时,溶液1应每次都新鲜。

      表3.溶液1(丙酮酸激酶反应混合物)的组成


    4. 用于淀粉合酶活性测定的溶液2(葡萄糖-6-磷酸脱氢酶反应混合物)(表4)
      注意:在进行测定时,溶液2应每次清新。

      表4.溶液2(G6PDH反应混合物)的组成

致谢

这项工作得到了加拿大自然科学和工程研究理事会给予BTA的资助。 作者 感谢Nina Kulichikhina女士提供的技术援助。 该协议改编自Nakamura等人。 (1989)和Schaffer和Petreikov(1997)。

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 72:248-254。
  2. Ghosh,HP和Preiss,J.(1966)。  腺苷二磷酸葡萄糖焦磷酸化酶。在菠菜叶绿体中淀粉生物合成中的调节酶。 J Biol Chem 241(19):4491-4504。
  3. Mukherjee,S.,Liu,A.,Deol,KK,Kulichikhin,K.,Stasolla,C.,Brule-Babel,A.and Ayele,BT(2015)。  在谷物灌浆期间蔗糖转运和蔗糖到淀粉代谢相关基因的转录协调和脱落酸介导的调节( Triticum aestivum L.)。 Plant Sci 240:143-160。
  4. Nakamura,Y.,Yuki,K.,Park,SY和Ohya,T。(1989)。  在稻谷发育的胚乳中的碳水化合物代谢。植物和细胞生理学 30(6):833-839。
  5. Schaffer,AA和Petreikov,M。(1997)。  淀粉代谢在番茄果实中经历瞬时淀粉积累。植物生理学113(3):739-746。
  6. Vos-Scheperkeuter,GH,de Boer,W.,Visser,RG,Feenstra,WJand Witholt,B。(1986)。  在马铃薯块茎中鉴定颗粒结合的淀粉合酶。植物生理学82(2):411-416。
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引用:Kulichikhin, K., Mukherjee, S. and Ayele, B. T. (2016). Extraction and Assays of ADP-glucose Pyrophosphorylase, Soluble Starch Synthase and Granule Bound Starch Synthase from Wheat (Triticum aestivum L.) Grains . Bio-protocol 6(18): e1929. DOI: 10.21769/BioProtoc.1929.
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