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Determination of Enzyme Kinetic Parameters of UDP-glycosyltransferases
UDP糖基转移酶动力学常数的测定   

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Abstract

The determination of enzyme kinetic parameters, such as the Km and kcat values, is an essential part of the characterization of newly discovered enzymes. This protocol describes the determination of enzyme kinetic parameters of the Barbarea vulgaris UDP-glycosyltransferases (UGTs) UGT73C11 and UGT73C13 toward the sapogenins oleanolic acid and hederagenin as sugar acceptor substrates. UGTs catalyze the transfer of glycosyl residues. They generally use uridine sugar nucleotides as their sugar donor substrates, whereas sugar acceptor substrates arise from structurally diverse sets of metabolite classes. This protocol is based on the quantification of 14C-labeled glycosides following thin layer chromatography (TLC)-based separation. The dependence of the measured signal on a universal radioactively-labeled sugar donor substrate allows the potential application of the protocol in combination with a wide range of different sugar acceptor substrates. However, since the here described TLC separation procedure has been optimized for the separation of sapogenins and their glycosides, some modifications may become necessary when investigating other compound classes.


Figure 1. Glucosylation reaction catalyzed by UGT73C10-UGT73C13 from Barbarea vulgaris (Augustin et al., 2012). All four enzymes utilize uridine diphosphate glucose (UDP-glc) as glucosyl-moiety donor substrate and different sapogenins such as the oleanane sapogenins oleanolic acid and hederagenin as glucosyl-moiety acceptor substrates.


Materials and Reagents

  1. Uridine-5'-diphosphoglucose (UDP-Glc) (e.g. Sigma-Aldrich, catalog number: S451649 )
  2. Uridine-5'-diphosphate-[14C]glucose (UDP-[14C]Glc) (e.g. PerkinElmer, catalog number: NEC403050UC )
  3. N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) (e.g. Sigma-Aldrich, catalog number: T5130 )
  4. Bovine serum albumin (BSA) (e.g. Sigma-Aldrich, catalog number: A7906 )
  5. Dithiothreitol (DTT) (e.g. Sigma-Aldrich, catalog number: D0632 )
  6. Silica gel 60 F254  plates (e.g. EMD Millipore, catalog number: 1055540001 )
  7. Appropriate E. coli protein expression strain (e.g. strain XJb(DE)) (Zymo Research, catalog number: T5051 )
  8. FRETWorks S-tag assay kit (EMD Millipore, catalog number: 70724 )
  9. TAPS buffer
  10. 1.6 mM oleanolic acid stock solution (see Recipes)
  11. 1.6 mM hederagenin stock solution (see Recipes)
  12. Pre-master mix for 36 UGT73C11 enzyme assay reactions (see Recipes)
  13. Pre-master mix for 37 UGT73C13 enzyme assay reactions (see Recipes)

Equipment

  1. Vacuum centrifuge (e.g. Labogene, catalog number: 7.008.100.777 )
  2. TLC Developing Chamber (e.g. VWR international, catalog number: 21432-739 )
  3. Storage phosphor screens (e.g. GE healthcare, catalog number: 28-9564-74 )
  4. Phosphorimager (e.g. Molecular Dynamics, model: STORM 840 )

Software

  1. ImageQuant 5.0 (Molecular Dynamics) or similar
  2. SigmaPlot 11.0 (Systat Software, Inc.) or similar

Procedure

I.   Preparation of radiolabeled sapogenin-glucoside standards and TLC plates with reference dilution series

  1. 100 μl (6.623 nmol [74 kBq]) UDP-[14C]Glc were evaporated to dryness in a 1.5 ml microcentrifugation tube using a vacuum centrifuge. The same microcentrifugation tube was used to prepare 200 μl of a glucosylation reaction master mix that was used to generate the reference glucosides.
  2. Final reaction conditions of the glucosylation reaction were adjusted to 25 mM TAPS pH 8.6, 1 mM DTT, 33.12 μM UDP-[14C]Glc (by dissolving the dried 6.623 nmol) and 467 μM non-radioactive UDP-Glc (additionally added).
  3. Frozen (-80 °C) E. coli cells resuspended in 10 mM TAPS buffer pH 8.0 were lysed by thawing (see Notes) and insoluble cell debris were removed by centrifugation for 20 min at 20,000 x g, 4 °C.
  4. The concentration of the recombinant UGTs in the E. coli lysate was determined by applying the FRETWorks S-tag assay kit according to manufacturer's instructions.
  5. E. coli lysate containing recombinant UGT73C11 was added to the master mix to a final concentration of 200 ng/μl UGT73C11. 60 μl of this master mix were transferred to 0.5 ml microcentrifugation tubes.
  6. Enzymatic reactions were started by adding 4 μl of a 1.6 mM aglycone stock solution. In our case the applied aglycones were the sapogenins oleanolic acid and hederagenin that had been solubilized in 100% DMSO (thus, final concentrations of the aglycone and DMSO in the enzymatic reaction were 100 μM and 6.25%, respectively).
  7. Complete conversion of the supplied aglycones to their corresponding glucosides was achieved by incubating the reactions overnight (18 h) at 37 °C (shaking is not necessary for this step).
  8. Conversion efficiency was evaluated by TLC analysis. For this purpose both sapogenin-glucosides and possibly remaining sapogenins were 4 times extracted with 50 μl ethyl acetate from a 20 μl aliquot of the complete reaction.
  9. The merged ethyl acetate fractions were evaporated to dryness in a vacuum centrifuge.
  10. The dried extracts were dissolved in 20 μl 96% ethanol and this solution stepwise (3.5 μl per step) spotted to a silica gel TLC plate. The TLC plate was pre-run for 1-2 min using methanol as mobile phase until the solvent front was approximately 1 cm above the loading line.
  11. The methanol was left to evaporate in a fume hood and the dry plate subsequently developed using dichloromethane: methanol: water (80:19:1) as mobile phase. After evaporation of the mobile phase the developed plate was sprayed with 10% sulfuric acid in methanol and heated to 100 °C until sapogenin and sapogenin-glucosides became visible as reddish bands on the TLC plate.
  12. The stained TLC plate was evaluated under visible as well as under long wave UV (366 nm) light.
  13. To generate standard curves of the 14C-labled sapogenin-glucosides, sequential dilutions of the glucosidation reactions were made.
  14. The highest chosen sapogenin-glucoside concentration for the standard curve was a 1:5 dilution of the overnight reaction with 62.5% ethanol, corresponding to a 20 μM sapogenin-glucoside concentration.
  15. This dilution was 11 times sequentially diluted with 50% ethanol, thereby generating dilutions with sapogenin-glucoside concentrations of 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.313 μM, 0.156 μM, 0.078 μM, 0.039 μM, 0.020 μM and 0.010 μM.
  16. 20 μl of each dilution were in 5 μl steps loaded to a silica gel TLC plate, which corresponds to sapogenin-glucoside amounts per spot of 400 nmol, 200 nmol, 100 nmol, 50 nmol, 25 nmol, 12.5 nmol, 6.25 nmol, 3.13 nmol, 1.56 nmol, 0.78 nmol, 0.39 nmol and 0.20 nmol, respectively.
  17. After evaporation of all solvents the TLC plates were pre-run for 1-2 min using methanol as mobile phase until the solvent front was approximately 1 cm above the loading line.
  18. The methanol was left to evaporate in a fume hood, and the TLC plates were afterwards developed with dichloromethane: methanol: water (80:19:1) as mobile phase.
  19. All solvents were left to evaporate and 1.5 μl, 1.0 μl and 0.5 μl of the original master mix of the glucosylation reaction (before addition of the sapogenins) were spotted on an unused side lane of the TLC plate. These spots would allow at a later point normalization of variation in the radioactivity between different reaction master mixes.
    Notes:
    1. Local regulations may require you to work with radioactive compounds in specialized areas. Please inform yourself about regulations and guidelines of working with radioactive compounds that apply at your workplace.
    2. UDP-[14C]Glc was dried out prior to usage, since Perkin Elmer provides this reagent solubilized in 70% ethanol, which serves as a sugar acceptor substrate for the investigated UGTs itself.
    3. Recombinant UGTs were in this case expressed in the BL21(DE) derivative XJb(DE). This strain expresses a viral endolysin protein and thus allows to be lysed by simply thawing after being frozen.
    4. Quantification with the FRETWorks S-tag assay kit is based on regeneration of RNaseS activity due the interaction of the S protein (included in the kit) and the S-tag N-terminally fused to the recombinant expressed UGTs. See kit manual for additional information.
    5. As references for the unmodified sapogenins were 2 nmol oleanolic acid and hederagenin loaded to the TLC plate that was used to determine the conversion efficiency.
    6. Sapogenin/saponin-glucoside evaluation on sulfuric acid stained TLC plates under long wave UV light is approximately 10 times more sensitive than under visible light.


    Figure 2. TLC plate with aliquots of the reactions to generate radiolabled (1) 3-O-[14C]glc-oleanolic acid (oa-glc) or (3) 3-O-[14C]glc-hederagenin (he-glc) standards.
    The TLC plate was evaluated under (A) visible (colored) as well as under (B) long wave UV light (366 nm, black/white). For comparison purpose were authentic (oa) oleanolic acid and (he) hederagenin (2 nmol each) loaded to lane (2).


II.  Preparation of enzyme assays for the determination of enzyme kinetic parameters

  1. In preparation of the UGT73C11 enzyme assays were 228 μl UDP-[14C]Glc (15.10 nmol [168.72 kBq]) evaporated to dryness in a 1.5 ml microcentrifugation tube using a vacuum centrifuge.
  2. 2.39 μl and 2.18 μl of a 1 mM UDP-Glc (non-radioactive) solution were evaporated to dryness in two additional 1.5 ml microcentrifugation tubes. Simultaneously, were for the UGT73C13 assays 86.4 μl UDP-[14C]Glc (5.72 nmol [63.94 kBq]), 1.99 μl 0.5 mM UDP-Glc (non-radioactive) and 1.59 μl 0.25 mM UDP-Glc (non-radioactive) dried out in 1.5 ml microcentrifugation tubes.
  3. Of the acceptor substrates, oleanolic acid and hederagenin, were for the UGT73C11 assays stock solutions with concentrations of 128 μM, 96 μM, 64 μM, 32 μM, 16 μM, 8 μM, 4 μM and 2 μM in 100% DMSO prepared.
  4. For the UGT73C13 assays were acceptor substrate stock solutions of 1,600 μM, 1,200 μM, 800 μM, 400 μM, 200 μM, 100 μM, 50 μM and 25 μM prepared.
  5. Frozen (-80 °C) aliquots of E. coli cells resuspended in 10 mM TAPS buffer pH 8.0 were lysed by thawing (see Notes) and insoluble cell debris removed by centrifugation for 20 min at 20,000 x g, 4 °C.
  6. The concentration of the recombinant UGTs in the E. coli lysate was determined by applying the FRETWorks S-tag assay kit according to manufacturer's instructions.
  7. The E. coli lysates were diluted to either 5 ng/μl (UGT73C11) or 50 ng/μl (UGT73C13) recombinant protein with 10 mg/ml BSA in 10 mM TAPS buffer pH 8.0.


III. Enzyme assays for UGT73C11

  1. Preparation of pre-master mix for 36 enzyme assay reactions as described in the recipe section.
  2. 427.4 μl of this pre-master mix were added to the microcentrifuge tube that contained the dried UDP-[14C]Glc to prepare a reaction master mix that would result with concentrations of the sugar donor substrate of 33.12 μM UDP-[14C]Glc (0.37 kBq/μl) and 467 μM non-radioactive UDP-Glc in the final enzyme assay.
  3. 96 μl of this master mix were transferred to the tube in which 2.39 μl 1 mM UDP-Glc had been dried out and mixed with 144 μl of the original pre-master mix. Enzyme assays set up with this master mix would have a sugar donor substrate concentration of 13.25 μM UDP-[14C]Glc (0.15 kBq/μl) and 487 μM non-radioactive UDP-Glc.
  4. 82 μl of the latter master mix were transferred to the tube in which 2.18 μl 1 mM UDP-Glc had been dried out, and mixed with 82 μl of the original pre-master mix. This last master mix is for preparing enzyme assays with a concentration of 6.62 μM UDP-[14C]Glc (0.08 kBq/μl) and 493 μM non-radioactive UDP-Glc.
  5. 18.75 μl aliquots of the master mixes were transferred to 1.5 ml microcentrifuge tubes on ice.
  6. For performing the actual assays, tubes with master mix aliquots were pre-incubated for 3 min at 30 °C and the enzymatic reaction started by addition of 1.25 μl acceptor substrate stock solutions prepared in step B-3.
    1. The master mix with the highest concentration of UDP-[14C]Glc (33.12 μM) was used to assay UGT73C11 with final sugar acceptor substrate concentrations of 1 μM, 0.5 μM, 0.25 μM and 0.125 μM, by adding 1.25 μl of the 16 μM, 8 μM, 4 μM and 2 μM oleanolic acid or hederagenin stock solutions.
    2. Similarly was the 13.25 μM UDP-[14C]Glc master mix used for final concentrations of 4 and 2 μM oleanolic acid and hederagenin, and the 6.62 μM UDP-[14C]Glc master mix for final concentrations of 8 and 6 μM of the two acceptor substrates. The corresponding stock solutions in these cases were 64 μM, 32 μM, 128 μM and 96 μM.
  7. After addition of the acceptor substrate, enzymatic reactions were allowed to take place by incubation for 3 min at 30 °C, and subsequently stopped by addition of 50 μl ethyl acetate and vigorous mixing for 10 sec.


  • IV.
  • Enzyme assays for UGT73C13 (in general similar to procedure C, however, enzyme and substrate concentrations differ, as UGT73C13 is less efficient in catalyzing the investigated reaction)
  1. Preparation of pre-master mix for 37 enzyme assay reactions as described in the recipe section.
  2. 405 μl of the pre-master mix were transferred to the tube with the dried UDP-[14C]Glc to prepare a reaction master mix for enzyme assays with a final concentration of 13.25 μM UDP-[14C]Glc (0.15 kBq/μl) and 487 μM non-radioactive UDP-Glc.
  3. 150 μl of this master mix were mixed with 150 μl of the pre-master mix in the microcentrifugation tube in which 1.99 μl 0.5 mM UDP-Glc had been dried out. This master mix was applied for reactions with a final sugar donor substrate concentrations of 6.62 μM UDP-[14C]Glc (0.08 kBq/μl) and 493 μM non-radioactive UDP-Glc.
  4. For a master mix for enzyme assays with a final UDP-[14C]Glc concentration of 3.31 μM (0.04 kBq/μl) and 497 μM non-radioactive UDP-Glc 120 μl of the previous master mix were diluted with 120 μl of the pre-master mix in the tube that contained the dried out 1.59 μl 0.25 mM UDP-Glc. The 13.25 μM UDP-[14C]Glc master mix was applied for assays with final oleanolic acid and hederagenin concentrations of 1.56 μM, 3.12 μM and 6.25 μM.
  5. 1.25 μl of the 25 μM, 50 μM and 100 μM sugar acceptor substrate stock solutions were added to 18.75 μl master mix to adjust these sugar acceptor substrate concentrations. Similarly, was the 6.62 μM UDP-[14C]Glc master mix used to assay final sugar acceptor substrate concentration of 12.5 μM and 25 μM (200 μM and 400 μM stock solutions), and the 3.31 μM UDP-[14C]Glc master mix for sugar acceptor substrate concentrations of 50 μM, 75 μM and 100 μM (800 μM, 1,200 μM and 1,600 μM stock solutions).


V.  Analysis of the enzyme assays

  1. Stopped assays were 4 times extracted with 50 μl ethyl acetate and the merged ethyl acetate extracts evaporated to dryness in a vacuum centrifuge.
  2. The dried extracts were dissolved in 20 μl 96% ethanol and in 5 μl steps loaded to silica gel TLC plates.
  3. Potential remainders of the radioactive sapogenin-glucosides were washed out with another 20 μl 96% ethanol and loaded to the same spots on the TLC plates.
  4. The TLC plates were pre-run for 1-2 min using methanol as mobile phase until the solvent front was approximately 1 cm above the loading line.
  5. The methanol was left for evaporation in a fume hood and the dried TLC plates were afterwards developed using dichloromethane: methanol: water (80:19:1) as mobile phase.
  6. After evaporation of the mobile phase, 0.5 μl, 1.0 μl and 1.5 μl of the master mix with the highest UDP-[14C]Glc were spotted on an unused lane of the TLC plate. Additionally, were also 1 μl of the two remaining master mixes with the lower UDP-[14C]Glc spotted to each TLC plate.
  7. The dried TLC plates were together with one of the prepared reference dilution series TLC plates of the same sapogenin-glucoside exposed for several days to a storage phosphor screen.
  8. The exposed phosphor screens were scanned with a STORM 840 Phosphorimager and signal intensities in the digital scans quantified using ImageQuant 5.0.
  9. To compensate for the 10-fold lower concentration of the radioactive sugar donor substrate as compared to the master mix used for generating the reference glucosides, were all sapogenin-glucoside intensities resulting from assays with a 3.31 μM UDP-[14C]Glc master mix prior to their further evaluation multiplied by factor 10. Similarly, were sapogenin-glucoside intensities resulting from 13.25 μM and 6.62 μM UDP-[14C]Glc master mixes multiplied by 2.5 and 5, respectively.
  10. To further address unintended variation in the amounts of UDP-[14C]Glc in the master mixes used for generating the reference glucosides and the actual enzyme assays, all glucoside intensities of the assays were multiplied with the ratio between the intensities of the corresponding master mix spots on each TLC plate.
  11. Km and Vmax  values were calculated using SigmaPlot 11.0 to perform non-linear regression according to the Michaelis-Menten equation or the velocity equation for substrate inhibition (please refer to the SigmaPlot manual for an instruction how to perform regressions).
    Notes:
    1. Recombinant UGTs were in this case expressed in the BL21(DE) derivative XJb(DE). This strain expresses a viral endolysin protein and thus allows to be lysed by simply thawing after being frozen.
    2. Upon lysis of the E. coli cells all lysates, lysate dilutions and master mixes were constantly kept on ice.
    3. Quantification with the FRETWorks S-tag assay kit is based on regeneration of RNaseS activity due the interaction of the S protein (included in the kit) and the S-tag N-terminally fused to the recombinant expressed UGTs. See kit manual for additional information.
    4. The E. coli lysates were diluted with a BSA solution instead of pure buffer, since the specific activity of the recombinant UGTs was seen to decrease upon reduction of total protein concentration.
    5. The final amount of the investigated enzyme should be chosen based on its activity. If too much of the acceptor substrate is used up within the applied incubation time, the determined reaction velocity will not be in the initial linear range anymore.
    6. The intention behind using master mixes with different ratios of radiolabeled to non-labeled UDP-Glc was to use as low amounts of the radiolabeled UDP-[14C]Glc as possible, while still ensuring  sufficient signal strength for assays with the lowest acceptor substrate concentrations. The final total concentration of 500 μM UDP-Glc was kept throughout the experiment to provide saturating conditions of the sugar donor substrate.
    7. The different pH in assays of UGT73C11 and UGT73C13 were chosen due to different pH optima of the two enzymes.
    8. Which concentrations of the acceptor substrate are tested, depends on the investigated enzyme. For deciding optimal concentrations, it is helpful to estimate the Km value in pre-experiments and choose concentrations below and above the estimated Km value for the final experiment.
    9. The optimal reaction time is dependent on the enzyme activity and the enzyme amount. Time course experiments have to be performed to determine if the reaction velocity is still in the initial linear range after the chosen incubation time. If too much of the acceptor substrate is converted at the lowest applied concentrations, resulting v/S-characteristics are typically sigmoid instead of hyperbolic.


    Figure 3. Autoradiogram of TLC plates to determine kinetic parameters of UGT73C11 towards hederagenin.
    The TLC plates with the ethyl acetate extracts from the actual enzyme assays are on the top as well as on the bottom, whereas the co-exposed TLC plate with the [14C]Glc-hederagenin standard curve is located in the middle. The band representing 3-O-[14C]Glc-hederagenin is for each plate marked with an arrow and the label he-glc. The concentrations below the he-glc bands of the UGT73C11 enzyme assay extracts represent the hederagenin concentration in the corresponding enzyme assay, while I and II mark duplicates. Substance amounts below each standard curve lane represent the amount of [14C]Glc-hederagenin present in the corresponding lane. Master mix aliquots to account for variation in the amount of UDP-[14C]Glc in the master mix preparation (MM1, MM2, MM3) were spotted either right or left of lanes with the actual assays or standards.

Recipes

  1. 1.6 mM oleanolic acid stock solution
    Dissolve 7.30 mg oleanolic acid in 10 ml DMSO
  2. 1.6 mM hederagenin stock solution
    Dissolve 7.56 mg hederagenin in 10 ml DMSO
  3. Pre-master mix for 36 UGT73C11 enzyme assay reactions
    36 μl 500 mM TAPS buffer pH 8.6
    3.6 μl 200 mM DTT
    36 μl 9.34 mM UDP-Glc
    72 μl 10 mg/ml BSA
    72 μl 5 ng/μl UGT73C11 (diluted E. coli lysate)
    455.4 μl water
  4. Pre-master mix for 37 UGT73C13 enzyme assay reactions
    37 μl 500 mM TAPS buffer pH 7.9
    37 μl 200 mM DTT
    37 μl 9.34 mM UDP-Glc 
    74 μl 10 mg/ml BSA
    74 μl 50 ng/μl UGT73C13 (diluted E. coli lysate)
    465.05 μl water

Acknowledgments

This protocol was adapted and modified from various, previous 14C-UDP-glucose-based UGT enzyme assay protocols commonly applied in the Section for Plant Biochemistry – Department for Plant Biochemistry Biotechnology – Faculty of Life Sciences – University of Copenhagen and preceding organizations. This work was supported by the Danish Council for Independent Research, Technology, and Production Sciences (grant nos. 09–065899/FTP and 274–06–0370), by the Villum Kann Rasmussen Foundation to Pro-Active Plants, and by a PhD stipend from the Faculty of Life Sciences, University of Copenhagen (to J.M.A.).

References

  1. Augustin, J. M., Drok, S., Shinoda, T., Sanmiya, K., Nielsen, J. K., Khakimov, B., Olsen, C. E., Hansen, E. H., Kuzina, V., Ekstrom, C. T., Hauser, T. and Bak, S. (2012). UDP-glycosyltransferases from the UGT73C subfamily in Barbarea vulgaris catalyze sapogenin 3-O-glucosylation in saponin-mediated insect resistance. Plant Physiol 160(4): 1881-1895.

简介

测定酶动力学参数,如Km和kcat值,是新发现的酶的表征的重要组成部分。该方案描述了通过将Barbarea vulgaris UDP-糖基转移酶(UGTs)UGT73C11和UGT73C13的酶动力学参数作为糖受体底物朝向皂甙元酸齐墩果酸和雄蕊草素的测定。 UGTs催化糖基残基的转移。他们通常使用尿苷糖核苷酸作为其供体底物,而糖受体底物来自结构不同的代谢物类别。该方案基于以薄层色谱(TLC)为基础的分离后14C标记的糖苷的定量。测量信号对通用放射性标记的糖供体底物的依赖性允许将该方案与广泛范围的不同糖受体底物结合使用。然而,由于这里描述的TLC分离程序已被优化用于分离皂角苷及其糖苷,所以在研究其它化合物类时可能需要进行一些修饰。


(Augustin等人,2012)。所有四种酶都利用尿苷二磷酸葡萄糖(uridine diphosphate glucose)。来自Barbarea vulgaris的UGT73C10-UGT73C13催化的葡糖基化反应 (UDP-glc)作为葡糖基部分供体底物和不同的皂苷元,例如齐墩果烷皂甙元齐墩果醇酸和常春藤苷配基作为葡糖基部分受体底物。


材料和试剂

  1. 尿苷-5'-二磷酸葡萄糖(UDP-Glc)(例如Sigma-Aldrich,目录号:S451649)
  2. 尿苷-5'-二磷酸 - [14 C]葡萄糖(UDP- [14 C] Glc)(例如,PerkinElmer,目录号:NEC403050UC )
  3. N-三(羟甲基)甲基-3-氨基丙磺酸(TAPS)(例如Sigma-Aldrich,目录号:T5130)
  4. 牛血清白蛋白(BSA)(例如Sigma-Aldrich,目录号:A7906)
  5. 二硫苏糖醇(DTT)(例如Sigma-Aldrich,目录号:D0632)
  6. 硅胶60F 254 板(例如 EMD Millipore,目录号:1055540001)
  7. 适当的E。 大肠杆菌蛋白质表达菌株(例如菌株XJb(DE))(Zymo Research,目录号:T5051)
  8. FRETWorks S标签测定试剂盒(EMD Millipore,目录号:70724)
  9. TAPS缓冲区
  10. 1.6 mM齐墩果酸储备溶液(见配方)
  11. 1.6 mM常春配基原液(见配方)
  12. 用于36UGT73C11酶测定反应的预制备混合物(参见配方)
  13. 用于37 UGT73C13酶测定反应的预主混合物(参见Recipes)

设备

  1. 真空离心机(例如Labogene,目录号:7.008.100.777)
  2. TLC显影室(例如,VWR international,目录号:21432-739)
  3. 储存荧光屏(如 GE healthcare,目录号:28-9564-74)
  4. 磷光体(例如 Molecular Dynamics,型号:STORM 840)

软件

  1. ImageQuant 5.0(Molecular Dynamics)或类似的
  2. SigmaPlot 11.0(Systat Software,Inc.)或类似的

程序

I.   放射性标记的皂苷元 - 葡萄糖苷标准品和具有参考稀释系列的TLC板的制备

  1. 将100μl(6.623nmol [74kBq])UDP- [14 C] Glc蒸发至干   在1.5ml微量离心管中使用真空离心机。 的 使用相同的微量离心管制备200μl的a 葡萄糖化反应主混合物用于产生 参考葡萄糖苷。
  2. 最终反应条件 葡糖基化反应调节至25mM TAPS pH 8.6,1mM DTT, 33.12μMUDP- [14 C] Glc(通过溶解干燥的6.623nmol)和467μM 非放射性UDP-Glc(另外加入)。
  3. 冷冻(-80℃)。 将重悬浮于10mM TAPS缓冲液pH 8.0中的大肠杆菌细胞通过解冻裂解   (参见注释),并通过离心除去不溶性细胞碎片 在20000xg,4℃下20分钟。
  4. 浓度的 重组UGT。 大肠杆菌裂解物 FRETWorks S标签测定试剂盒根据制造商的说明。
  5. E。 将含有重组UGT73C11的大肠杆菌裂解物加入到原模中 混合至终浓度200ng /μlUGT73C11。 60微升的这个主人 混合物转移到0.5ml微量离心管中。
  6. 通过加入4μl的1.6mM糖苷配基开始酶促反应 储备溶液。 在我们的情况下,应用的苷元是皂苷元 齐墩果酸和常春配基,其已溶解于100%DMSO中(因此,酶的糖苷配基和DMSO的最终浓度 反应分别为100μM和6.25%)
  7. 完成转换 的所提供的苷元与其相应的葡糖苷   通过在37℃下孵育反应过夜(18小时)(不振荡 此步骤必需)。
  8. 通过TLC评价转化效率   分析。 为此目的,皂草苷 - 葡糖苷和可能 剩余的皂甙元用50μl乙酸乙酯萃取4次 从完全反应的20μl等分试样。
  9. 合并的乙酸乙酯级分在真空离心机中蒸发至干。
  10. 将干燥的提取物溶于20μl96%乙醇中 溶液逐步(3.5μl/步)点样到硅胶TLC板上。 使用甲醇作为流动相将TLC板预运行1-2分钟 直到溶剂前沿高于装载线约1cm。
  11. 将甲醇在通风橱和干燥板中蒸发 随后用二氯甲烷:甲醇:水(80:19:1)展开   流动相。 蒸发后的流动相为展开板   用10%硫酸的甲醇溶液喷雾并加热至100℃ 直到皂苷元和皂苷元 - 葡糖苷变为可见的带红色带   在TLC板上。
  12. 在可见光以及长波UV(366nm)光下评价染色的TLC板
  13. 为了产生14 C标记的皂苷元 - 葡萄糖苷的标准曲线, 进行葡糖苷化反应的连续稀释。
  14. 的 最高选择的皂苷元 - 葡萄糖苷浓度为标准曲线 是与62.5%乙醇的过夜反应的1:5稀释, 对应于20μM的皂苷元 - 葡萄糖苷浓度。
  15. 这个 稀释液用50%乙醇依次稀释11倍 用皂草苷 - 葡萄糖苷浓度为10μM生成稀释液   μM,2.5μM,1.25μM,0.625μM,0.313μM,0.156μM,0.078μM,0.039μM, 0.020μM和0.010μM。
  16. 每个稀释液20μl为5μl步骤 加载到硅胶TLC板上,相当于 每个斑点的皂苷配基 - 葡糖苷量为400nmol,200nmol,100nmol,50   nmol,25nmol,12.5nmol,6.25nmol,3.13nmol,1.56nmol,0.78nmol, 分别为0.39nmol和0.20nmol。
  17. 蒸发后全部 溶剂,将TLC板预先运行1-2分钟,使用甲醇作为 流动相直到溶剂前沿约1cm以上 装载线。
  18. 将甲醇在通风橱中蒸发, 并随后用TLC板显影 二氯甲烷:甲醇:水(80:19:1)作为流动相。
  19. 所有 使溶剂蒸发,并加入1.5μl,1.0μl和0.5μl 原始主混合物的葡糖基化反应(加入前) 皂苷元)点在TLC板的未使用的侧泳道上。 这些斑点将允许在稍后的点的变化的归一化 不同反应主混合物之间的放射性 注意:
    1. 当地法规可能要求您在专门领域使用放射性化合物。请告知自己有关在工作场所使用的放射性化合物的使用规定和准则。
    2. 因为Perkin Elmer提供了溶解在70%乙醇中的这种试剂,所以在使用前将Glc干燥,其用作所研究的UGT本身的糖受体底物。
    3. 在这种情况下,重组UGT在BL21(DE)衍生物XJb(DE)中表达。该菌株表达病毒内溶素蛋白,因此允许通过在冷冻后简单解冻而溶解。
    4. 使用FRETWorks S标签测定试剂盒的定量基于RNaseS活性的再生,因为S蛋白(包含在试剂盒中)和N末端融合到重组表达的UGT的S标签的相互作用。有关其他信息,请参阅工具包手册。
    5. 作为未修饰的皂苷元的参考文献,是用于测定转化效率的2nmol齐墩果酸和常春配基加载到TLC板上。
    6. 在长波UV光下硫酸染色的TLC板上的皂苷配基/皂苷 - 葡萄糖苷评价比在可见光下的灵敏度大约高10倍。


    图2.具有产生可放射性的(1)3-O- [14 C] glc-齐墩果酸(oa-glc)或(3)3-O- [ 14 C] glc-hederagenin(he-glc)标准品。 在(A)可见(有色)以及(B)长波紫外光(366nm,黑/白)下评价TLC板。为了比较目的,将加载到泳道(2)的真正(oa)齐墩果酸和(he)常春配基(每种2nmol)。


II。 制备用于测定酶动力学参数的酶测定法

  1. 在UGT73C11酶的制备中,使用真空离心机在1.5ml微量离心管中将228μlUDP- [14 C] Glc(15.10nmol [168.72kBq])蒸发至干。
  2. 在另外两个1.5ml微量离心管中将2.39μl和2.18μl的1mM UDP-Glc(非放射性)溶液蒸发至干。同时,对于UGT73C13测定,将86.4μlUDP- [14 C] Glc(5.72nmol [63.94kBq]),1.99μl0.5mM UDP-Glc(非放射性)和1.59μl0.25mM UDP -Glc(非放射性)在1.5ml微量离心管中干燥。
  3. 受体底物,齐墩果酸和常春配基,用于在100%DMSO中制备浓度为128μM,96μM,64μM,32μM,16μM,8μM,4μM和2μM的UGT73C11测定储备溶液。
  4. 对于UGT73C13测定,制备了1,600μM,1,200μM,800μM,400μM,200μM,100μM,50μM和25μM的受体底物储备溶液。
  5. 冷冻(-80℃)E的等分试样。 通过解冻(参见注释)裂解重悬浮于10mM TAPS缓冲液pH 8.0中的大肠杆菌细胞,并通过在20,000xg,4℃下离心20分钟除去不溶性细胞碎片。
  6. 重组UGT在E中的浓度。 通过应用FRETWorks S标签测定试剂盒根据制造商的说明书测定大肠杆菌裂解物。
  7. E。 用在10mM TAPS缓冲液pH 8.0中的10mg/ml BSA将大肠杆菌裂解物稀释至5ng /μl(UGT73C11)或50ng /μl(UGT73C13)重组蛋白。


III。 UGT73C11的酶测定

  1. 制备用于36种酶测定反应的预先混合物,如配方部分所述
  2. 将427.4μl该预先混合物加入到含有干燥的UDP- [14 C] Glc的微量离心管中以制备反应主混合物,其将导致糖供体底物的浓度为33.12 μMUDP- [14 C] Glc(0.37kBq /μl)和467μM非放射性UDP-Glc。
  3. 将96μl该主混合物转移到其中已干燥2.39μl1mM UDP-Glc并与144μl原始预制备混合物混合的试管中。用该主混合物建立的酶测定将具有13.25μMUDP- [14 C] Glc(0.15kBq /μl)和487μM非放射性UDP-Glc的糖供体底物浓度。
  4. 将82μl后面的主混合物转移到其中2.18μl1mM UDP-Glc已经干燥的管中,并与82μl的原始预先混合物混合。该最后的主混合物用于制备浓度为6.62μMUDP- [14 C] Glc(0.08kBq /μl)和493μM非放射性UDP-Glc的酶测定。
  5. 将18.75μl等份的主混合物转移到冰上的1.5ml微量离心管中。
  6. 为了进行实际测定,将具有主混合物等分试样的管在30℃下预温育3分钟,并通过加入在步骤B-3中制备的1.25μl受体底物储备溶液开始酶反应。
    1. 使用具有最高浓度的UDP- [14 C] Glc(33.12μM)的主混合物来测定具有1μM,0.5μM,0.25μM和0.125μM的最终糖受体底物浓度的UGT73C11,通过加入1.25μl的16μM,8μM,4μM和2μM齐墩果酸或常春配基储备溶液。
    2. 类似地,用于4和2μM齐墩果酸和常春配基的最终浓度的13.25μMUDP- [14 C] Glc主混合物和6.62μMUDP- [14 C] C] Glc主混合物,最终浓度为8和6μM的两种受体底物。在这些情况下,相应的储备溶液为64μM,32μM,128μM和96μM。
  7. 在加入受体底物后,通过在30℃温育3分钟使酶促反应发生,随后通过加入50μl乙酸乙酯并剧烈混合10秒停止。


  • IV。
  • UGT73C13的酶测定(通常类似于程序C,但是酶和底物浓度不同,因为UGT73C13在催化所研究的反应中效率较低)
  1. 制备用于37种酶测定反应的预主混合物,如配方部分所述
  2. 将405μl预 - 主混合物转移到具有干燥的UDP- [14 C] Glc的管中,以制备用于酶测定的反应主混合物,终浓度为13.25μMUDP- [ 14 C] Glc(0.15kBq /μl)和487μM非放射性UDP-Glc。
  3. 将150μl该主混合物与150μl预 - 主混合物在已经干燥了1.99μl0.5mM UDP-Glc的微量离心管中混合。将该主混合物用于具有6.62μMUDP- [14 C] Glc(0.08kBq /μl)和493μM非放射性UDP-Glc的最终糖供体底物浓度的反应。
  4. 对于具有3.31μM(0.04kBq /μl)和497μM非放射性UDP-Glc的最终UDP- [14 C] Glc浓度的酶测定的主混合物,120μl之前的主混合物用含有干燥的1.59μl0.25mM UDP-Glc的试管中的120μl预 - 主混合物稀释。应用13.25μMUDP- [14 C] Glc预混合物用于最终齐墩果酸和常春藤苷酸浓度为1.56μM,3.12μM和6.25μM的测定。
  5. 将1.25μl的25μM,50μM和100μM糖受体底物储备溶液加入到18.75μl主混合物中以调节这些糖受体底物浓度。类似地,用于测定12.5μM和25μM(200μM和400μM储备溶液)的最终糖受体底物浓度的6.62μMUDP- [14 C] Glc主混合物和3.31μM用于糖受体底物浓度为50μM,75μM和100μM(800μM,1,200μM和1,600μM储备溶液)的UDP- [ 14 C] Glc主混合物。


V. 酶测定的分析

  1. 终止的测定用50μl乙酸乙酯萃取4次,合并的乙酸乙酯萃取物在真空离心机中蒸发至干。
  2. 将干燥的提取物溶于20μl96%乙醇中,并以5μl步骤加载到硅胶TLC板上。
  3. 将潜在的剩余的放射性皂苷元 - 葡萄糖苷用另外20μl96%乙醇洗出并加载到TLC板上的相同点。
  4. 使用甲醇作为流动相将TLC板预运行1-2分钟,直到溶剂前沿在装载线上方约1cm。
  5. 将甲醇在通风橱中蒸发,干燥的TLC板随后用二氯甲烷:甲醇:水(80:19:1)作为流动相显色。
  6. 蒸发流动相后,将0.5μl,1.0μl和1.5μl具有最高UDP- [14 C] Glc的主混合物点在TLC板的未使用的泳道上。另外,还有1μl的两个剩余的主混合物,其中较低的UDP- [14 C] Glc点样到每个TLC板。
  7. 将干燥的TLC板与相同皂苷元 - 葡萄糖苷的制备的参比稀释系列TLC板之一一起暴露于储存磷光体屏幕数天。
  8. 用STORM 840磷光成像仪扫描曝光的荧光屏,使用ImageQuant 5.0对数字扫描中的信号强度进行定量。
  9. 为了补偿与用于产生参考葡萄糖苷的主混合物相比较低10倍的放射性糖供体底物浓度,所有浓度都是由3.31μMUDP- [在将其进一步评估之前乘以10倍。类似地,由13.25μM和6.62μMUDP- [14 C] Glc主混合物产生的皂苷元 - 葡萄糖苷强度乘以2.5和5。
  10. 为了进一步解决用于产生参考葡萄糖苷的主混合物中UDP- [14 C] Glc的量的无意的变化和实际的酶测定,将测定的所有葡萄糖苷强度乘以比率在每个TLC板上的相应主混合物斑点的强度之间。
  11. K sub和V sub max,使用SigmaPlot 11.0计算值,根据Michaelis-Menten方程式或底物抑制的速度方程式进行非线性回归(有关如何执行回归的指令,请参阅SigmaPlot手册)。
    注意:
    1. 在这种情况下,重组UGT在BL21(DE)衍生物XJb(DE)中表达。该菌株表达病毒内溶素蛋白,因此允许通过在冷冻后简单解冻而溶解。
    2. 大肠杆菌 细胞所有裂解物,裂解物稀释液和主混合物都保持在冰上。
    3. 使用FRETWorks S标签测定试剂盒的定量基于RNaseS活性的再生,因为S蛋白(包含在试剂盒中)和N末端融合到重组表达的UGT的S标签的相互作用。有关其他信息,请参阅工具包手册。
    4. 大肠杆菌裂解物用BSA溶液代替纯的缓冲液稀释,因为重组UGT的比活性在总蛋白浓度降低时可以看出降低。
    5. 所研究的酶的最终量应根据其活性来选择。如果在所应用的孵育时间内用尽过多的受体底物,则确定的反应速度将不再处于初始线性范围内。
    6. 使用具有不同比例的放射性标记的未标记的UDP-Glc的主混合物的意图是使用少量的放射性标记的UDP- [ 14 sup> C] Glc,同时仍然确保 用于具有最低受体底物浓度的测定的足够的信号强度。在整个实验期间保持500μMUDP-Glc的最终总浓度,以提供糖供体底物的饱和条件。
    7. 选择UGT73C11和UGT73C13的测定中的不同pH是由于两种酶的最佳pH不同。
    8. 测试受体底物的哪些浓度,取决于所研究的酶。为了确定最佳浓度,有助于在预实验中估计Km值,并选择低于和高于估计的K m 最后的实验。
    9. 最佳反应时间取决于酶活性和酶量。必须进行时程实验以确定在选择的温育时间之后反应速度是否仍然在初始线性范围内。如果太多的受体底物在最低应用浓度下转化,则所得到的v/S特性通常是S形的而不是双曲线的。


    图3.TLC板的放射自显影图,以确定UGT73C11对常春配基的动力学参数具有来自实际酶测定的乙酸乙酯提取物的TLC板在顶部以及底部,而共同暴露的TLC具有[14 C] Glc-常春配基标准曲线的平板位于中间。表示3-O- [14 C] Glc-常春藤苷酸的带是每个用箭头标记的平板和标记物his-glc。低于UGT73C11酶测定提取物的he-glc条带的浓度表示相应酶测定中的常春配基浓度,而I和II标记重复。每个标准曲线下面的物质量表示存在于相应泳道中的[14 C] Glc-常春藤苷酸的量。将主混合物等分试样以说明主混合制备物(MM1,MM2,MM3)中UDP- [14 C] Glc的量的变化,利用实际测定在泳道的右边或左边点样,或者标准

食谱

  1. 1.6mM齐墩果酸储备液
    将7.30mg齐墩果酸溶于10ml DMSO中
  2. 1.6mM常春配基原液
    将7.56mg常春配基在10ml DMSO中溶解
  3. 用于36UGT73C11酶测定反应的预制混合物
    36微升500mM TAPS缓冲液,pH8.6 3.6μl200mM DTT
    36μl9.34 mM UDP-Glc
    72μl10mg/ml BSA
    72μl5ng /μlUGT73C11(稀释的大肠杆菌裂解物)
    455.4μl水
  4. 用于37 UGT73C13酶测定反应的前主混合物
    37μl500mM TAPS缓冲液pH7.7 37μl200 mM DTT
    37μl9.34mM UDP-Glc<
    74μl10mg/ml BSA
    74μl50ng /μlUGT73C13(稀释的大肠杆菌裂解物)
    465.05μl水

致谢

该协议适应和修改从各种,以前基于14C-UDP葡萄糖的UGT酶分析协议,通常应用于植物生物化学部门 - 植物生物化学生物系 - 生命科学系 - 哥本哈根大学和前面的组织。这项工作得到了丹麦独立研究,技术和生产科学委员会(授予号09-065899/FTP和274-06-0370)的支持,由Villum Kann Rasmussen基金会授予Pro-Active植物,并通过博士哥本哈根大学生命科学学院(至JMA)的津贴。

参考文献

  1. Augustin,JM,Drok,S.,Shinoda,T.,Sanmiya,K.,Nielsen,JK,Khakimov,B.,Olsen,CE,Hansen,EH,Kuzina,V.,Ekstrom,CT,Hauser, Bak,S。(2012)。 来自寻常型巴布达UGT73C亚家族的UDP-糖基转移酶催化皂苷元3- O-葡糖基化在皂苷介导的昆虫抗性中。植物生理学 160(4):1881-1895。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Augustin, J. M. and Bak, S. (2013). Determination of Enzyme Kinetic Parameters of UDP-glycosyltransferases. Bio-protocol 3(14): e825. DOI: 10.21769/BioProtoc.825.
  2. Augustin, J. M., Drok, S., Shinoda, T., Sanmiya, K., Nielsen, J. K., Khakimov, B., Olsen, C. E., Hansen, E. H., Kuzina, V., Ekstrom, C. T., Hauser, T. and Bak, S. (2012). UDP-glycosyltransferases from the UGT73C subfamily in Barbarea vulgaris catalyze sapogenin 3-O-glucosylation in saponin-mediated insect resistance. Plant Physiol 160(4): 1881-1895.
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