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Determination of Fructokinase Activity from Zobellia galactanivorans
检测Zobellia galactanivorans的果糖激酶活性   

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Abstract

Mannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. Several pathways have been described for the metabolism of mannitol by bacteria, including the phosphoenolpyruvate-dependent phosphotransferase system (PST) and a M2DH-based catabolic pathway. The latter involves two enzymes, a mannitol-2-dehydrogenase (EC 1.1.1.67) and a fructokinase (EC 2.7.1.4), and has been identified in different bacteria, e.g.,, the marine Bacteroidetes Zobellia galactanivorans (Zg) which had recently gained interest to study the degradation of macroalgal polysaccharides. This protocol describes the biochemical characterization of a recombinant fructokinase (FK) of Zobellia galactanivorans. The ZgFK enzyme catalyzes the conversion of fructose to fructose-6-phosphate using ATP as a cofactor.
[Principle] Fructokinase (FK) activity was determined by an enzyme-coupled assay (Figure 1). ADP formed through the FK reaction, i.e., phosphorylation of fructose to fructose-6-phosphate (F6P), is used by the pyruvate kinase (PK) which transforms phosphoenolpyruvate (PEP) to pyruvate. Then, lactate dehydrogenase (LDH) converts pyruvate to lactate using NADH as a cofactor. FK activity is measured by following the decrease in absorbance at 340 nm which corresponds to the transformation of NADH to NAD+.


Figure 1. Enzyme-coupled reaction used for determination of fructokinase (FK) activity

Keywords: Mannitol degradation(甘露醇降解), Fructokinase(果糖激酶), Zobellia galactanivorans(zobellia galactanivorans), Flavobacteria(黄杆菌)

Materials and Reagents

  1. UV-Star® PS Microplate (96 Well) (Greiner Bio-One GmbH, catalog number: 655801 )
  2. Purified recombinant His-tagged ZgFK
    Note: This protein was produced in Escherichia coli BL21 (DE3) containing the recombinant pFO4_ZgFK vector, as described by Groisillier et al. (2010). This recombinant protein was purified by affinity chromatography using a HisPrep FF 16/10 column (GE Healthcare Dharmacon) onto an Äkta avant system (GE Healthcare Dharmacon). The complete purification protocol is described in details in Groisillier et al. (2015).
  3. Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle (Sigma-Aldrich, catalog number: P0294 )
  4. MilliQ water
  5. Phospho(enol)pyruvic acid tri(cyclohexylammonium) salt (Sigma-Aldrich, catalog number: P7252 )
  6. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  7. 4-morpholineethane-sulfonic acid (MES) (Sigma-Aldrich, catalog number: M2933 )
  8. Bis-Tris (Sigma-Aldrich, catalog number: B9754 )
  9. β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate (NADH) (Sigma-Aldrich, catalog number: N8129 )
  10. Examples of chemicals to be tested to assess substrate specificity:
    1. D-(-)-fructose (Sigma-Aldrich, catalog number: F0127 )
    2. D- (+)-glucose (Sigma-Aldrich, catalog number: G8270 )
    3. D-(+)-mannose (Sigma-Aldrich, catalog number: M4625 )
    4. D-mannitol (Sigma-Aldrich, catalog number: M9647 )
    5. D-sorbitol (Sigma-Aldrich, catalog number: S1876 )
    6. D-mannitol-1-phosphate (Sigma-Aldrich, catalog number: 92416 )
    7. D-fructose-1-phosphate (Sigma-Aldrich, catalog number: S408689 )
    8. α-D-Glucose 1-phosphate disodium salt hydrate (Sigma-Aldrich, catalog number: G9380 )
    9. D-Mannose 6-phosphate sodium salt (Sigma-Aldrich, catalog number: M3655 )
    10. D-Glucose 6-phosphate sodium salt (Sigma-Aldrich, catalog number: G7879 )
    11. D-Fructose 6-phosphate disodium salt hydrate (Sigma-Aldrich, catalog number: F3627 )
    12. Adenosine 5’-triphosphate (ATP) disodium salt hydrate (Sigma-Aldrich, catalog number: A1852 )
  11. 1 M Tris-HCl (pH 7.5) (see Recipes)
  12. 100 mM MgCl2 (see Recipes)
  13. 1 M KCl (see Recipes)
  14. 100 mM PEP (see Recipes)
  15. 100 mM ATP (see Recipes)
  16. 10 mM NADH (see Recipes)
  17. PK/LDH (see Recipes)

Equipment

  1. Safire2 UV spectrophotometer microplate reader (Tecan Trading AG)
  2. NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific)

Software

  1. Hyper 32 (Hyper32.software.informer.com)
  2. Microsoft Excel

Procedure

  1. The reaction mixture contains 100 mM Tris-HCl (pH 7.5), 1 mM fructose, 1 mM ATP, 100 mM KCl, 1.5 mM MgCl2, 1 mM phosphoenolpyruvate (PEP), 0.5 mM freshly prepared NADH, 0.2 µl Pyruvate kinase/lactate dehydrogenase (PK/LDH) enzymes (stored at -20 °C before use), and 1 to 10 µg of purified recombinant enzyme, in a volume of 100 µl. Blank corresponds to reaction mixture where substrate is substituted by MilliQ water (Table 1).

    Table 1. Composition of blank and reaction mixture for determination of ZgFK activity
    Stock solutions
    µl added in blank
    µl added in reaction mix (test)
    Tris-HCl (pH 7.5) (1 M)
    10
    10
    Fructose (25 mM)
    0
    4
    ATP (100 mM)
    1
    1
    MgCl2 (100 mM)
    1.5
    1.5
    KCl (1 M)
    10
    10
    PEP (100 mM)
    1
    1
    NADH (10 mM)
    2
    2
    PK/LDH (600-1,500 units/ml)
    0.2
    0.2
    ZgFK (1 µg/µl)
    1
    1
    MilliQ water
    73.3
    69.3

  2. The continuous assay reaction is started by adding the substrate, and activity is assayed by following changes in absorbance at 340 nm which corresponds to the conversion of NADH to NAD+, in a Safire2 UV spectrophotometer microplate reader. The assay is performed at 25 °C for up to 20 min. Only the early linear part of the curves is used to calculate activity (Figure 2).
  3. Calculate the consumption of NADH using the formula:
    [(ΔA340nm test-ΔA340nm blank)/(t*6220*0.3*0.0001)]
    Where
    ΔA340nm= variation of absorbance during the duration of incubation
    t=duration of incubation (min)
    6220= extinction coefficient for NADH (L mol-1 cm-1)
    0.3= optical path (cm)
    0.0001= assay volume (L)
    The rate of decrease of NADH is directly proportional to the rate of formation of lactate and thus to the FK activity. One unit (U) of FK activity corresponds to 1 µmol of NADH oxidized per min.


    Figure 2. Early linear parts of curves representing changes in absorbance (340 nm) monitored as a function of time (sec) in presence of fructose. The curves represent two series of triplicates at 0 and 5 mM of fructose, respectively.

  4. To calculate specific activities, divide the value obtained in the equation above by the quantity of ZgFK proteins present in the sample. Perform three replicates for each assay and determine the average ± S.E. (cf. Notes) of these three replicates. This applies also to the experiments described below. As an example, typical value for specific activity of ZgFK was 0.49 U/mg in presence of 5 mM fructose.
  5. To determine substrate specificity, assess ZgFK activity in presence of each of the substrates listed in the “Materials and Reagents” section, using concentration ranging from 1 mM to at least 100 mM.
  6. To determine the optimal pH, replace the 100 mM Tris-HCl (pH 7.5) buffer used in step 1 of the procedure by other buffers prepared at different pHs. As an indication, it is possible to use:
    100 mM MES for pH 5.5, 6, 6.5
    100 mM Bis-Tris propane for pH 6.5, 7, 7.5, 8, 8.5, 9, 9.5
    100 mM Tris-HCl for pH 7, 7.5, 8, 8.5, 9
  7. To determine the optimal temperature, incubate the reaction mixtures described in step 1 at temperatures ranging, for instance, from 10 °C to 50 °C, with incremental of 5 or 10 °C.
  8. To examine the influence of NaCl, add in the reaction mixture described in step 1 sodium chloride to obtain final concentrations ranging from 0 to 2 M.
  9. To estimate the kinetic parameters of the enzyme for a selected substrate S, run individual enzyme reactions in presence of at least five different concentrations of this substrate and a fixed concentration of ATP. Determine the initial reaction rate for each reaction and plot 1/V versus 1/[S] to obtain a Lineweaver-Burk plot, from which Km and Vm for S can be calculated.


    Figure 3. Lineweaver-Burk plot used to determine the Km (10 mM) and Vm (2.55 U/mg) of ZgFK for fructose. [S] is the fructose concentration (in mM) and V is the reaction rate (in µmol/min/mg of protein).

Notes

  1. PK/LDH enzymes activities must be compared under the selected assay conditions (temperature, pH, salt concentrations…) with activities recommended by the supplier (one unit convert 1.0 µmole of substrate per minute at pH 7.6 at 37 °C). If these activities change, do the protocol in two steps: first, perform the fructokinase reaction under selected condition (without PK/LDH enzymes, PEP and NADH) for different times of incubation; then, in a second step, measure [NADH] after these different times of incubation and plot the results as illustrated in Figure 2.
  2. Before determining Km and Vm for a given substrate S, be sure that cofactor (ATP) is in excess, i.e., that V does not increase with increasing quantities of cofactor in the reaction mixture; in the same vein, before determining Km and Vm for the cofactor, be sure that S is in excess, i.e., that V does not increase with increasing quantities of S in the reaction mixture. In theory, saturating concentration is equivalent to 100 Km, but 10 Km is usually sufficient (Bisswanger, 2014). It is then necessary to adjust the ZgFK concentration and the incubation time to obtain a linear decrease of absorbance at 340 nm, i.e., a linear consumption of NADH.
  3. S. E. corresponds to standard error calculated in Excel.

Recipes

  1. 1 M Tris-HCl (pH 7.5)
    Stored at room temperature
  2. 100 mM MgCl2
    Stored at room temperature
  3. 1 M KCl
    Stored at room temperature
  4. 100 mM PEP
    Prepared fresh in MilliQ water on the week of use and stored at -20 °C
  5. 100 mM ATP
    Prepared fresh in MilliQ water on the day of use
  6. 10 mM NADH
    Prepared fresh in MilliQ water on the day of use
  7. PK/LDH
    Stored at -20 °C

Acknowledgments

This protocol was performed by Groisillier et al. (2015). This work was supported by the French National Research Agency via the investment expenditure program IDEALG (ANR-10-BTBR-02). We also acknowledge funding from the Émergence-UPMC-2011 research program.

References

  1. Bisswanger, H. (2014). Enzymes assays. Perspective in Sciences 1: 41-55.
  2. Groisillier, A., Herve, C., Jeudy, A., Rebuffet, E., Pluchon, P. F., Chevolot, Y., Flament, D., Geslin, C., Morgado, I. M., Power, D., Branno, M., Moreau, H., Michel, G., Boyen, C. and Czjzek, M. (2010). MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms. Microb Cell Fact 9: 45.
  3. Groisillier, A., Labourel, A., Michel, G. and Tonon, T. (2015). The mannitol utilization system of the marine bacterium Zobellia galactanivorans. Appl Environ Microbiol 81(5): 1799-1812.

简介

甘露醇是在多种活生物体中发生的多元醇,其中它实现不同的生理作用。已经描述了用于由细菌代谢甘露醇的几种途径,包括磷酸烯醇丙酮酸依赖性磷酸转移酶系统(PST)和基于M2DH的分解代谢途径。后者涉及两种酶,甘露醇-2-脱氢酶(EC 1.1.1.67)和果糖激酶(EC 2.7.1.4),并且已经在不同的细菌中鉴定,例如海洋拟杆菌Zobellia galactanivorans ( Zg ),其最近获得了研究大分子多糖降解的兴趣。该方案描述了Zobellia 半乳糖厌氧杆菌的重组果糖激酶(FK)的生物化学表征。 < strong> [原理] 果糖激酶(FK)活性通过以下方法确定:果糖激酶(FK)活性通过使用ATP作为辅因子来催化果糖转化成果糖-6-磷酸。酶偶联测定(图1)。通过FK反应(即果糖磷酸化成果糖-6-磷酸(F6P))形成的ADP由将磷酸烯醇丙酮酸(PEP)转化为丙酮酸的丙酮酸激酶(PK)使用。然后,乳酸脱氢酶(LDH)使用NADH作为辅因子将丙酮酸转化为乳酸。通过跟随在340nm处的吸光度的降低来测量FK活性,其对应于NADH转化为NAD +


图1.用于测定果糖激酶(FK)活性的酶偶联反应

关键字:甘露醇降解, 果糖激酶, zobellia galactanivorans, 黄杆菌

材料和试剂

  1. UV-Star microplate(96孔)(Greiner Bio-One GmbH,目录号:655801)
  2. 纯化的重组His标记的< em> Zg FK
    注意:该蛋白质在含有重组pFO4_ZgFK载体的大肠杆菌BL21(DE3)中产生,如Groisillier等人,(2010)所述。通过使用HisPrep FF 16/10柱(GE Healthcare Dharmacon)的亲和层析将该重组蛋白质纯化到?ktaavant系统(GE Healthcare Dharmacon)上。完整的纯化方案在Groisillier等人(2015)中有详细描述。
  3. 来自兔肌肉的丙酮酸激酶/乳酸脱氢酶(Sigma-Aldrich,目录号:P0294)
  4. MilliQ水
  5. 磷酸(烯醇)丙酮酸三(环己基铵)盐(Sigma-Aldrich,目录号:P7252)
  6. (Sigma-Aldrich,目录号:T1503)
  7. 4-吗啉乙磺酸(MES)(Sigma-Aldrich,目录号:M2933)
  8. Bis-Tris(Sigma-Aldrich,目录号:B9754)
  9. β-烟酰胺腺嘌呤二核苷酸,还原二钠盐水合物(NADH)(Sigma-Aldrich,目录号:N8129)
  10. 要测试以评估底物特异性的化学品的实例:
    1. D - ( - ) - 果糖(Sigma-Aldrich,目录号:F0127)
    2. D-(+) - 葡萄糖(Sigma-Aldrich,目录号:G8270)
    3. D - (+) - 甘露糖(Sigma-Aldrich,目录号:M4625)
    4. D-甘露醇(Sigma-Aldrich,目录号:M9647)
    5. D-山梨醇(Sigma-Aldrich,目录号:S1876)
    6. D-甘露醇-1-磷酸酯(Sigma-Aldrich,目录号:92416)
    7. D-果糖-1-磷酸(Sigma-Aldrich,目录号:S408689)
    8. α-D-葡萄糖-1-磷酸二钠盐水合物(Sigma-Aldrich,目录号:G9380)
    9. D-甘露糖-6-磷酸钠盐(Sigma-Aldrich,目录号:M3655)
    10. D-葡萄糖-6-磷酸钠盐(Sigma-Aldrich,目录号:G7879)
    11. D-果糖-6-磷酸二钠盐水合物(Sigma-Aldrich,目录号:F3627)
    12. 腺苷5'-三磷酸(ATP)二钠盐水合物(Sigma-Aldrich,目录号:A1852)
  11. 1 M Tris-HCl(pH 7.5)(见配方)
  12. 100mM MgCl 2(参见配方)
  13. 1 M KCl(见配方)
  14. 100 mM PEP(参见配方)
  15. 100 mM ATP(参见配方)
  16. 10 mM NADH(参见配方)
  17. PK/LDH(参见配方)

设备

  1. Safire2紫外分光光度计酶标仪(Tecan Trading AG)
  2. NanoDrop 2000分光光度计(Thermo Fisher Scientific)

软件

  1. Hyper 32(Hyper32.software.informer.com)
  2. Microsoft Excel

程序

  1. 反应混合物含有100mM Tris-HCl(pH7.5),1mM果糖,1mM ATP,100mM KCl,1.5mM MgCl 2,1mM磷酸烯醇丙酮酸(PEP),0.5mM新鲜制备的NADH ,0.2μl丙酮酸激酶/乳酸脱氢酶(PK/LDH)酶(使用前保存于-20℃)和1?10μg纯化的重组酶,体积为100μl。空白对应于反应混合物,其中底物被MilliQ水代替(表1)
    表1.用于测定Zg FK活性的空白和反应混合物的组成
    库存解决方案
    μl以空白
    添加
    μl加入反应混合物(试验)
    Tris-HCl(pH7.5)(1M) 10
    10
    果糖(25mM)
    0
    4
    ATP(100mM)
    1
    1
    MgCl 2(100mM)
    1.5
    1.5
    KCl(1 M)
    10
    10
    PEP(100mM) 1
    1
    NADH(10mM) 2
    2
    PK/LDH(600-1,500单位/ml)
    0.2
    0.2
    Zg FK(1μg/μl)
    1
    1
    MilliQ水
    73.3
    69.3

  2. 通过加入底物开始连续测定反应,通过在Safire2UV分光光度计酶标仪中在340nm处的吸光度的变化(其对应于NADH到NAD +的转化)测定活性。该测定在25℃下进行最多20分钟。只有曲线的早期线性部分用于计算活动(图2)。
  3. 使用公式计算NADH的消耗:
    [(ΔA340nm测试-ΔA340nm空白)/(t * 6220 * 0.3 * 0.0001)]
    其中
    ΔA340nm <=孵育期间的吸光度变化
    t =培养持续时间(min)
    6220 = NADH的消光系数(L mol -1 cm -1
    0.3 =光程(cm)
    0.0001 =测定体积(L)
    NADH的减少速率与乳酸盐的形成速率成正比,因此与FK活性成正比。 FK活性的一个单位(U)对应于每分钟氧化的1μmolNADH

    图 ?曲线的早期线性部分表示吸光度的变化(340 ?nm)在果糖存在下作为时间(秒)的函数监测。曲线分别表示在0和5mM果糖下的两个系列的三次重复。

  4. 为了计算比活性,将在上述等式中获得的值除以样品中存在的 Zg FK蛋白的量。对每个测定进行三次重复,并确定平均值±S.E。 (参见注释)。这也适用于下述实验。作为实例,在5mM果糖存在下,Zg/FK的比活性的典型值为0.49U/mg。
  5. 为了测定底物特异性,在"材料和试剂"部分列出的每种底物存在下,使用1mM至至少100mM的浓度评价Zg/FK活性。
  6. 为了确定最佳pH,用在不同pH下制备的其它缓冲液替换在步骤1中使用的100mM Tris-HCl(pH 7.5)缓冲液。作为指示,可以使用:
    100mM MES,pH 5.5,6,6.5
    100mM Bis-Tris丙烷,pH6.5,7,7.5,8,8.5,9,9.5。
    100mM Tris-HCl,pH 7,7.5,8,8.5,9mM
  7. 为了确定最佳温度,将步骤1中所述的反应混合物在例如10℃至50℃的温度范围内,以5℃或10℃的增量温育。
  8. 为了检查NaCl的影响,在步骤1中描述的反应混合物中加入氯化钠以获得范围从0至2μM的终浓度。
  9. 为了估计所选底物S的酶的动力学参数,在至少五种不同浓度的该底物和固定浓度的ATP的存在下进行单独的酶反应。确定每个反应的初始反应速率,并绘制1/V对1/[S],以获得Lineweaver-Burk图,其中对于S来说K sub和V sub可以计算。


    图3.用于测定K m(10mM)和V m(2.55U/mg)的Lineweaver-Burk图, [S]是果糖浓度(以mM计),V是反应速率(以μmol/min/mg蛋白质计)。

笔记

  1. PK/LDH酶活性必须在选择的测定条件(温度,pH,盐浓度...)下与供应商推荐的活性(一个单位在pH 7.6在37℃下转化1.0μmole底物每分钟)进行比较。如果这些活动发生改变,请按照两个步骤进行操作:首先,在选择的条件下(无PK/LDH酶,PEP和NADH)进行果糖激酶反应不同的温育时间;然后,在第二步中,在这些不同的孵育时间之后测量[NADH],并绘制如图2所示的结果。
  2. 在确定给定底物S的K m和V m之前,确保辅因子(ATP)过量,即 V不是随着反应混合物中辅因子的量增加而增加;在确定辅因子的Km和Vm之前,确保S是过量的,即V不随反应混合物中S的量增加而增加。在理论上,饱和浓度相当于100K m,但是10K m/s通常是足够的(Bisswanger,2014)。然后需要调节Fg浓度和孵育时间以获得在340nm处的吸光度的线性降低,即, NADH的线性消耗
  3. S.E对应于在Excel中计算的标准误差

食谱

  1. 1 M Tris-HCl(pH 7.5)
    在室温下贮存
  2. 100mM MgCl 2/v/v 在室温下贮存
  3. 1 M KCl
    在室温下贮存
  4. 100mM PEP
    在使用一周后在MilliQ水中新鲜制备并储存在-20℃下
  5. 100 mM ATP
    在使用当天在MilliQ水中新鲜制备
  6. 10mM NADH
    在使用当天在MilliQ水中新鲜制备
  7. PK/LDH
    储存于-20°C

致谢

该方案由Groisillier等人(2015)进行。这项工作得到了法国国家研究机构通过投资支出计划IDEALG(ANR-10-BTBR-02)的支持。我们还感谢émergence-UPMC-2011研究计划的资助。

参考文献

  1. Bisswanger,H.(2014)。 酶学测定。 1:41- 55.
  2. Gro?illier,A.,Herve,C.,Jeudy,A.,Rebuffet,E.,Pluchon,PF,Chevolot,Y.,Flament,D.,Geslin,C.,Morgado,IM,Power,D.,Branno, M.,Moreau,H.,Michel,G.,Boyen,C.and Czjzek,M。(2010)。 MARINE-EXPRESS:利用高通量克隆和表达策略进行海洋后基因组分析 Microb Cell Fact 9:45。
  3. Groisillier,A.,Labourel,A.,Michel,G.and Tonon,T。(2015)。 海洋细菌Zobellia galactanivorans的甘露醇利用系统。 Appl Environ Microbiol 81(5):1799-1812。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Groisillier, A. and Tonon, T. (2015). Determination of Fructokinase Activity from Zobellia galactanivorans. Bio-protocol 5(21): e1633. DOI: 10.21769/BioProtoc.1633.
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