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Determination of Recombinant Mannitol-1-phosphatase Activity from Ectocarpus sp.
水云属中重组甘露醇-1-磷酸酶活性的测定   

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Abstract

Brown algae belong to a phylogenetic lineage distantly related to green plants and animals, and are found predominantly, but not exclusively, in the intertidal zone, a harsh and frequently changing environment. Because of their unique evolutionary history and of their habitat, brown algae feature several peculiarities in their metabolism. One of these is the mannitol cycle, which plays a central role in their physiology, as mannitol acts as carbon storage, osmoprotectant, and antioxidant. This polyol is derived directly from the photoassimilate fructose-6-phosphate via the action of a mannitol-1-phosphate dehydrogenase (M1PDH, EC 1.1.1.17) and a mannitol-1-phosphatase (M1Pase, EC 3.1.3.22). This protocol describes the biochemical characterization of a recombinant M1Pase of Ectocarpus sp. The M1Pase enzyme catalyzes the conversion of mannitol-1-phosphate to mannitol (Figure 1).


Figure 1. Reaction catalyzed by a mannitol-1-phosphatase

Keywords: Mannitol cycle(甘露醇周期), Mannitol-1-phosphatase(甘露醇- 1 -磷酸酶), Ectocarpus sp.(水云属sp。), Brown algae(褐海藻)

Materials and Reagents

  1. UV-Star® PS microplate (96 well) (Greiner Bio-One GmbH, catalog number: 655801 )
  2. 0.22 µm filter
  3. Escherichia coli BL21 (DE3)
  4. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  5. Purified recombinant His-tagged M1Pase
    Note: This protein was produced in Escherichia coli BL21 (DE3) containing the recombinant pFO4_ M1Pase 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) onto an Äkta avant system (GE Healthcare). The complete purification protocol is described in details in Groisillier et al. (2014).
  6. MgCl2 (Sigma-Aldrich, catalog number: M8266 )
  7. MilliQ water
  8. Malachite green phosphate assay kit (Gentaur, catalog number: POMG-25H )
    Note: Malachite green phosphate assay protocol is available at https://www.bioassaysys.com/Datasheet/POMG.pdf.
  9. Examples of chemicals to be tested to assess substrate specificity
    1. D-mannitol-1-phosphate (Sigma-Aldrich, catalog number: 92416 )
    2. D-fructose-1-phosphate (Sigma-Aldrich, catalog number: F1127 )
    3. D-glucose-1-phosphate (Sigma-Aldrich, catalog number: G9380 )
    4. D-mannose-6-phosphate (Sigma-Aldrich, catalog number: M3655 )
    5. D-glucose-6-phosphate (Sigma-Aldrich, catalog number: G7879 )
    6. D-fructose-6-phosphate (Sigma-Aldrich, catalog number: F3627 )
  10. NaCl (Sigma-Aldrich, catalog number: 71380 )
  11. 1 M Tris-HCl (pH 7.5) (see Recipes)
  12. 100 mM MgCl2 (see Recipes)
  13. 5 M NaCl (see Recipes)

Equipment

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

Software

  1. Hyper32 (Informer Technologies, http://hyper32.software.informer.com/)
  2. Microsoft Excel

Procedure

  1. The standard reaction mixture contains 100 mM Tris-HCl (pH 7.5), 1 mM M1P, 5 mM MgCl2 and 6 to 78 µg of purified recombinant enzyme quantified by NanoDrop (based on the extinction coefficient of 18,700 M-1 cm-1 calculated for the protein of interest), in a final volume of 450 µl. Inactive boiled purified recombinant enzyme is used as a negative control (Table 1).

    Table 1. Composition of negative control and reaction mixture for determination of M1Pase activity


  2. The mix is prepared with water, Tris-HCl, MgCl2 and enzyme, then the reaction is started by adding the substrate, and incubation is conducted for 12 min, at 30 °C, with sampling every 3 min. For this, the volume of the reaction mixture is 450 μl, and 80 μl reaction mixture is sampled at t0, t3min, t6min, t9min and t12min. Each condition is tested in three replicates (Figure 2A).


    Figure 2. Measurement of absorbance at 620 nm as a function of time (min) in the presence of 1 mM M1P. A. The curves represent two series of triplicates containing 24 µg of purified M1Pase and 24 µg of boiled purified M1Pase, respectively. Abs 620 nm at t0 is taken just after the addition of M1P in the reaction mix. B. The table contains values of absorbance measured under both conditions tested and at different times of experiment.

    In each sample, reaction is stopped by adding 20 μl of malachite green reagent for subsequent determination of the quantity of phosphate released. To this aim, absorbance in each sample is read at 620 nm in a Safire2 UV spectrophotometer microplate reader after 20 min of incubation at room temperature (Figure 2B). Values are then compared with a standard curve of free phosphate prepared according to the malachite green phosphate assay kit manufacturer’s instructions (Figure 3).


    Figure 3. Use of the malachite green phosphate assay kit to determine free phosphate concentrations. A. Color formation was observed after 20 min of incubation at room temperature for different concentrations of phosphate (from well 1 to 8, 2 wells for each concentration): 0 μM, 4 μM, 8 μM, 12 μM, 16 μM, 24 μM, 32 μM and 40 μM (buffer only, 100 mM Tris-HCl pH 7.5, 5 mM MgCl2, final concentrations). B. Standard curve representing absorbance monitored at 620 nm as a function of variable quantities of free phosphate (Pi). In this example, the linear regression curve was Abs 620 nm = [0.0001 x (pmol Pi in 100 μl)] -0.0085, and R2 = 0.9985. C. Table corresponding to values of absorbance measured for different quantities of free phosphate.

  3. Phosphatase activities, based on production of free phosphate, are calculated using the linear regression curve determined as in Figure 3:
    [(ΔA620 nm test - ΔA620 nm negative control) + 0.0085]/(0.0001*t)
    Where
    ΔA620 nm = variation of absorbance during the duration of incubation
    t = time of incubation (min)
    One unit (U) of activity corresponds to 1 nmol of Pi released per min.
  4. To calculate specific activities, divide the value obtained in the equation above by the quantity of proteins present in the sample. Perform three replicates for each assay, and determine the average ± standard error (S.E.) of these three replicates. From values given in Figure 2, specific activity of M1Pase was 11.30 U/mg in presence of 1 mM M1P.
  5. To determine substrate specificity, test M1Pase activity in the presence of each substrate listed in the “Materials and Reagents” section, using concentration ranging from 0.05 mM to 1 mM, at room temperature.
  6. To determine the optimal temperature, incubate the reaction mixtures used in step 1 at temperatures ranging, for instance, from 10 °C to 50 °C, with incremental of 5 or 10 °C. The experiments described below were performed at the optimal temperature of 30 °C.
  7. The effect of pH on the enzymatic activity of purified M1Pase is determined by using 100 mM Tris/HCl buffer whose pH ranged from 5.8 to 9, in presence of 5 mM MgCl2.
  8. To examine the influence of NaCl, add NaCl in the reaction mixture described in step 1 to obtain final concentrations ranging from 0 to 1 M.
  9. To estimate the kinetic parameters of the enzyme for a selected substrate S, run individual enzyme reactions in the presence of at least five different concentrations of this substrate (for example: 0.08 mM, 0.12 mM, 0.18 mM, 0.27 mM, 0.4 mM, and 0.6 mM). 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 4). This calculation can be done using the Hyper32 software.


    Figure 4. Lineweaver-Burk plot used to determine the Km (0.67 mM) and Vm (25.25 U/mg) of M1Pase for M1P. [S] is the M1P concentration (in mM) and V is the reaction rate (in U/mg of protein). Three replicates were performed for each assay.

Notes

  1. Respect the incubation time for each reaction. Samples taken from the reaction mixture at different times of incubations are kept on the ice until the end of the experiment. Then malachite is added for 20 min, before reading absorbance at 620 nm.
  2. Respect a time of 20 min at room temperature between the addition of malachite green and the reading of absorbance at 620 nm.
  3. Perform a standard curve for each change in the composition of the reaction mix, e.g., buffer, pH.
  4. This protocol can be used to detect M1Pase activity in a whole cell extract. However, it is necessary to be cautious when using the malachite green kit to quantify phosphate released by enzymes contained in cellular extracts because of possible interference caused by chemicals present in these extracts. It is also important to consider that cellular extracts may contain free phosphate that will be quantified by the malachite green kit.

Recipes

  1. 1 M Tris-HCl (pH 7.5)
    1. Dissolve 121.14 g of Trizma® base in around 800 ml of MilliQ water
    2. Adjust to pH 7.5 with HCl
    3. Complete to 1 liter with MilliQ water
    4. Filter through a 0.22 µm filter and store at room temperature
  2. 100 mM MgCl2
    1. Dissolve 95.21 mg of MgCl2 in 10 ml of MilliQ water
    2. Filter through a 0.22 µm filter and store at room temperature
  3. 5 M NaCl
    1. Dissolve 95.21 mg of NaCl in 10 ml of MilliQ water
    2. Filter through a 0.22 µm filter and store at room temperature

Acknowledgments

This work was supported by the French National Research Agency via the investment expenditure program IDEALG (ANR-10-BTBR-02). The authors also acknowledge funding from the Émergence-UPMC-2011 research program.

References

  1. 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.
  2. Groisillier, A., Shao, Z., Michel, G., Goulitquer, S., Bonin, P., Krahulec, S., Nidetzky, B., Duan, D., Boyen, C. and Tonon, T. (2014). Mannitol metabolism in brown algae involves a new phosphatase family. J Exp Bot 65(2): 559-570.

简介

褐藻属于与绿色植物和动物遥远相关的系统发生谱系,并且主要发现于但不限于潮间带,一种苛刻且频繁变化的环境。由于它们独特的进化史和它们的栖息地,褐藻具有其代谢中的一些特性。其中之一是甘露醇循环,其在其生理学中发挥中心作用,因为甘露糖醇充当碳储存,渗透保护剂和抗氧化剂。该多元醇通过甘露醇-1-磷酸酯脱氢酶(M1PDH,EC 1.1.1.17)和甘露醇-1-磷酸酶(M1Pase,EC 3.1.3.22)的作用直接从光生酸酯果糖-6-磷酸衍生。该方案描述了Ectocarpus的重组M1P酶的生物化学表征。 M1P酶催化甘露醇-1-磷酸转化为甘露醇(图1)。



图1.甘露醇-1-磷酸酶催化的反应

关键字:甘露醇周期, 甘露醇- 1 -磷酸酶, 水云属sp。, 褐海藻

材料和试剂

  1. UV-Star PS微孔板(96孔)(Greiner Bio-One GmbH,目录号:655801)
  2. 0.22μm过滤器
  3. 大肠杆菌 BL21(DE3)
  4. (Sigma-Aldrich,目录号:T1503)
  5. 纯化的重组His标签的M1P酶
    注意:该蛋白质在含有重组pFO4_ M1Pase载体的大肠杆菌BL21(DE3)中产生,如Groisillier等人 (2010)。 该重组蛋白通过亲和层析纯化 His GErep FF 16/10柱(GE Healthcare)上,在Äktaavant系统(GE Healthcare)上。 完整的纯化方案详细描述于Groisillier et al。 (2014)。
  6. MgCl 2(Sigma-Aldrich,目录号:M8266)
  7. MilliQ水
  8. 孔雀绿磷酸盐测定试剂盒(Gentaur,目录号:POMG-25H)
    注意:孔雀绿磷酸盐测定方案可在 https://www.bioassaysys.com/Datasheet/POMG.pdf
  9. 要测试以评估底物特异性的化学品的实例
    1. D-甘露醇-1-磷酸酯(Sigma-Aldrich,目录号:92416)
    2. His GErep FF 16/10柱(GE Healthcare)上,在Äktaavant系统(GE Healthcare)上。 完整的纯化方案详细描述于Groisillier et al。 (2014)。
    3. MgCl 2(Sigma-Aldrich,目录号:M8266)
    4. MilliQ水
    5. 孔雀绿磷酸盐测定试剂盒(Gentaur,目录号:POMG-25H)
      注意:孔雀绿磷酸盐测定方案可在 https://www.bioassaysys.com/Datasheet/POMG.pdf
    6. 要测试以评估底物特异性的化学品的实例
      1. D-甘露醇-1-磷酸酯(Sigma-Aldrich,目录号:92416)
      2. 5 M NaCl(见配方)

      设备

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

      软件

      1. Hyper32(Informer Technologies, http://hyper32.software.informer.com/
      2. Microsoft Excel

      程序

      1. 标准反应混合物含有100mM Tris-HCl(pH7.5),1mM M1P,5mM MgCl 2和6至78μg通过NanoDrop定量的纯化重组酶(基于18,700的消光系数 (对于目标蛋白质计算的),最终体积为450μl。 无活性煮沸的纯化重组酶用作阴性对照(表1)
        表1.用于测定M1Pase活性的阴性对照和反应混合物的组成


      2. 用水,Tris-HCl,MgCl 2和酶制备混合物,然后通过加入底物开始反应,并且在30℃下进行温育12分钟,每3 min。为此,反应混合物的体积为450μl,并且在t 0℃,t 3min,t 6min下对80μl反应混合物进行取样, ,t <9min和t <12min。每个条件以三次重复测试(图2A)。


        图2.在1mM M1P存在下,620nm处吸光度的测量作为时间(分钟)的函数。曲线表示两个系列的一式三份,其含有24μg纯化的M1Pase和24μg的煮沸的纯化的M1Pase。在反应混合物中加入M1P之后立即进行在t 0下的Abs 620nm。 B.该表包含在测试的两个条件下和在不同的实验时间测量的吸光度值
        在每个样品中,通过加入20μl孔雀绿试剂来终止反应,用于随后测定释放的磷酸盐的量。为此目的,在室温下孵育20分钟后,在Safire2UV分光光度计酶标仪中在620nm读取每个样品的吸光度(图2B)。然后将数值与根据孔雀绿磷酸盐测定试剂盒制造商的说明制备的游离磷酸盐的标准曲线进行比较(图3)。


        图3.使用孔雀石绿磷酸盐测定试剂盒测定游离磷酸盐浓度 A.在室温下,对于不同浓度的磷酸盐(从孔1至8,每个浓度2个孔):0μM,4μM,8μM,12μM,16μM,24μM,32μM和40μM(仅缓冲液,100mM Tris-HCl pH 7.5,5mM MgCl 2, /终浓度)。 B.代表在620nm处监测的吸光度的标准曲线,作为游离磷酸盐(Pi)的可变量的函数。在该实施例中,线性回归曲线为Abs 620nm = [0.0001×(ppm Pi in100μl)] -0.0085,和R sup2 = 0.9985。 C.对应于对不同量的游离磷酸盐测量的吸光度值的表
      3. 使用如图3中确定的线性回归曲线计算基于游离磷酸盐产生的磷酸酶活性:
        [(ΔA620nm测试-ΔA620nm阴性对照)+ 0.0085] /(0.0001 * t)
        其中
        ΔA620nm=孵育期间的吸光度变化
        t =培养时间(min)
        一个活性单位(U)对应于每分钟释放的1nmol Pi
      4. 为了计算比活性,将上式中获得的值除以样品中存在的蛋白质的量。对每个测定进行三次重复,并确定这三个重复的平均值±标准误差(S.E。)。从图2中给出的值,在1mM M1P存在下,M1Pase的比活性为11.30U/mg。
      5. 为了测定底物特异性,在"材料和试剂"部分中列出的每种底物的存在下测试M1Pase活性,使用 浓度范围为0.05mM至1mM,在室温下
      6. 为了确定最佳温度,在步骤1中使用的反应混合物在例如10℃至50℃的温度范围内,以5或10℃的增量孵育。下述实验在30℃的最佳温度下进行
      7. 在5mM MgCl 2存在下,通过使用pH范围为5.8至9的100mM Tris/HCl缓冲液测定pH对纯化的M1P酶的酶活性的影响。
      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NaCl,以获得范围为0至1μM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存在下进行单个酶反应(例如:0.08mM,0.12mM,0.18mM,0.27mM,0.4mM和0.6mM)。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4)。此计算可以使用Hyper32软件完成。


        图4.用于测定M1P的M1 mase的K m(0.67mM)和V m(25.25U/mg)的Lineweaver-Burk图。强> [S]是M1P浓度(以mM计),V是反应速率(以U/mg蛋白质计)。对每次测定进行三次重复

      笔记

      1. 尊重每个反应的孵育时间。 在不同的孵育时间从反应混合物取出的样品保持在冰中直到实验结束。 然后加入孔雀石20分钟,然后读取620nm处的吸光度。
      2. 在室温下加入孔雀石绿和读取620nm处的吸光度之间的时间为20分钟。
      3. 对反应混合物的组成,例如缓冲液,pH的每个变化执行标准曲线。
      4. 该方案可用于检测全细胞提取物中的M1Pase活性。 但是,这是必要的 在使用孔雀石绿色试剂盒以量化由细胞提取物中所含酶释放的磷酸盐时要谨慎,因为这些提取物中存在的化学物质可能引起干扰。 同样重要的是考虑细胞提取物可以含有游离磷酸盐,其将由孔雀绿试剂盒定量。

      食谱

      1. 1M Tris-HCl(pH7.5)
        1. 将121.14g Trizma底溶于约800ml MilliQ水中
        2. 用HCl调节pH 7.5,
        3. 用MilliQ水补足至1升
        4. 通过0.22μm过滤器过滤并在室温下贮存
      2. 100mM MgCl 2
        1. 将95.21mg MgCl 2溶解在10ml MilliQ水中
        2. 通过0.22μm过滤器过滤并在室温下贮存
      3. 5 M NaCl
        1. 将95.21mg NaCl溶于10ml MilliQ水中
        2. 通过0.22μm过滤器过滤并在室温下贮存

      致谢

      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。 作者还感谢Émergence-UPMC-2011研究计划的资助。

      参考文献

      1. Großillier,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生物的后基因组分析。 Microb Cell Fact 9:45.
      2. Groisillier,A.,Shao,Z.,Michel,G.,Goulitquer,S.,Bonin,P.,Krahulec,S.,Nidetzky,B.,Duan,D.,Boyen,C.and Tonon, 2014)。  褐藻中的甘露醇代谢涉及新的磷酸酶 65(2):559-570。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Groisillier, A. and Tonon, T. (2016). Determination of Recombinant Mannitol-1-phosphatase Activity from Ectocarpus sp.. Bio-protocol 6(16): e1896. DOI: 10.21769/BioProtoc.1896.
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