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Determination of Recombinant Mannitol-1-phosphate Dehydrogenase Activity from Ectocarpus sp.
水云属中重组甘露醇-1-磷酸脱氢酶活性的测定   

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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 the recombinant catalytic domain of one of the three M1PDHs identified in Ectocarpus sp. This recombinant catalytic domain, named hereafter M1PDHcat, catalyzes the reversible conversion of fructose-6-phosphate (F6P) to mannitol-1-phosphate (M1P) using NAD(H) as a cofactor. M1PDHcat activity was assayed in both directions i.e., F6P reduction and M1P oxidation (Figure 1).


Figure 1. Reversible reaction of mannitol-1-phosphate dehydrogenase

Materials and Reagents

  1. UV-Star® PS microplate (96 well) (Greiner Bio One International, catalog number: 655801 )
  2. 0.22 µm filter
  3. Purified recombinant His-tagged M1PDHcat
    Note: This protein was produced in Escherichia coli BL21 (DE3) containing the recombinant pFO4_M1PDHcat 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) and then by gel filtration using a Superdex 200 (GE Healthcare) onto an Äkta avant system (GE Healthcare). The complete purification protocol is described in details in Bonin et al. (2015).
  4. MilliQ water
  5. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  6. 4-morpholineethane-sulfonic acid (MES) (Sigma-Aldrich, catalog number: M2933 )
  7. HEPES (Sigma-Aldrich, catalog number: H3375 )
  8. Bis-Tris propane (Sigma-Aldrich, catalog number: B6755 )
  9. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 71380 )
  10. Examples of chemicals to be tested to assess substrate and co-factor specificity:
    1. D-mannitol-1-phosphate (Sigma-Aldrich, catalog number: 92416 )
    2. D-fructose-1-phosphate (Sigma-Aldrich, catalog number: F1127 )
    3. α-D-glucose-1-phosphate disodium salt hydrate (Sigma-Aldrich, catalog number: G9380 )
    4. D-mannose-6-phosphate sodium salt (Sigma-Aldrich, catalog number: M3655 )
    5. D-glucose-6-phosphate sodium salt (Sigma-Aldrich, catalog number: G7879 )
    6. D-fructose-6-phosphate disodium salt hydrate (Sigma-Aldrich, catalog number: F3627 )
    7. β-NAD (Sigma-Aldrich, catalog number: N1636 )
    8. β-NADP (Sigma-Aldrich, catalog number: N5755 )
    9. β-NADH (Sigma-Aldrich, catalog number: N8129 )
    10. β-NADPH (Sigma-Aldrich, catalog number: N5130 )
  11. 1 M Tris-HCl (see Recipes)
  12. 5 M NaCl (see Recipes)
  13. 10 mM NADH (see Recipes)
  14. 10 mM NAD+ (see Recipes)

Equipment

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

Software

  1. Hyper 32 (Informer Technologies, model: hyper32)
  2. Microsoft Excel

Procedure

  1. The standard F6P reduction reaction mixture contains 50 mM Tris-HCl (pH 7.0), 1 mM F6P, 0.2 mM NADH and 0.1 to 10 µg of purified recombinant M1PDHcat, in a final volume of 100 µl. The standard M1P oxidation reaction mixture contains 50 mM Tris-HCl (pH 9.0), 1 mM M1P, 0.5 mM NAD+ and 0.1 to 10 µg of purified recombinant M1PDHcat in 100 µl. Quantities of proteins are determined with NanoDrop at 280 nm based on the extinction coefficient of 37,525 M-1 cm-1 calculated for the protein of interest. The negative control corresponds to the reaction mixture in which substrate is substituted by MilliQ water (Table1).

    Table 1. Composition of negative control and reaction mixture for determination of M1PDHcat activity in the direction of F6P reduction and M1P oxidation


  2. The mix is prepared with water, Tris-HCl, NAD(H) and enzyme, then the reaction is started by adding the substrate and the activity is monitored by following changes in absorbance at 340 nm, which corresponds to the consumption (F6P • M1P) or production (M1P • F6P) of NADH, in a Safire2 UV spectrophotometer microplate reader. The plate is subjected to 10 sec of orbital shaking before measurements. Values are recorded every 13 sec approximately, and this interval of time can be modified according to the activity of the enzyme under consideration. The assay is performed at 30 °C for up to 20 min. Only the early linear part of the curves is used to calculate activity (Figures 2 and 3).
  3. F6P reduction and M1P oxidation activities, based on consumption or production of NADH respectively, are calculated using the formula:
    [(ΔA340 nm test - ΔA340 nm negative control) x 0.0001/(t x 6220 x 0.3)]
    Where,
    ΔA340 nm = 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). This value depends of the plate/cuvette used,
    0.0001 = assay volume (L),
    One unit (U) of activity, F6P reduction or M1P oxidation, corresponds to 1 µmole of co-factor oxidized or reduced per min respectively.


    Figure 2. Measurement of absorbance (340 nm) monitored as a function of time (sec) in presence or in absence of M1P. A. The curves represent two series of triplicates obtained in absence or in presence of 1 mM of M1P and 0.2 µg of enzyme. B. The table contains values of absorbance measured under both conditions tested and at different times of experiment.


    Figure 3. Measurement of absorbance (340 nm) monitored as a function of time (sec) in presence or in absence of F6P. A. The curves represent two series of triplicates obtained in absence or in presence of 1 mM of F6P and 0.04 µg of enzyme. B. The table contains values of absorbance measured under both conditions tested and at different times of experiment.

  4. To calculate specific activities, divide the values obtained in the equation above by the quantity of M1PDHcat proteins present in the sample. Perform three replicates for each condition, and use the average calculated in absence and in presence of substrate to determine the specific activity of each reaction. This applies also to the experiments described below. From values of Figures 2 and 3, specific activities of M1PDHcat are 11.3 U/mg in presence of 1 mM M1P and 39.0 U/mg in presence of 1 mM F6P.
  5. To determine substrate specificity, test M1PDHcat activity in the presence of each substrate listed in the ‘Materials and Reagents’ section, using concentration ranging from 1 mM to at least 50 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 °C or 10 °C. The experiments described below are performed at the optimal temperature of 30 °C.
  7. To determine the optimal pH, replace the 50 mM Tris-HCl pH 7.0 or pH 9.0 buffer used in step 1 by other buffers prepared at different pHs. As an indication, it is possible to use:
    1. 50 mM MES for pH 5.5, 6, 6.5
    2. 50 mM Bis-Tris propane for pH 6.5, 7, 7.5, 8, 8.5, 9, 9.5
    3. 50 mM Tris-HCl for pH 7, 7.5, 8, 8.5, 9
    4. 50 mM Tris-acetate for 6, 6.5, 7
    5. 50 mM HEPES for 7, 7.5, 8, 8.5
  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 0.1 mM, 0.25 mM, 1 mM, 4 mM, 20 mM final concentration), and a fixed concentration of NAD+ or NADH. 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) using the hyper 32 software.


    Figure 4. Lineweaver-Burk plots used to determine in A) the Km (0.19 mM) and Vm (46 U/mg) of M1PDHcat for F6P and in B) the Km (0.38 mM) and Vm (15.6 U/mg) of M1PDHcat for the M1P. [S] is the substrate concentration (in mM) and V is the reaction rate (in U/mg of protein). Three replicates were performed for each assay.

Notes

  1. Before determining Km and Vm for a given substrate S, be sure that cofactor 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. The theory is that this saturating concentration is equivalent to 100Km, but 10Km is usually enough (Bisswanger, 2014). It is then necessary to adjust the amount of M1PDHcat and the incubation time to obtain a linear variation of absorbance at 340 nm, i.e., a linear change of NADH quantity.

Recipes

  1. 1 M Tris-HCl
    Dissolve 121.14 g of Trizma® base in about 800 ml of MilliQ water, adjust pH with HCl then complete to 1 liter. Filter through a 0.22 µm filter and store at room temperature.
  2. 5 M NaCl
    Dissolve 95.21 mg of NaCl in 10 ml of MilliQ water. Filter through a 0.22 µm filter and store at room temperature.
  3. 10 mM NADH
    Dissolve 6.63 mg of NADH in 1 ml of MilliQ water. Filter through a 0.22 µm filter and store at room temperature. Prepare fresh on the day of use.
  4. 10 mM NAD+
    Dissolve 6.63 mg of NAD+ in 1 ml of MilliQ water. Filter through a 0.22 µm filter and store at room temperature. Prepare fresh on the week of use and store at -20 °C.

Acknowledgments

This protocol was performed by Bonin et al. (2015). This work was supported by the French National Research Agency via the investment expenditure program IDEALG (ANR-1 0-BTBR-02). The authors also acknowledge funding from the Émergence-UPMC-2011 research program.

References

  1. Bisswanger, H. (2014). Enzymes assays. Perspec Sci 1: 41-55.
  2. Bonin, P., Groisillier, A., Raimbault, A., Guibert, A., Boyen, C. and Tonon, T. (2015). Molecular and biochemical characterization of mannitol-1-phosphate dehydrogenase from the model brown alga Ectocarpus sp. Phytochemistry 117: 509-520.
  3. 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.

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。该协议描述了在Ectocarpus中鉴定的三种M1PDH之一的重组催化结构域的生物化学表征。该重组催化结构域(下文称为M1PDHcat)使用NAD(H)作为辅因子催化果糖-6-磷酸(F6P)向甘露醇-1-磷酸(M1P)的可逆转化。 M1PDHcat活性在两个方向上测定,即,F6P还原和M1P氧化(图1)。

src =
图1.甘露醇-1-磷酸脱氢酶的可逆反应


材料和试剂

  1. UV-Star PS微孔板(96孔)(Greiner Bio One International,目录号:655801)
  2. 0.22μm过滤器
  3. 纯化的重组His标签的M1PDHcat
    注意:该蛋白质在含有重组pFO4_M1PDHcat载体的大肠杆菌BL21(DE3)中产生,如Groisillier等人(2010)。使用HisPrep FF 16/10柱(GE Healthcare)通过亲和层析,然后使用Superdex 200(GE Healthcare)通过凝胶过滤将该重组蛋白纯化到?ktaavant系统(GE Healthcare)上。完整的纯化方案详细描述于Bonin et al。 (2015)。
  4. MilliQ水
  5. (Sigma-Aldrich,目录号:T1503)
  6. 4-吗啉乙磺酸(MES)(Sigma-Aldrich,目录号:M2933)
  7. HEPES(Sigma-Aldrich,目录号:H3375)
  8. Bis-Tris丙烷(Sigma-Aldrich,目录号:B6755)
  9. 氯化钠(NaCl)(Sigma-Aldrich,目录号:71380)
  10. 要测试以评估底物和辅因子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. D-果糖-1-磷酸(Sigma-Aldrich,目录号:F1127)
    3. α-D-葡萄糖-1-磷酸二钠盐水合物(Sigma-Aldrich,目录号:G9380)
    4. D-甘露糖-6-磷酸钠盐(Sigma-Aldrich,目录号:M3655)
    5. D-葡萄糖-6-磷酸钠盐(Sigma-Aldrich,目录号:G7879)
    6. D-果糖-6-磷酸二钠盐水合物(Sigma-Aldrich,目录号:F3627)
    7. β-NAD(Sigma-Aldrich,目录号:N1636)
    8. β-NADP(Sigma-Aldrich,目录号:N5755)
    9. β-NADH(Sigma-Aldrich,目录号:N8129)
    10. β-NADPH(Sigma-Aldrich,目录号:N5130)
  11. 1 M Tris-HCl(参见配方)
  12. 5 M NaCl(见配方)
  13. 10 mM NADH(参见配方)
  14. 10 mM NAD + (参见配方)

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)

软件

  1. Hyper 32(Informer Technologies,型号: hyper32
  2. Microsoft Excel

程序

  1. 标准F6P还原反应混合物含有50mM Tris-HCl(pH 7.0),1mM F6P,0.2mM NADH和0.1至10μg纯化的重组M1PDHcat,终体积为100μl。标准M1P氧化反应混合物在100μl中含有50mM Tris-HCl(pH 9.0),1mM M1P,0.5mM NAD +和0.1至10μg纯化的重组M1PDHcat。基于针对目的蛋白质计算的37,525M sup-1 cm -1消耗系数,用280nm的NanoDrop测定蛋白质的量。阴性对照对应于其中底物被MilliQ水替代的反应混合物(表1)
    表1.用于测定在F6P还原和M1P氧化方向上M1PDHcat活性的阴性对照和反应混合物的组成


  2. 用水,Tris-HCl,NAD(H)和酶制备混合物,然后通过加入底物开始反应,通过在340nm处的吸光度变化监测活性,其对应于消耗(F6P·M1P )或生产(M1P?F6P)的NADH,在Safire2紫外分光光度计酶标仪。在测量之前,使板经受10秒的轨道摇动。每13秒记录一次值,并且可以根据所考虑的酶的活性来修改该时间间隔。该测定在30℃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和图3)
  3. F6P还原和M1P氧化活性,分别基于NADH的消耗或生产,使用以下公式计算:
    [(ΔA340nm测试-ΔA340nm阴性对照)×0.0001 /(t×6220×0.3)]
    在哪里,
    ΔA340nm=培养期间的吸光度变化,
    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)。该值取决于所使用的板/比色皿,
    0.0001 =测定体积(L),
    一个活性单位(U),F6P还原或M1P氧化,分别相当于每分钟氧化或还原的1微摩尔辅因子。


    图2.在存在或不存在M1P的情况下作为时间(秒)的函数监测的吸光度(340nm)的测量.A。曲线表示在不存在或存在下的两个系列的一式三份1mM的M1P和0.2μg的酶。 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

    图3.在存在或不存在F6P的情况下作为时间(秒)的函数监测的吸光度(340nm)的测量。所述曲线表示在不存在或存在或不存在下获得的两个系列的一式三份1mM F6P和0.04μg酶。 B.该表包含在两种测试条件下和在不同实验时间测量的吸光度值
  4. 为了计算比活性,将上式中获得的值除以样品中存在的M1PDHcat蛋白的量。对每种条件进行三次重复,并使用在不存在和存在底物时计算的平均值,以确定每个反应的比活性。这也适用于下述实验。从图2和图3的值,M1PDHcat的比活性在1mM M1P存在下为11.3U/mg,在1mM F6P存在下为39.0U/mg。
  5. 为了测定底物特异性,在室温下,在"材料和试剂"部分中列出的每种底物存在下,使用浓度范围为1mM至至少50mM,测试M1PDHcat活性。
  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℃的增量温育。下述实验在30℃的最佳温度下进行
  7. 为了确定最佳pH,用在不同pH下制备的其它缓冲液替换步骤1中使用的50mM Tris-HCl pH 7.0或pH 9.0缓冲液。作为指示,可以使用:
    1. 50 mM MES,pH 5.5,6,6.5
    2. 50mM Bis-Tris丙烷,pH6.5,7,7.5,8,8.5,9,9.5。
    3. 50mM Tris-HCl(pH 7),7.5,8,8.5,9,
    4. 50mM Tris-乙酸盐,6,6.5,7mM
    5. 50 mM HEPES,7,7.5,8,8.5
  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(例如0.1mM,0.25mM,1mM,4mM,20mM终浓度)存在下进行单独的酶反应, ,和固定浓度的NAD +或NADH。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可以使用hyper 32软件计算(图4)

    图4.用于在A)中测定F6P的M1 subHcat(0.19mM)和V m1(46U/mg)的MIIRHcat的Lineweaver-Burk图和B)M1P的M1PDHcat的K m(0.38mM)和V m(15.6U/mg)。是底物浓度(mM),V是反应速率(U/mg蛋白质)。对每次测定进行三次重复

笔记

  1. 在确定给定衬底S的K m和V m之前,确保辅因子过量,即,即,V不随着增加反应混合物中辅因子的量;在相同的静脉中,在确定辅因子的K sub和V sub之前,确保S是过量的,即,e不随着反应混合物中S的量的增加而增加。理论上,该饱和浓度相当于100K m,但10K m/s通常就足够了(Bisswanger,2014)。然后需要调节M1PDHcat的量和孵育时间以获得在340nm处的吸光度的线性变化,即NADH量的线性变化。

食谱

  1. 1M Tris-HCl
    将121.14g Trizma底溶解在约800ml的MilliQ水中,用HCl调节pH,然后完成至1升。通过0.22μm过滤器过滤,在室温下储存
  2. 5 M NaCl
    将95.21mg NaCl溶解在10ml MilliQ水中。通过0.22μm过滤器过滤,在室温下储存
  3. 10mM NADH
    将6.63mg NADH溶解在1ml MilliQ水中。通过0.22μm过滤器过滤并在室温下储存。在使用当天准备新鲜。
  4. 10 mM NAD +
    将6.63mg的NAD +溶解在1ml的MilliQ水中。通过0.22μm过滤器过滤并在室温下储存。在使用周准备新鲜,并在-20°C下保存

致谢

该方案由Bonin等人进行。 (2015)。这项工作是由法国国家研究机构通过投资支出方案IDEALG(ANR-10-BTBR-02)支持的。作者还感谢émergence-UPMC-2011研究计划的资助。

参考文献

  1. Bisswanger,H.(2014)。  酶分析。 em> Perspec Sci 1:41-55。
  2. Bonint,P.,Groisillier,A.,Raimbault,A.,Guibert,A.,Boyen,C.and Tonon,T。(2015)。  来自模型棕色藻的外来物的甘露醇-1-磷酸脱氢酶的分子和生物化学表征 Phytochemistry 117:509-520。
  3. 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生物的后基因组分析。 Microb Cell Fact 9:45.
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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-phosphate Dehydrogenase Activity from Ectocarpus sp.. Bio-protocol 6(21): e1982. DOI: 10.21769/BioProtoc.1982.
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