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Measurement of Nucleotide Triphosphate Sugar Transferase Activity via Generation of Pyrophosphate
通过焦磷酸盐的产生测量核苷三磷酸糖转移酶的活性   

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参见作者原研究论文

本实验方案简略版
Journal of Bacteriology
Jul 2014

Abstract

Nucleotide triphosphate (NTP) transferases (EC. 2. 7. 7. X) transfer a nucleoside monophosphate moiety from NTP to another substrate. NTP sugar transferases form a large member of the NTP transferase. There are many variations for the substrate combination of the NTP sugar transferases. It is important to measure the precise enzymatic activity of such NTP sugar transferases by a simple and efficient method. In our method, we measure pyrophosphate as a byproduct of nucleotide diphosphate (NDP)-sugar generation using the pyrophosphate assay kit. The kit reagents include two enzymes that convert pyrophosphate to phosphate, and then phosphorolyze chromogenic substrate to allow color development at 360 nm (see details below). Thus, the NDP-sugar formation can be simply traced as production of pyrophosphate, which is monitored by absorbance at 360 nm. This method is reliable and versatile for measurements of various pyrophosphate-producing enzymes that include NTP sugar transferases.

[Principle and overview] NTP transferases catalyze the reversible reaction as follows: NTP + sugar-1P <-> NDP-sugar + PPi

The enzyme reaction can be monitored as generation of inorganic pyrophosphate (PPi). The EnzChek Pyrophosphate Assay kit (Molecular Probes, Life Technologies, Carlsbad, CA) includes two enzymes and sufficient materials for color development to quantitate pyrophosphate. The inorganic pyrophosphatase (component E in the kit) degrades pyrophosphate into phosphate. Purine nucleoside phosphorylase (PNP, component B) utilizes phosphate to cleave the colorgenic substrate 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG, component A) into ribose-1-phosphate and 2-amino-6-mercapto-7-methylpurine. The product 2-amino-6-mercapto-7-methylpurine has the absorption maximum at 360 nm. The component C is the dilution solution that includes minimal MgCl2 sufficient for the inorganic pyrophosphatase. Thus, NTP transferase activity can be monitored at 360 nm as generation of a byproduct pyrophosphate.

Typically, the nucleotidyl sugar transferase reactions have been measured by High Performance Liquid Chromatography (HPLC) (Kawano et al., 2014). There are several advantages and disadvantages in HPLC method and our enzymatic method (O: advantage, X: disadvantage).
{HPLC method}
O) Can measure the reaction in both directions (NDP-sugar formation and degradation)
O) Can measure with a small amount of the sample protein
X) Can not follow the real-time reaction
X) The peaks of substrates and products must be separated in the chromatogram
{Pyrophosphate assay method}
O) Can observe the reaction in real-time
O) Easy to use many kinds of substrates because of the simple detection at 360 nm
O) Can be used in other pyrophosphate or phosphate generating reactions such as adenylate cyclase, diguanylate cyclase ( Enomoto et al., 2014), and DNA polymerase
X) Can not measure the pyrophosphate-consuming direction of the reversible reaction of NDP-sugar pyrophosphorylase. Km and kcat for NDP-sugar and pyrophosphate are not obtained by the pyrophosphate assay method but by HPLC method.
X) Need certain amount of the experimental protein. The maximum activity that the kit reagents allow corresponds theoretically to the rate of color development for the positive control is 2 x 10-2 U in our case. The instruction states the minimum detection of 5 x 10-5 U. Of course, the minimum activity we can measure may depend on the background activity due to contaminants in the substrates or in the sample preparation.


Figure 1. Chromogenic reactions in the assay kit

Materials and Reagents

  1. EnzChek Pyrophosphate Assay kit (Molecular Probes, catalog number: E-6645 )
    1. The contents of the components A~E are shown below.
    2. 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG)
    3. Purine nucleoside phosphorylase (PNP)
    4. 20x reaction buffer: 1.0 mM Tris-HCl, 20 mM MgCl2 (pH 7.5)
    5. Pyrophosphate standard solution: 50 mM Na4P2O7
    6. Inorganic pyrophosphatase
  2. 1 M MgCl2
  3. 100 mM substrates (our examples in Table 1)
  4. Enzyme preparation (with or without imidazole, see note 5 below)

Equipment

  1. Block incubator (ASTEC, model: BI-516S )
  2. Spectrophotometer (Shimadzu, model: UV-2600 )
  3. Electronic cooling and heating cuvette holder (Shimadzu, model: S-1700 )
  4. Quarts cuvette with a lid

    Table 1. An example of substrate reagents
    Substrate
    Company
    Catalog number
    Adenosine triphosphate (ATP)
    Sigma-Aldrich
    A-2383
    Guanosine triphosphate (GTP)
    Sigma-Aldrich
    G-8877
    Cytidine triphosphate (CTP)
    Sigma-Aldrich
    C-1506
    Uridine triphosphate (UTP)
    Sigma-Aldrich
    U-6625
    Deoxythymidine triphosphate (dTTP)
    Sigma-Aldrich
    T-0251
    Glucose-1-phosphate (Glc-1P)
    Sigma-Aldrich
    G-9380
    N-acetylglucosamine-1-phosphate (GlcNAc-1P)
    Sigma-Aldrich
    A-2142
    Galactose-1-phosphate (Gal-1P)
    Sigma-Aldrich
    G-0380
    Mannose-1-phosphate (Man-1P)
    Santa Cruz
    Biotechnology
    SC-284868

Procedure

  1. Measurement of enzyme activity
    1. Set up the spectrophotometer and electronic cooling and heating cuvette holder. Hold a cuvette in the holder to keep the temperature (usually 37 °C).
    2. Prepare the assay kit solutions as written in the product manual and keep them on ice. The instruction states component A should be used within 4 h when melted.
    3. Prepare reaction mixtures according to Table 2.
    4. Preincubate the mixture at 22 °C for 20 min to react the contaminated pyrophosphate and phosphate in the block incubator. We usually incubate the mixture for another 10 min at 37 °C (the measuring temperature) to warm up the mixture. The original instruction recommends the preincubation for 10 min at 22 °C.
    5. Put the reaction mixture into the cuvette in the spectrophotometer and record the absorbance at 360 nm in a time-scan mode for 5 ~ 10 min. Usually, the linear time course of the color development was obtained often initial lag time of 10 ~ 20 sec. Therefore, it may be sufficient to record time points every 30 ~ 60 sec.

  2. Data analysis
    1. Measure the rate of absorbance change (∆A360/sec).
    2. Convert the reaction rate (∆A360/sec) to the velocity (molar concentration/sec) by the difference of final A360 between the positive control and the negative control 1 (see the formula below).

      V (M / sec) =
      ∆A360 (cm-1) / sec - ∆A360 (cm-1) / sec (negative control)
      ε (M-1 cm-1)

      We experimentally determine the molar extinction coefficient of ~ 11,000 (M-1 cm-1) at 360 nm every time based on the final absorption of the positive control as follows.

      ε (M-1 cm-1) =
      final A360 (cm-1, positive control) - final A360 (cm-1, negative control 1)
      pyrophosphate concentration (M, positive control)

      The kit provides standard solution of 50 mM pyrophosphate (component D). We obtained fairy reproducible value for ε in our hands although the instruction does not mention about the known ε value, the original literature (Upson et al., 1996) states ε (M-1 cm-1) = 11,000 at pH 7.6.

      Table 2. Preparation of reaction mixtures


      The asterisk represent the kit components A~E. I~IV: The order of addition for preparation of reaction mixtures.

Representative data

Here are representative data of cyanobacterial UDP-glucose pyrophosphorylase expressed and purified from E. coli cells (Maeda et al., 2014). The initial slope of the graph was used for calculation. We usually use enzymes at 50 ~ 200 nM, and substrates [(d)NTP and sugar-1P] at 50 µM ~ 2 mM.

Table 3. Preparation of reaction mixtures in our example



Figure 2. An example of measurements for polyhistidine-tagged cyanobacterial UDP-glucose pyrophosphorylase, which was purified by Ni-affinity chromatography

Notes

  1. Before measurement, it is essential to check the reaction capacity of kit. Add 50~75 µM sodium pyrophosphate to the kit to confirm the maximum activity of pyrophosphorylase and purine nucleoside phosphorylase in the kit. We cannot measure NTP transferase activities higher than the maximum activity of the kit (Figure 2).
  2. To check the quality of the assay kit, we must measure the positive control first.
  3. Negative control 2 gives the level of contamination of phosphate and pyrophosphate. Negative control 3 and 4 give background enzyme activities to degrade (d)NTP and sugar-1P, respectively. Phosphate and pyrophosphate in the sample solutions or substrate reagents increase the background absorbance at 360 nm. We experienced some reagents such as mannose-1-phosphate contained too much phosphate. Such impurities may depend on the lot of the reagent or product manufacturer.
  4. A simple representative example for an enzyme sample is shown in Figure 2. The slope of the sample should be between the pyrophosphate positive control and the negative control. The initial slope of the graph was used for calculation according to the Data analysis.
  5. The imidazole (up to final 100 mM in the assay mixture) has little effect on the assay kit activity. This means that the kit accepts usual preparations of His-tagged enzymes, which are (partially) purified by Ni-affinity chromatography.
  6. Manganese ion (Mn2+) inhibits the assay kit activity at final 5 mM or even lower concentration.
  7. Reproducibility of the measurements is pretty high, though the maximum activity of the kit depends on the freshness of the kit.
  8. According to the kit instructions, the reagents of the kit are stable for six months to one year at < -20 °C. Reconstituted MESG (component A) may be stored at < -20 °C for at least one month. Reconstituted purine nucleoside phosphorylase (component B) may be stored at 4 °C for at least one month. Diluted inorganic pyrophosphatase (component E) may be stored at 4 °C for at least one week.

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research from MEXT and PRESTO from JST (to R. N.) and by Grants-in-Aid for Scientific Research and the GCOE program “From the Earth to Earths” from MEXT, and CREST from JST (to M. I.).

References

  1. Enomoto, G., Nomura, R., Shimada, T., Ni Ni, W., Narikawa, R., and Ikeuchi, M. (2014). Cyanobacteriochrome SesA is a diguanylate cyclase that induces cell aggregation in Thermosynechococcus. J Biol Chem 289(36): 24801-9.
  2. Kawano, Y., Sekine, M. and Ihara, M. (2014). Identification and characterization of UDP-glucose pyrophosphorylase in cyanobacteria Anabaena sp. PCC 7120. J Biosci Bioeng 117(5): 531-538.
  3. Maeda, K., Narikawa, R., and Ikeuchi, M. (2014). CugP is a novel ubiquitous non-GalU-type bacterial UDP-glucose pyrophosphorylase found in cyanobacteria. J Bacteriol 196 (13): 2348-54.
  4. Upson, R. H., Haugland, R. P., Malekzadeh, M. N. and Haugland, R. P. (1996). A spectrophotometric method to measure enzymatic activity in reactions that generate inorganic pyrophosphate. Anal Biochem 243(1): 41-45.

简介

核苷三磷酸(NTP)转移酶(EC.2.7.7.X)将核苷单磷酸部分从NTP转移到另一底物。 NTP糖转移酶形成NTP转移酶的大成员。 NTP糖转移酶的底物组合有许多变化。重要的是通过简单有效的方法测量这种NTP糖转移酶的精确酶活性。在我们的方法中,我们使用焦磷酸测定试剂盒测量焦磷酸作为核苷酸二磷酸(NDP)糖产生的副产物。试剂盒试剂包括将焦磷酸盐转化为磷酸盐,然后磷酸化发色底物以允许在360nm显色的两种酶(见下文详述)。因此,NDP-糖形成可以简单地追溯为焦磷酸盐的产生,其通过在360nm处的吸光度监测。该方法对于测量包括NTP糖转移酶的各种产生焦磷酸的酶是可靠的和多用途的。

[原理和概述] NTP转移酶催化可逆反应如下:NTP +糖-1P - NDP-糖+ PPi
酶反应可以监测为无机焦磷酸盐(PPi)的产生。 EnzChek焦磷酸测定试剂盒(Molecular Probes,Life Technologies,Carlsbad,CA)包括两种酶和用于显色以定量焦磷酸的足够材料。无机焦磷酸酶(试剂盒中的组分E)将焦磷酸盐降解为磷酸盐。嘌呤核苷磷酸化酶(PNP,组分B)利用磷酸将生色底物2-氨基-6-巯基-7-甲基嘌呤核糖核苷(MESG,组分A)切割成核糖-1-磷酸和2-氨基-6-巯基-7 - 甲基嘌呤。产物2-氨基-6-巯基-7-甲基嘌呤在360nm处具有最大吸收。组分C是包含对于无机焦磷酸酶足够的最小MgCl 2的稀释溶液。因此,可以在360nm监测NTP转移酶活性,作为副产物焦磷酸盐的产生。
通常,通过高效液相色谱(HPLC)测量核苷酸糖转移酶反应(Kawano等人,2014)。 HPLC方法和我们的酶法有几个优点和缺点(O:优点,X:缺点)。
{HPLC method}
O)可以测量两个方向的反应降解)
O)可用少量样品蛋白测量
X)不能跟踪实时反应
X)底物和产物的峰必须在色谱图中分离
{焦磷酸测定方法}
O)可实时观察反应
O)由于在360nm处的简单检测,易于使用多种底物
O)可用于其他产生焦磷酸盐或磷酸盐的反应,如腺苷酸环化酶,二鸟苷酸环化酶(Enomoto等人,2014)和DNA聚合酶
X)无法测量焦磷酸盐消耗方向的NDP-糖焦磷酸化酶的可逆反应。 NDP-糖和焦磷酸盐的Km和kcat不是通过焦磷酸测定法而是通过HPLC方法获得的。
X)需要一定量的实验蛋白。试剂盒试剂允许的最大活性在理论上对应于阳性对照的显色速率在我们的情况下为2×10 -2 -2U。该指令表明最小检测为5×10 -5 U。当然,我们可以测量的最小活性可以取决于由于底物中或样品制备中的污染物引起的背景活性。


图1.测定试剂盒中的显色反应

材料和试剂

  1. EnzChek焦磷酸测定试剂盒(Molecular Probes,目录号:E-6645)
    1. 组件A〜E的内容如下所示。
    2. 2-氨基-6-巯基-7-甲基嘌呤核糖核苷(MESG)
    3. 嘌呤核苷磷酸化酶(PNP)
    4. 20倍反应缓冲液:1.0mM Tris-HCl,20mM MgCl 2(pH7.5)
    5. 焦磷酸盐标准溶液:50mM Na 4 P 2 O 7
    6. 无机焦磷酸酶
  2. 1 M MgCl 2
  3. 100mM底物(表1中的实施例)
  4. 酶制剂(含或不含咪唑,见下文注5)

设备

  1. 块孵化器(ASTEC,型号:BI-516S)
  2. 分光光度计(Shimadzu,型号:UV-2600)
  3. 电子冷却和加热试管座(Shimadzu,型号:S-1700)
  4. 用盖子排空比杯杯

    表1.底物试剂的例子
    基板
    公司
    目录号码
    三磷酸腺苷(ATP)
    Sigma-Aldrich
    A-2383
    鸟苷三磷酸(GTP)
    Sigma-Aldrich
    G-8877
    胞苷三磷酸(CTP)
    Sigma-Aldrich
    C-1506
    尿苷三磷酸(UTP)
    Sigma-Aldrich
    U-6625
    脱氧胸苷三磷酸(dTTP)
    Sigma-Aldrich
    T-0251
    葡萄糖-1-磷酸(Glc-1P)
    Sigma-Aldrich
    G-9380
    N-乙酰葡糖胺-1-磷酸(GlcNAc-1P)
    Sigma-Aldrich
    A-2142
    半乳糖-1-磷酸(Gal-1P)
    Sigma-Aldrich
    G-0380
    甘露糖-1-磷酸(Man-1P)
    圣克鲁斯
    生物技术
    SC-284868

程序

  1. 测定酶活性
    1. 设置分光光度计和电子冷却和加热试管 持有人持有持有人的比色杯以保持温度(通常为37℃) °C)
    2. 按照产品中的说明准备试剂盒解决方案 手动将其保存在冰上。指令表示组件A 应在融化后4 h内使用。
    3. 根据表2制备反应混合物
    4. 将混合物在22℃预温育20分钟以反应 污染的焦磷酸盐和磷酸盐在块培养箱中。我们 通常在37℃下将混合物再孵育10分钟(测量) 温度)加热混合物。原始说明推荐 在22°C预温育10分钟。
    5. 放反应混合物 在分光光度计中进入比色皿,并记录吸光度 360 nm以时间扫描模式进行5〜10 min。通常,线性时间 颜色发展的过程通常是初始滞后时间 10〜20秒因此,每隔30〜60秒记录一次时间就足够了。

  2. 数据分析
    1. 测量吸光度变化率(ΔA<360 /秒)。
    2. 转换 反应速度(ΔA360℃/秒)与速度(摩尔浓度/秒)之比 最终的A< 360>之间的差异在正面对照和 负控制1(见下面的公式)。

      V(M/sec)=
      360度(cm -1)/sec-ΔA360(cm -1)/sec(cm -1)阴性对照)
      ε(M -1 cm -1

      我们通过实验来确定〜的摩尔消光系数 基于最终吸收值,每次360nm,每次11,000(M -1 cm -1 ) 积极的控制如下
      ε(M -1 cm -1 )=
      最终A 360(cm -1 ,阳性对照) - 最终A 360(cm -1 ,阴性对照1)
      焦磷酸盐浓度(M,阳性对照)

      该试剂盒提供标准溶液50mM焦磷酸盐(组分 D)。我们手中虽然可以获得童话的可重现价值, 原来没有提到已知的ε值 文献(Upson等人,1996)在pH 7.6下表示ε(M -1 cm -1 )= 11,000。

      表2.反应混合物的制备


      星号代表套件组件A〜E。 I〜IV:反应混合物的添加顺序。

代表数据

这里是从E表达和纯化的蓝细菌UDP-葡萄糖焦磷酸化酶的代表性数据。大肠杆菌细胞(Maeda等人,2014)。图的初始斜率用于计算。我们通常使用50〜200nM的酶,底物[(d)NTP和糖-1P]在50μM〜2mM。

表3.在我们的例子中的反应混合物的制备
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图2.多组氨酸标记的蓝细菌UDP-葡萄糖焦磷酸化酶的测量实例,其通过Ni-亲和层析纯化

笔记

  1. 在测量之前,必须检查试剂盒的反应能力。向试剂盒中加入50〜75μM焦磷酸钠,以确定试剂盒中焦磷酸化酶和嘌呤核苷磷酸化酶的最大活性。我们不能测量高于试剂盒最大活性的NTP转移酶活性(图2)
  2. 要检查试剂盒的质量,我们必须首先测量阳性对照。
  3. 负对照2给出磷酸盐和焦磷酸盐的污染水平。阴性对照3和4分别提供背景酶活性以降解(d)NTP和糖-1P。样品溶液或底物试剂中的磷酸盐和焦磷酸盐增加了360nm处的背景吸光度。我们经历了一些试剂,如甘露糖-1-磷酸盐含有太多的磷酸盐。这些杂质可能取决于试剂或产品制造商的数量。
  4. 酶样品的简单代表性实例如图2所示。样品的斜率应在焦磷酸盐阳性对照和阴性对照之间。根据数据分析,使用曲线图的初始斜率进行计算。
  5. 咪唑(在测定混合物中达到最终100mM)对测定试剂盒活性几乎没有影响。这意味着试剂盒可以接受通过Ni-亲和层析纯化的His-标记的酶的常规制备。
  6. 锰离子(Mn 2 + )抑制最终5mM或甚至更低浓度的测定试剂盒活性。
  7. 测量的重现性相当高,尽管套件的最大活动取决于套件的新鲜度。
  8. 根据试剂盒说明书,试剂盒的试剂在6个月至1年内稳定, -20°C。重组的MESG(组分A) -20°C至少一个月。重组嘌呤核苷磷酸化酶(组分B)可以在4℃下储存至少一个月。稀释的无机焦磷酸酶(组分E)可以在4℃下储存至少一周

致谢

这项工作得到MEXT和PRESTO科学研究资助项目的支持,这些科学研究由JST(到RN)和科学研究资助机构以及MEXT的GCOE计划"地球到地球"和来自MEXT的CREST JST(MI)。

参考文献

  1. Enomoto,G.,Nomura,R.,Shimada,T.,Ni Ni,W.,Narikawa,R。和Ikeuchi,M。(2014)。 蓝细菌色素SesA是一种二氢叶酸环化酶,其在热球感球菌中诱导细胞聚集。/a> J Biol Chem 289(36):24801-9。
  2. Kawano,Y.,Sekine,M。和Ihara,M。(2014)。 蓝藻中UDP-葡萄糖焦磷酸化酶的鉴定和表征 sp。 PCC 7120. Biosci Bioeng 117(5):531-538。
  3. Maeda,K.,Narikawa,R。和Ikeuchi,M。(2014)。 CugP是一种在蓝细菌中发现的新型无处不在的非GalU型细菌UDP-葡萄糖焦磷酸化酶。 a> J Bacteriol 196(13):2348-54。
  4. Upson,R.H.,Haugland,R.P.,Malekzadeh,M.N。和Haugland,R.P。(1996)。 测量产生无机焦磷酸盐的反应中的酶活性的分光光度法。 Anal Biochem 243(1):41-45。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Maeda, K., Narikawa, R. and Ikeuchi, M. (2015). Measurement of Nucleotide Triphosphate Sugar Transferase Activity via Generation of Pyrophosphate. Bio-protocol 5(8): e1450. DOI: 10.21769/BioProtoc.1450.
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当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。