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Campylobacter jejuni γ-glutamyltranspeptidase Activity Assay
空肠弯曲杆菌γ-谷氨酰胺转肽酶活性鉴定   

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Abstract

The enzyme γ-glutamyltranspeptidase (GGT, EC 2.3.2.2) is highly conserved among eukaryotic and prokaryotic organisms (Heisterkamp et al., 2008) and has a key function in glutathione metabolism. Although the enzyme is highly conserved and found throughout organisms ranging from bacteria to plants and animals several major difference between eukaryotic and prokaryotic GGT can be noticed. They mainly concern the enzyme localization and posttranslational modification. Eukaryotic GGT is cell membrane anchored and highly glycosylated whereas prokaryotic GGT does not undergo this posttranslational modification and is a soluble periplasmic protein. GGT amino acids sequences of diverse origin exhibit high amino acid similarity (Ong et al., 2008). The prokaryotic GGT enzymes are produced as proenzyme, equipped with a typical prokaryotic signal sequence and transported through the inner membrane into the periplasm where the enzyme undergoes autocatalytic cleavage. This proteolysis yields a mature dimer which transfers the γ-glutamyl moieties from extracellular glutathione and related compounds to amino acids or peptides (Hanigan et al., 1998). The GGT enzyme activity can be easily measured as it catalyzes the transfer of a γ-glutamyl group from a colorless substrate, L-γ-glutamyl-3-carboxy-4-nitroanilide, to the acceptor, glycylglycine with leads to the production of yellow colored product, p-nitroaniline (Figure 1) which can be measured by a spectrophotometer (Figure 2). Here we describe a protocol to measure the GGT activity in the Gram-negative bacterium Campylobacter jejuni, with some minor modifications this protocol works also for other Gram-negative bacterial species.



Figure 1. Yellow colored product, p-nitroaniline formed during the GGT enzyme assay


Figure 2. Spectral curve of pNA in Tris/HCl buffer, recorded on a Biodrop µLite (Isogen)

Keywords: Campylobacter(空肠弯曲菌), Bacterial GGT assay(细菌GGT的检测), Glutamyltranspetidase(谷氨酰转肽酶), Periplasmic enzym assay(周质酶的测定), Glutathione(谷胱甘肽)

Materials and Reagents

  1. 25 mm culture flasks with vent cap (Corning, catalog number: 430639 )
  2. 96 well plate flat bottom (Corning, Costar®, catalog number: 3599 )
  3. Campylobacter culture
  4. UltraPure™ Tris Buffer (powder format) (Thermo Fisher Scientific, Invitrogen™, catalog number: 15504-020 )
  5. Lysozyme from chicken egg white (Sigma-Aldrich, catalog number: L-6876 )
  6. L-Glutamic acid γ-(3-carboxy-4-nitroanilide) ammonium salt (Sigma-Aldrich, catalog number: 49525 )
  7. Glycylglycine (Sigma-Aldrich, catalog number: G3915 )
  8. Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 23227 )
  9. 4-nitroaniline (Sigma-Aldrich, catalog number: 185310 )
  10. Hearth Infusion medium (Thomas Scientific, Oxoid, catalog number: CM1032B )
  11. Tris/HCl (pH 7.6 and pH 8.2)
  12. Lysis buffer (see Recipes)
  13. Reaction buffer (see Recipes)
  14. pNA stock solution (see Recipes)

Equipment

  1. Absorbance microplate reader (such as BMG Labtech GmbH, Fluorstar Omega orequivalent)
  2. Vibra-Cell™ Sonicater (Sonics & Materials, model: VC40 )
  3. Refrigerated table top centrifuge
  4. The anoxomat system (MART Microbiology)

Procedure

  1. Dilute a Campylobacter preculture in 5 ml Heart Infusion (HI) broth to an optical density 600 nm of 0.05. Growth the culture in vertically standing 25 mm culture flasks with vent cap in a microaerobic atmosphere (MART) at 42 °C, 160 rpm in HI broth for until stationary phase ~20 h.
    Note: If other bacterial species are assayed use species specific growth conditions.
  2. Pellet ~1.109 bacteria of the bacterial culture, remove the supernatant and store the pellet at -80 °C for at least 1 h.
    Note: The -80 °C freezing step will help to disrupt the cells, shorter incubation will lead to less disrupted cells.
  3. Resuspend the pellet in ice cold 250 μl lysis buffer, and incubated for 30 min on ice.
  4. Sonicate 6 times (5 sec each, with 15 sec intervals on ice) at 40 mV and the output control set at 80%.
  5. Centrifuge the cell lysate for 10 min at 12,000 x g at 4 °C.
  6. Harvest the supernatant and keep on ice before use.
  7. Measure the protein concentration of 20 μl supernatant (BCA kit with standard) in a 96 wells plate according to the kit’s protocol.
  8. Make a serial dilution in a range between 0 and 200 µM of the standard pNA [4-nitroaniline (stock solution 1 mM)] in 50 mM Tris/HCl (pH 7.6).
  9. Mix 20 μl supernatant or standard pNA in a 96 wells plate with 180 μl of reaction buffer.
  10. Measure the optical density of the samples and standard in the 96 wells plate at a wavelength of 405 nm (OD405) every 60 s during an incubation period of 30 min at 37 °C (Figures 3 and 4).
  11. Calculate the slope of all values in a linear range of the obtained graphs (see below).
  12. GGT activity is expressed as nmol min-1 mg protein-1.


Figure 3. A typical pNA standard curve


Figure 4. Graph showing in time the pNA increase in the bacterial lysates made of C.jejuni wt (red) and racRS mutant (blue)

To calculate the GGT activity first plot the pNA standard curve. Calculate the Δabsorbance (A time x - A time y) in a linear range. Next compare the calculated Δabsorbance of each sample to the standard pNA curve to determine the amount of pNA generated between time x and time y (nmol pNA).

The GGT activity is calculated by the formula: (nmol pNA)
T x M
T = the time incubated (in min)
M = mg protein determined by BCA protein kit
GGT activity is reported as nmole/min-1/ mg protein-1

Example:
Δabsorbance(wt) = 0.58 (15 min) - 0.43 (5 min)=0.15
Δabsorbance 0.15 = 40 nmole pNA
Time x - time y = 10 min
M = 0.02 mg protein
GGT activity is: 40/(10 x 0.02) = 200 nmole/min-1/ mg protein-1

Recipes

  1. Lysis buffer
    50 mM Tris/HCl (pH 7.6)
    1 μg/ml lysozyme
  2. Reaction buffer
    2.9 mM L-γ-glutamyl-3-carboxy-4-nitroanilide
    100 mM glycylglycine
    100 mM Tris/HCl (pH 8.2)
  3. pNA stock solution
    1 mM 4-nitroaniline
    50 mM Tris/HCl (pH 7.6)
    Can be stored at -20 °C

Acknowledgments

The γ-glutamyltranspeptidase activity protocol described here and in, van der Stel et al. (2015), is modified from a protocol described in Chevalier et al. (1999). This work was supported by the NWO-ECHO grant 711.012.007 to MW.

References

  1. Chevalier, C., Thiberge, J. M., Ferrero, R. L. and Labigne, A. (1999). Essential role of Helicobacter pylori γ-glutamyltranspeptidase for the colonization of the gastric mucosa of mice. Mol Microbiol 31(5): 1359-1372.
  2. Hanigan, M. H. (1998). gamma-Glutamyl transpeptidase, a glutathionase: its expression and function in carcinogenesis. Chem Biol Interact 111-112: 333-342.
  3. Heisterkamp, N., Groffen, J., Warburton, D. and Sneddon, T. P. (2008). The human gamma-glutamyltransferase gene family. Hum Genet 123(4): 321-332.
  4. Ong, P. L., Yao, Y. F., Weng, Y. M., Hsu, W. H. and Lin, L. L. (2008). Residues Arg114 and Arg337 are critical for the proper function of Escherichia coli gamma-glutamyltranspeptidase. Biochem Biophys Res Commun 366(2): 294-300.
  5. van der Stel, A. X., van Mourik, A., Laniewski, P., van Putten, J. P., Jagusztyn-Krynicka, E. K. and Wosten, M. M. (2015). The Campylobacter jejuni RacRS two-component system activates the glutamate synthesis by directly upregulating gamma-glutamyltranspeptidase (GGT). Front Microbiol 6: 567.

简介

酶γ-谷氨酰转肽酶(GGT,EC 2.3.2.2)在真核和原核生物体中是高度保守的(Heisterkamp等人,2008),并且在谷胱甘肽代谢中具有关键功能。虽然酶是高度保守的,并且在从细菌到植物和动物的整个生物体中发现,可以注意到真核和原核GGT之间的几个主要区别。它们主要涉及酶定位和翻译后修饰。真核GGT是细胞膜锚定和高度糖基化,而原核GGT不经历这种翻译后修饰,并且是可溶性周质蛋白。不同来源的GGT氨基酸序列显示高氨基酸相似性(Ong等人,2008)。原核GGT酶作为酶原产生,装备有典型的原核信号序列,并通过内膜转运到周质中,其中酶经历自催化切割。这种蛋白水解产生成熟的二聚体,其将γ-谷氨酰部分从胞外谷胱甘肽和相关化合物转移到氨基酸或肽(Hanigan等人,1998)。可以容易地测量GGT酶活性,因为其催化γ-谷氨酰基从无色底物L-γ-谷氨酰基-3-羧基-4-硝基苯胺转移至受体甘氨酰甘氨酸,导致产生黄色有色产物,对硝基苯胺(图1),其可以通过分光光度计(图2)测量。在这里我们描述了一种测定革兰氏阴性细菌空肠弯曲杆菌中的GGT活性的方案,其中一些微小的修饰,该方案也适用于其他革兰氏阴性菌/em>细菌种。



黄色产物,在GGT酶测定期间形成的对硝基苯胺


图2.在BiodropμLite(Isogen)上记录的pNA在Tris/HCl缓冲液中的光谱曲线<

关键字:空肠弯曲菌, 细菌GGT的检测, 谷氨酰转肽酶, 周质酶的测定, 谷胱甘肽

材料和试剂

  1. 带有通风帽的25mm培养瓶(Corning,目录号:430639)
  2. 96孔板平底(Corning,Costar ,目录号:3599)
  3. 弯曲杆菌培养物
  4. UltraPure TM Tris缓冲液(粉末形式)(Thermo Fisher Scientific,Invitrogen TM,目录号:15504-020)
  5. 来自鸡蛋白的溶菌酶(Sigma-Aldrich,目录号:L-6876)
  6. L-谷氨酸γ-(3-羧基-4-硝基苯胺)铵盐(Sigma-Aldrich,目录号:49525)
  7. 甘氨酰甘氨酸(Sigma-Aldrich,目录号:G3915)
  8. Pierce TM BCA蛋白测定试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,目录号:23227)
  9. 4-硝基苯胺(Sigma-Aldrich,目录号:185310)
  10. 炉底输液介质(Thomas Scientific,Oxoid,目录号:CM1032B)
  11. Tris/HCl(pH 7.6和pH 8.2)
  12. 裂解缓冲液(见配方)
  13. 反应缓冲液(参见配方)
  14. pNA储备溶液(见配方)

设备

  1. 吸光度酶标仪(例如BMG Labtech GmbH,Fluorstar Omega等)
  2. Vibra-Cell TM Sonicater(Sonics& Materials,型号:VC40)
  3. 冷冻台式离心机
  4. anoxomat系统(MART Microbiology)

程序

  1. 在5ml心浸液(HI)肉汤中稀释弯曲杆菌预培养物至0.05的光密度600nm。在具有通气帽的垂直放置的25mm培养烧瓶中,在微量厌氧气氛(MART)中,在42℃,160rpm,HI肉汤中培养培养物直至静止期约20小时。
    注意:如果其他细菌种类的测定使用物种的特定生长条件。
  2. 细胞培养物的细胞,去除上清液并将沉淀物在-80℃下储存至少1小时。
    注意:-80°C冷冻步骤有助于破坏细胞,更短的温育将导致更少的细胞破坏。
  3. 将沉淀重悬于冰冷的250μl裂解缓冲液中,并在冰上孵育30分钟
  4. 在40mV下超声6次(每次5秒,在冰上15秒间隔),输出控制设定为80%。
  5. 在4℃下,以12,000×g离心细胞裂解液10分钟。
  6. 收获上清液,使用前保存在冰上。
  7. 根据试剂盒的方案,在96孔板中测量20μl上清液(标准的BCA试剂盒)的蛋白质浓度。
  8. 在50mM Tris/HCl(pH 7.6)中,在标准pNA [4-硝基苯胺(储备溶液1mM)]的0至200μM范围内进行系列稀释。
  9. 将20μl上清液或标准pNA在96孔板中与180μl反应缓冲液混合
  10. 在37℃下孵育30分钟期间,每60秒在405nm波长(OD 405)下测量96孔板中的样品和标准品的光密度(图3和图4 )。
  11. 计算所获得图的线性范围内所有值的斜率(见下文)。
  12. GGT活性表示为nmol min <-1> mg蛋白 -1


图3.典型的pNA标准曲线


图4.以时间显示由空肠弯曲杆菌(红色)和外显子R 突变体(蓝色)制成的细菌裂解物中pNA增加的图。

为了计算GGT活性,首先绘制pNA标准曲线。计算线性范围内的Δ吸光度(A时间x - A时间y)。接下来将每个样品的计算的Δ吸光度与标准pNA曲线进行比较,以确定在时间x和时间y之间产生的pNA的量(nmol pNA)。

GGT活性由下式计算:(nmol pNA)
T x M
T =孵育时间(以分钟计)
M =通过BCA蛋白试剂盒确定的mg蛋白质
GGT活性报告为nmole/min -1 /mg蛋白 -1

示例:
Δ吸光度(wt)= 0.58(15min)-0.43(5min)= 0.15
Δ吸光度0.15 = 40nmole pNA
时间x - 时间y = 10分钟
M = 0.02mg蛋白质
GGT活性为:40 /(10×0.02)= 200nmole/min /mg蛋白

食谱

  1. 裂解缓冲液
    50mM Tris/HCl(pH7.6)
    1μg/ml溶菌酶
  2. 反应缓冲液
    2.9mM L-γ-谷氨酰基-3-羧基-4-硝基苯胺
    100mM甘氨酰甘氨酸 100mM Tris/HCl(pH8.2)
  3. pNA储液
    1mM 4-硝基苯胺 50mM Tris/HCl(pH7.6)
    可以储存在-20°C

致谢

本文和van der Stel等人(2015)中描述的γ-谷氨酰转肽酶活性方案从Chevalier等人(1999)中描述的方案修改。这项工作由NWO-ECHO拨款711.012.007支持MW。

参考文献

  1. Chevalier,C.,Thiberge,J.M.,Ferrero,R.L。和Labigne,A。(1999)。 幽门螺杆菌的基本作用γ-谷氨酰转肽酶用于胃的定植粘膜的小鼠。 Mol Microbiol 31(5):1359-1372。
  2. Hanigan,M.H。(1998)。 γ-谷氨酰转肽酶,谷胱甘肽:其在癌发生中的表达和功能。 Chem Biol Interact 111-112:333-342。
  3. Heisterkamp,N.,Groffen,J.,Warburton,D。和Sneddon,T.P。(2008)。 人类γ-谷氨酰转移酶基因家族。 Hum Genet 123(4):321-332。
  4. Ong,P.L.,Yao,Y.F.,Weng,Y.M.,Hsu,W.H.and Lin,L.L。(2008)。 残基Arg114和Arg337对于大肠杆菌 gamma的正常功能至关重要-glutamyltranspeptidase。 Biochem Biophys Res Commun 366(2):294-300。
  5. van der Stel,A.X.,van Mourik,A.,Laniewski,P.,van Putten,J.P.,Jagusztyn-Krynicka,E.K.and Wosten,M.M。 空肠弯曲杆菌RacRS双组分系统通过直接上调γ-谷氨酰转肽酶(GGT)激活谷氨酸合成, 。 Front Microbiol 6:567.
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:van der Stel, A. and Wösten, M. M. (2016). Campylobacter jejuni γ-glutamyltranspeptidase Activity Assay. Bio-protocol 6(5): e1747. DOI: 10.21769/BioProtoc.1747.
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