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Spectrophotometric Determination of Glutamine Synthetase Activity in Cultured Cells
分光光度测定培养细胞的谷氨酰胺合成酶的活性   

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Abstract

Glutamine synthetase (GS), which catalyzes the conversion of glutamate and ammonia to glutamine, is widely distributed in animal tissues and cell culture lines. The importance of this enzyme is suggested by the fact that glutamine, the product of GS-catalyzed de novo synthesis reaction, is the most abundant free amino acid in blood (Smith and Wilmore, 1990). Glutamine is involved in many biological processes including serving as the nitrogen donor for biosynthesis, as an exchanger for the import of essential amino acids, as a means to detoxifying intracellular ammonia and glutamate, and as a bioenergetics nutrient to fuel the tricarboxylic acid (TCA) cycle (Bott et al., 2015). The method for the assay of GS enzymatic activity relies on its γ-glutamyl transferase reaction by measuring γ-glutamylhydroxamate synthesized from glutamine and hydroxylamine, and the chromatographic separation of the reaction product from the reactants (Deuel et al., 1978). An overview of the GS glutamyl transferase reaction can be found in Figure 1. GS activity was measured by a spectrophotometric assay at a specific wavelength of 560 nm using a microplate reader. The method is simple, and has a comparable sensitivity with those methods applying radioactively labelled substrates. This modified procedure has been applied to assay/determine GS activity in cultured cell lines including the human mammary epithelial MCF10A cells and the murine pre-B FL5.12 cells, and could be used to measure GS activity in other cell lines.


Figure 1. An overview of the GS glutamyl transferase reaction

Keywords: Glutamine synthetase(谷氨酰胺合成酶), Activity assay(活性测定), Glutamate ammonia ligase(谷氨酸氨连接酶), GLUL(glul), GS(GS)

Materials and Reagents

  1. 0.05% trypsin-EDTA (1x) (Thermo Fisher Scientific, GibcoTM, catalog number: 25300-054 )
  2. PBS (1x) (Corning, catalog number: 21-031-CV )
  3. Imidazole (Sigma-Aldrich, catalog number: I0250 )
  4. L-glutamine (Sigma-Aldrich, catalog number: G3126 )
  5. Hydroxylamine hydrochloride (Sigma-Aldrich, catalog number: 55459 )
  6. Sodium arsenate dibasic heptahydrate (Sigma-Aldrich, catalog number: S9663 )
  7. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Sigma-Aldrich, catalog number: 221279 )
  8. Adenosine 5’-diphosphate sodium salt (ADP) (Sigma-Aldrich, catalog number: A2754 )
  9. Iron(III) chloride hexahydrate (FeCl3) (Sigma-Aldrich, catalog number: 31232 )
  10. Trichloroacetic acid (Sigma-Aldrich, catalog number: 522082 )
    Note: This product has been discontinued.
  11. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
  12. L-glutamic acid γ-monohydroxamate (γ-glutamylhydroxamate) (Sigma-Aldrich, catalog number: G2253 )
  13. Lysis buffer (see Recipes)
  14. 1x assay buffer (see Recipes)
  15. 1x stop buffer (see Recipes)
  16. γ-glutamylhydroxamate standard stock (see Recipes)

Equipment

  1. AccumetTM AB15 Basic and BioBasicTM pH/mV/°C Meters (Thermo Fisher Scientific, Fisher Scientific, catalog number: 13-636-AB15B )
  2. Sonicator (model: CL-18/120 ) (Thermo Fisher Scientific, Fisher ScientificTM, catalog number: FB120110 )
  3. -80 °C freezer
  4. 37 °C incubator
  5. Microcentrifuge (Eppendorf, model: 5418 )
  6. Microplate (96-well plate clear bottom) reader (Molecular Devices, model: SpectraMax M5 )

Software

  1. SoftMaxPro software

Procedure

  1. Collect ~8 x 106 cells in a 10 cm dish cells by using 1x trypsin.
  2. Rinse the cells with cold 1x PBS and then remove the PBS twice.
  3. Add certain amount (i.e., 300-500 μl) of lysis buffer (50 mM imidazole-HCl, pH 6.8) and lyse the cells with sonicator at level 4 output control, 50% duty cycle for 30 sec and pause for 30 sec for 5 cycles.
  4. Incubate the cells at -80 °C for at least 4 h for the freeze/thaw cell lysis procedure.
  5. Thaw the cells and centrifuge the cells at 16,000 x g at 4 °C for 15 min.
  6. Collect the supernatant and measure the protein concentration by BCA protein assay (He, 2011).
  7. Add 20-40 μg of protein, then add lysis buffer to raise volume to 50 μl.
  8. Add equal volume (50 μl) of 1x assay buffer and then incubate at 37 °C for 2~6 h (Please see the notes).
  9. Add equal volume (100 μl) of 1x stop buffer to observe brown color products and centrifuge at 16,000 x g at 4 °C for 10 min.
  10. The absorbance of γ-glutamylhydroxamate in the supernatant of samples was measured at 560 nm. The blank was 50 μl lysis buffer + 50 μl 1x assay buffer + 100 μl 1x stop buffer.
  11. A serial dilution of 25, 12.5, 6.25, 3.125, 1.5623, 0.781, and 0.391 mM γ-glutamylhydroxamate (100 μl) were added with 100 μl of 1x stop buffer and centrifuge at 16,000 x g at 4 °C for 10 min. The absorbance of γ-glutamylhydroxamate in the supernatant was measured at 560 nm at room temperature as a standard curve (Figure 2).


    Figure 2. A representative standard curve. The standard curve is generated by performing a serial dilution of γ-glutamylhydroxamate. The upper boundary of the standard starts at a known concentration of 25 mM, and this is diluted by half to reach the lower boundary at 0.39 mM. A blank with no standard is included. These concentrations are then input into SoftMaxPro software and the absorbance is read at 560 nm. The readings from known concentrations are determined and a 4 parameter fit is applied. In this way the standard curve is generated within a large working range, unknown samples can be tested, and the absorbance of unknowns can be plotted to determine concentration.

Representative data

The GS activity was reported as nmol min-1 g protein-1. The GS activity was expressed by the formula: γ-glutamylhydroxamate concentration (mM) x 100 μl/incubation time (min)/protein amount (g). For example, the concentration of γ-glutamylhydroxamate calculated from MCF10A vector cells was 2.378 mM as compared with standard curve. The added protein amount was 10.36 μg, and the incubation time at 37 °C was 120 min. The GS activity would be 2.378 x 100/120/10.36 x10-6 = 1.91 x 105 nmol min-1 g protein-1 as indicated (Figure 3).


Figure 3. Myc induces GS activity. c-Myc was stably expressed in MCF10A cells. GS activity was determined and is shown as the mean plus SEM of at least five independent experiments. *P < 0.05. Student’s t-test was used to determine the P value (Bott et al., 2015).

Notes

The various incubation time (2-6 h) at 37 °C was determined by the amount of active GS expressed in different cell lines. Higher active GS expressed leads to shorter incubation time to reach the optimal amount of γ-glutamylhydroxamate. The optimal incubation time used in the assay may be adjusted for different cell lines.

Recipes

  1. Lysis buffer (50 mM imidazole-HCl, pH 6.8)
    Add 170.2 mg imidazole in 50 ml ddH2O and adjust pH to 6.8 with HCl.
  2. 1x assay buffer
    1. 5x stock 250 mM imidazole-HCl, pH 6.8
      Add 851 mg imidazole in 50 ml ddH2O and adjust pH to 6.8 with HCl.
    2. 5x stock 250 mM L-glutamine, 250 mM imidazole-HCl
      Add 1.8275 g L-glutamine in 50 ml 5x stock 250 mM imidazole-HCl.
    3. 5x stock 125 mM hydroxylamine
      Add 434.3 mg hydroxylamine in 50 ml ddH2O.
    4. 5x stock 125 mM sodium arsenate
      Add 1.95 g sodium arsenate in 50 ml ddH2O.
    5. 5x stock 10 mM MnCl2
      Add 99.2 mg MnCl2 in 50 ml ddH2O.
    6. 5x stock 0.8 mM ADP
      Add 17.09 mg ADP in 50 ml ddH2O.
    Then mix equal amount of (b)-(f) to get 1x assay buffer (final concentration: 50 mM imidazole-HCl, 50 mM L-glutamine, 25 mM hydroxylamine, 25 mM sodium arsenate, 2 mM MnCl2 and 0.16 mM ADP).
  3. 1x stop buffer
    3x stop buffer (270 mM FeCl3, 5.4 N HCl, 4.35% trichloroacetic acid)
    Use 7.3 g FeCl3, 45 ml 12 N HCl as well as 4.35 ml 100% trichloroacetic acid and add ddH2O to 100 ml to get 3x stop buffer.
    Dilute 3x stop buffer with ddH2O to get 1x stop buffer (final concentration: 90 mM FeCl3, 1.8 N HCl and 1.45% trichloroacetic acid) when doing the assay.
  4. 100 mM γ-glutamylhydroxamate standard stock
    Add 16.214 mg γ-glutamylhydroxamate in 1 ml ddH2O.

Acknowledgments

This protocol was adapted and modified from a previous study (Deuel et al., 1978). It was described in (Bott et al., 2015). This work was supported by grants from NIH (R01CA129536 and R01GM97355 to WXZ).

References

  1. Bott, A. J., Peng, I. C., Fan, Y., Faubert, B., Zhao, L., Li, J., Neidler, S., Sun, Y., Jaber, N., Krokowski, D., Lu, W., Pan, J. A., Powers, S., Rabinowitz, J., Hatzoglou, M., Murphy, D. J., Jones, R., Wu, S., Girnun, G. and Zong, W. X. (2015). Oncogenic Myc induces expression of glutamine synthetase through promoter demethylation. Cell Metab 22(6): 1068-1077.
  2. Deuel, T. F., Louie, M. and Lerner, A. (1978). Glutamine synthetase from rat liver. Purification, properties, and preparation of specific antisera. J Biol Chem 253(17): 6111-6118.
  3. He, F. (2011). BCA (bicinchoninic acid) protein assay. Bio-protocol 101: e44.
  4. Smith, R. J. and Wilmore, D. W. (1990). Glutamine nutrition and requirements. JPEN J Parenter Enteral Nutr 14(4 Suppl): 94S-99S.

简介

谷氨酰胺合成酶(GS),其催化谷氨酸和氨转化成谷氨酰胺,广泛分布在动物组织和细胞培养系中。该酶的重要性通过谷氨酰胺,GS-催化的从头合成反应的产物,是血液中最丰富的游离氨基酸的事实提示(Smith和Wilmore,1990)。谷氨酰胺参与许多生物过程,包括作为生物合成的氮供体,作为输入必需氨基酸的交换剂,作为解毒细胞内氨和谷氨酸的手段,以及作为生物能量营养物来给三羧酸(TCA)周期(Bott等人,2015)。用于测定GS酶活性的方法依赖于其γ-谷氨酰转移酶反应,通过测量由谷氨酰胺和羟胺合成的γ-谷氨酰羟肟酸酯,以及反应产物与反应物的色谱分离(Deuel等人 。,1978)。 GS谷氨酰转移酶反应的概述可以在图1中找到。通过分光光度测定法在560nm的特定波长下使用酶标仪测量GS活性。该方法简单,并且具有与应用放射性标记的底物的那些方法相当的灵敏度。该修改的方法已经应用于在包括人乳腺上皮MCF10A细胞和鼠前B FL5.12细胞的培养细胞系中测定/测定GS活性,并且可以用于测量其他细胞系中的GS活性。 >


图1 。GS glutamyl transferase reaction的概述

关键字:谷氨酰胺合成酶, 活性测定, 谷氨酸氨连接酶, glul, GS

材料和试剂

  1. 0.05%胰蛋白酶-EDTA(1x)(Thermo Fisher Scientific,Gibco TM ,目录号:25300-054)
  2. PBS(1x)(Corning,目录号:21-031-CV)
  3. 咪唑(Sigma-Aldrich,目录号:10202)
  4. L-谷氨酰胺(Sigma-Aldrich,目录号:G3126)
  5. 盐酸羟胺(Sigma-Aldrich,目录号:55459)
  6. 砷酸氢钠七水合物(Sigma-Aldrich,目录号:S9663)
  7. 氯化锰(II)四水合物(MnCl 2·4H 2 O)(Sigma-Aldrich,目录号:221279)
  8. 腺苷5'-二磷酸钠盐(ADP)(Sigma-Aldrich,目录号:A2754)
  9. 氯化铁(III)六水合物(FeCl 3)(Sigma-Aldrich,目录号:31232)
  10. 三氯乙酸(Sigma-Aldrich,目录号:522082)
    注意:此产品已停产。
  11. 盐酸(HCl)(Sigma-Aldrich,目录号:258148)
  12. L-谷氨酸γ-单羟肟酸酯(γ-谷氨酰羟肟酸酯)(Sigma-Aldrich,目录号:G2253)
  13. 裂解缓冲液(见配方)
  14. 1x测定缓冲液(参见配方)
  15. 1x停止缓冲区(参见配方)
  16. γ-谷氨酰异羟肟酸盐标准品(见配方)

设备

  1. Accumet TM AB15 Basic和BioBasic TM pH/mV /℃计(Thermo Fisher Scientific,Fisher Scientific,目录号:13-636-AB15B)
  2. 超声波仪(型号:CL-18/120)(Thermo Fisher Scientific,Fisher Scientific ,目录号:FB120110)
  3. -80°C冰箱
  4. 37℃孵育器
  5. 微量离心机(Eppendorf,型号:5418)
  6. 微孔板(96孔板透明底部)读数器(Molecular Devices,型号:SpectraMax M5)

软件

  1. SoftMaxPro软件

程序

  1. 通过使用1×胰蛋白酶在10cm皿细胞中收集?8×10 6个细胞。
  2. 用冷的1×PBS冲洗细胞,然后取出PBS两次
  3. 加入一定量(即,300-500μl)的裂解缓冲液(50mM咪唑-HCl,pH6.8),并用超声波仪在4级输出对照,50%占空比秒,暂停30秒,共5个周期
  4. 将细胞在-80℃孵育至少4小时用于冷冻/解冻细胞裂解程序
  5. 解冻细胞,并在4℃下以16000×g离心细胞15分钟。
  6. 收集上清液并通过BCA蛋白测定法测量蛋白质浓度(He,2011)
  7. 加入20-40μg蛋白质,然后加入裂解缓冲液将体积升至50μl。
  8. 加入等体积(50μl)的1x测定缓冲液,然后在37℃孵育2?6小时(请参见注释)。
  9. 加入等体积(100μl)的1x终止缓冲液以观察棕色产物,并在4℃下以16,000×g离心10分钟。
  10. 在560nm测量样品上清液中γ-谷氨酰羟肟酸的吸光度。空白是50μl裂解缓冲液+50μl1x测定缓冲液+100μl1x终止缓冲液
  11. 向系列稀释的25,12.5,6.25,3.125,1.5623,0.781和0.391mMγ-谷氨酰羟肟酸酯(100μl)中加入100μl1x终止缓冲液,并在4℃以16,000×g离心10分钟。在室温下在560nm测量上清液中γ-谷氨酰羟肟酸盐的吸光度作为标准曲线(图2)。


    图2.代表性标准曲线。通过进行γ-谷氨酰羟肟酸的系列稀释来产生标准曲线。标准品的上边界以25mM的已知浓度开始,并且将其稀释一半以达到0.39mM的下边界。包括没有标准的空白。然后将这些浓度输入SoftMaxPro软件中,并在560nm处读取吸光度。确定来自已知浓度的读数,并应用4参数拟合。这样,在大工作范围内产生标准曲线,可以测试未知样品,并绘制未知物的吸光度以确定浓度。

代表数据

GS活性报告为nmol min -1 g蛋白 -1 。 GS活性由下式表示:γ-谷氨酰羟肟酸浓度(mM)×100μl/孵育时间(min)/蛋白质量(g)。例如,由MCF10A载体细胞计算的γ-谷氨酰羟肟酸盐的浓度与标准曲线相比为2.378mM。添加的蛋白质量为10.36μg,在37℃下的孵育时间为120分钟。 GS活性将是2.378×100/120/10.36×10 6 -6·s -1 = 1.91×10 5 p nmol min -1 g蛋白 -1 ,如图3所示。


图3.Myc诱导GS活性。 c-Myc在MCF10A细胞中稳定表达。测定GS活性并显示为至少5次独立实验的平均值±SEM。 * P 0.05。使用学生的 t 测试来确定 P 值(Bott等人,2015)。

笔记

通过在不同细胞系中表达的活性GS的量确定在37℃下的各种培养时间(2-6小时)。更高的活性GS表达导致更短的温育时间以达到最佳量的γ-谷氨酰羟肟酸。可以针对不同的细胞系调整用于测定的最佳孵育时间。

食谱

  1. 裂解缓冲液(50mM咪唑-HCl,pH6.8) 在50ml ddH 2 O中加入170.2mg咪唑并用HCl调节pH至6.8。
  2. 1x测定缓冲液
    1. 5x母液250mM咪唑-HCl,pH 6.8
      在50ml ddH 2 O中加入851mg咪唑并用HCl调节pH至6.8。
    2. 5x母液250mM L-谷氨酰胺,250mM咪唑盐酸盐 在50ml 5×母液250mM咪唑-HCl中加入1.8275g L-谷氨酰胺
    3. 5x母液125mM羟胺
      在50ml ddH 2 O中加入434.3mg羟胺。
    4. 5x母液125mM砷酸钠
      在50ml ddH 2 O中加入1.95g砷酸钠
    5. 5×储备液10mM MnCl 2/v/v 在50ml ddH 2 O中加入99.2mg MnCl 2。
    6. 5x储液0.8mM ADP
      在50ml ddH 2 O中加入17.09mg ADP。
    然后混合等量的(b) - (f),得到1x测定缓冲液(最终浓度:50mM咪唑-HCl,50mM L-谷氨酰胺,25mM羟胺,25mM砷酸钠,2mM MnCl 2,和0.16mM ADP)。
  3. 1x停止缓冲区
    3x终止缓冲液(270mM FeCl 3,5.4N HCl,4.35%三氯乙酸)
    使用7.3g FeCl 3,45ml 12N HCl以及4.35ml 100%三氯乙酸,并将ddH 2 O加至100ml,得到3×终止缓冲液。 /> 当进行测定时,用ddH 2 O稀释3x终止缓冲液以得到1x终止缓冲液(终浓度:90mM FeCl 3,1.8N HCl和1.45%三氯乙酸)。
  4. 100 mMγ-谷氨酰羟肟酸盐标准品
    在1ml ddH 2 O中加入16.214mgγ-谷氨酰羟肟酸盐。

致谢

该方案从先前的研究(Deuel等人,1978)改编和修改。它在(Bott等人,2015年)中描述。这项工作是由来自NIH(R01CA129536和R01GM97355到WXZ)的拨款支持。

参考文献

  1. Bott,AJ,Peng,IC,Fan,Y.,Faubert,B.,Zhao,L.,Li,J.,Neidler,S.,Sun,Y.,Jaber,N.,Krokowski, W.,Pan,JA,Powers,S.,Rabinowitz,J.,Hatzoglou,M.,Murphy,DJ,Jones,R.,Wu,S.,Girnun,G.and Zong,WX(2015) 致癌性Myc通过启动子去甲基化诱导谷氨酰胺合成酶的表达。 Cell Metab 22(6):1068-1077。
  2. Deuel,TF,Louie,M。和Lerner,A。(1978)。  来自大鼠肝脏的谷氨酰胺合成酶。特异性抗血清的纯化,性质和制备。 J Biol Chem 253(17):6111-6118。
  3. 他,F.(2011)。  BCA(二金鸡宁酸)蛋白测定。 生物协议 101:e44
  4. Smith,RJ和Wilmore,DW(1990)。  谷氨酰胺营养和要求。 14(4 Suppl):94S-99S。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Peng, I., Bott, A. J. and Zong, W. (2016). Spectrophotometric Determination of Glutamine Synthetase Activity in Cultured Cells. Bio-protocol 6(19): e1959. DOI: 10.21769/BioProtoc.1959.
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