Assay of Ornithine Decarboxylase and Spermidine/Spermine N1-acetyltransferase Activities
鸟氨酸脱羧酶和亚精胺/精胺N¹-乙酰转移酶活性测定试验   

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本实验方案简略版
Nature
Apr 2014

 

Abstract

The polyamines, spermidine (Spd) and spermine, and their diamine precursor putrescine, are important regulators of various cellular functions, such as proliferation and differentiation. Polyamine homeostasis is tightly regulated on the level of uptake, excretion, biosynthesis, interconversion and terminal catabolism. The rate-controlling enzymes of polyamine biosynthesis and interconversion are ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SSAT), respectively. Here, we describe a protocol to assay ODC (Jänne and Williams-Ashman, 1971) and SSAT (Libby, 1978) activities from cell or tissue samples.

Keywords: 14C-L-ornithine (14C鸟氨酸), 14C-Acetylcoenzyme A (14C乙酰辅酶A), Polyamine biosynthesis (多胺生物合成), Polyamine acetylation (多胺的乙酰化)

Materials and Reagents

  1. EDTA (Sigma-Aldrich, catalog number: E9884 )
  2. Tris-HCl (Sigma-Aldrich, catalog number: T5941 )
  3. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  4. DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
  5. Protease inhibitor cocktail, such as Complete EDTA-free protease inhibitor tablets (Roche Diagnostics, catalog number: 11873580001 )
  6. L-ornithine monohydrochloride (Sigma-Aldrich, catalog number: O2375 )
  7. Acid-treated [14C]-L-ornithine (100 µCi/ml, 40-60 mCi/mmol) (PerkinElmer, catalog number: NEC710050UC )
  8. Pyridoxal 5’phosphate monohydrate (PLP) (Sigma-Aldrich, catalog number: 82870 )
  9. Citric acid (Sigma-Aldrich, catalog number: C0759 )
  10. SOLVABLE (PerkinElmer, catalog number: 6NE9100 )
  11. [Acetyl-1-14C]-Acetyl Coenzyme A (AcCoA) (20 µCi/ml, 40-60 mCi/mmol) (PerkinElmer, catalog number: NEC313050UC ) (Note 1)
  12. Spermidine trihydrochloride (Sigma-Aldrich, catalog number: S2501 )
  13. Hydroxylamine hydrochloride (Sigma-Aldrich, catalog number: 159417 )
  14. ddH2O
  15. 96% ethanol such as ETAX A (Altia Oyj, catalog number: 12210143 )
  16. Liquid scintillation cocktail, such as OptiPhase HiSafe 2 (PerkinElmer, catalog number: 1200-436 )
  17. Buffer A (see Recipes)
  18. 1 M Tris-HCl (see Recipes)
  19. 200 mM EDTA (pH 8.0) (see Recipes)
  20. 100 mM DTT (see Recipes)
  21. Acid-treated [14C]-L-ornithine (see Recipes)
  22. 10 mM or 25 mM L-ornithine (see Recipes)
  23. 20 mM PLP (see Recipes)
  24. 2 M citric acid (see Recipes)
  25. 100 mM Spd (see Recipes)
  26. 1 M hydroxylamine (see Recipes)

Equipment

  1. 10-ml glass tubes with rubber caps (Note 2)
  2. Tube rack for glass tubes
  3. WhatmanTM 3MM Chr chromatography paper (Thermo Fisher Scientific, catalog number: 05-713-336 )
    Note: Cut into 3 cm x 2 cm size and folded 4 times (Figure 1A).
  4. Long 18-21G needle
  5. 10-ml syringe
  6. Forceps
  7. Tissue homogenizer, such as 3-ml Potter-Elvehjem glass tube and pestle (Sigma-Aldrich, catalog number: P7734 ) and a drill to operate the pestle
  8. 0.5-ml microcentrifuge tubes
  9. Microcentrifuge
  10. Thermal cycler with block fitted for 0.5 ml tubes
  11. WhatmanTM Grade P81 Ion Exchange Cellulose Chromatography Paper (Thermo Fisher Scientific, catalog number: 05-171-2A ) (cut into 1.5 x 1.5 cm size)
  12. ParafilmTM (Sigma-Aldrich, catalog number: P7793 )
  13. Horizontal shaker
  14. A sheet of filter paper
  15. Wallac 4-ml plastic scintillation vials and caps (PerkinElmer, catalog number: 1200-421 )
  16. Liquid scintillation counter and plates fitted for 4-ml vials

Procedure

  1. Sample preparation
    1. Cell or tissue samples
      1. Cell samples
        Resuspend cell pellets to ice-cold buffer A by pipetting up-and-down until cell suspension is homogenous; keep the samples on ice (Note 3, Note 4).
      2. Tissue samples
        Weigh tissue and homogenize it on ice to 2-3x vol of buffer A by using Potter-Elvehjem homogenizer or other suitable homogenizer (Notes 3-4). For example, use 100 mg tissue and 200-300 µl of buffer A. Homogenize the sample until no tissue clumps remain. Transfer the homogenate to clean microcentrifuge tube with pipette.
    2. Incubate tubes for 20 min on ice.
    3. Centrifuge at 15,000 x g for 30 min at +4 °C.
    4. Transfer the supernatant to a clean microcentrifuge tube and discard the pellet. Keep the samples on ice.

  2. ODC activity assay
    1. Calculate the amount of tubes and reaction mixture needed. Each sample should be assayed in duplicate. Include also two blank reactions and two “total counts” (for determing the total radioactivity in the reaction mixture).
    2. Label the needed amount of glass tubes and assemble them into the tube rack. Place the rack on ice-waterbath.
    3. Prepare reaction mixture (Table 1) on ice.

      Table 1. Reaction mixture (one reaction) for ODC activity assay
      Component
      Amount
      1 M Tris-HCl (pH 7.4)
      25 µl
      200 mM EDTA
      5 µl
      100 mM DTT
      10 µl
      10 mM or 25 mM L-ornithine (Note 5)
      10 µl
      100 µCi/ml [14C]-L-ornithine
      2 µl
      20 mM PLP
      5 µl
      ddH2O
      143 µl
      Total
      200 µl

    4. Pipet 200 µl of reaction mixture carefully to the bottom of the test tube, taking care not to touch the walls of the tubes with pipet tip (Note 6).
    5. Add 50 µl of sample or blank (buffer A) to the bottom of the tube.
    6. Pipet 25 µl of SOLVABLE onto 3 MM folded paper (Note 7), put it to the top of a glass tube and close with rubber cap (Figure 1B).
    7. Incubate tubes (upright) at +37 °C water bath with gentle rocking for 30 min (tissue samples) or 60 min (cell samples).
    8. Take the tube rack back on ice-waterbath.
    9. In the fume hood, add 1 ml of 2 M citric acid to the wall of the tube by using long 18-21G needle attached to a 10-ml syringe (Note 2, Note 8) (Figure 1C, D).
    10. Continue incubation at +37 °C water bath with gentle rocking for 15 min.
    11. Put the folded papers into 4-ml scintillation vials using forceps (see also Figure 2C).
    12. Include also two “total count” vials-pipet 100 µl of reaction mixture to a folded paper and put it into a scintillation vial.
    13. Add 3 ml of scintillation cocktail to each vial and close with caps (see also Figure 2D-E).
    14. Measure [14C]-radioactivity with liquid scintillation counter (Note 9).
    15. Calculating results (see also Table 3):
      1. Check the label of [14C]-L-ornithine for specific activity and calculate the total molar amount of L-ornithine (labelled + unlabelled) in the reaction mixture (200 µl):
        Labelled L-ornithine:
        clabelled = radioactive c (mCi/ml)/specific activity (mCi/mmol)
        nlabelled = clabelled*Vlabelled
        ntotal = nlabelled+nunlabelled
      2. Next calculate how many cpm correspond to one pmol of L-ornithine:
        Relative specific activity (RSA) (pmol/cpm) = ntotal/[(average “total count” cpm)*2]
      3. Then calculate the result:
        1. For cells
          [(average sample cpm - average blank cpm) * RSA]/(mg protein per 50 µl sample) = pmol/h/mg protein
        2. For tissue
          {[(average sample cpm - average blank cpm) * RSA]/(mg protein per 50 µl sample)}/0.5 h = pmol/h/mg protein
          Activity can be expressed as per mg protein, mill. cells, µg DNA or mg tissue.
    16. For tissue and cell samples, dilution of the sample is generally needed when cpm-values exceed ~15,000.


      Figure 1. ODC activity assay. A. Folded Whatman 3 MM filter papers. B. ODC reaction tubes with folded papers in place. C. Stopping the reaction with the addition of 2 M citric acid, using needle-penetrable rubber caps and D normal rubber caps.

  3. SSAT activity assay
    1. Calculate the amount of tubes and reaction mixture needed. Each sample should be assayed in duplicate. Include also two blank reactions and two “total counts” for determing the total radioactivity in the reaction mixture.
    2. Label the needed amount of 0.5-ml microcentrifuge tubes and place them on ice.
    3. Prepare reaction mixture (Table 2) on ice.

      Table 2. Reaction mixture (one reaction) for SSAT activity assay
      Component
      Amount
      1 M Tris-HCl (pH 7.8)
      10 µl
      100 mM Spd
      3 µl
      100 mM DTT
      1 µl
      (20 µCi/ml) [14C]-AcCoA
      2.5 µl
      ddH2O
      73.3 µl
      Total
      90 µl

    4. Pipet 90 µl of reaction mixture to each tube.
    5. Pipet 10 µl of sample or blank (buffer A) to each tube.
    6. Incubate in a thermal cycler for 10 min at +37 °C (Note 10).
    7. Cool to +4 °C and add 20 µl of 1 M hydroxylamine.
    8. Heat at +100 °C for 3 min.
    9. Centrifuge tubes at 15,000 x g for 5 min at RT.
    10. Place P81 paper squares onto Parafilm and number them with pencil (Figure 2A).
    11. Pipet 40 µl of sample supernatant to each paper square and let dry at RT. Include also two “total counts”- pipet 45 µl of reaction mixture to filter paper.
    12. Place the “total count” paper squares into 4-ml scintillation vials using forceps, add 3 ml of scintillation cocktail to each vial and close them with caps (Figure 2C-E).
    13. Put sample and blank paper squares to 1 L Erlenmeyer flask.
    14. Wash paper squares with dH2O for 3 x 5 min and 1 x 10 min (500 ml each wash), shaking on a horizontal shaker at ~200 rpm (Notes 11-12) (Figure 2B).
    15. Wash paper squares once with 400 ml of 96% ethanol for 5 min, shaking on a horizontal shaker at ~200 rpm (Note 11).
    16. Pour paper squares with ethanol to a large container, pick them with forceps onto a big sheet of filter paper and let dry.
    17. Put the paper squares into 4-ml scintillation vials (Figure 2C).
    18. Add 3 ml of scintillation cocktail into each vial and close them with caps (Figure 2D-E).
    19. Measure [14C]-radioactivity with liquid scintillation counter (Note 9).
    20. Calculating the results (see also Table 3):
      1. Check the [14C]-AcCoA batch label for specific activity and calculate its molar amount in the reaction mixture (90 µl):
        c = radioactive c (mCi/ml)/specific activity (mCi/mmol)
        n = c * V
      2. Next calculate how many cpm correspond to one pmol of AcCoA:
        Relative specific activity (RSA) (pmol/cpm) = n/[(average “total count” cpm)*2]
      3. Then calculate the result:
        {[(average sample cpm - average blank cpm) * RSA] * (120 µl/40 µl)/(mg per 10 µl sample)} * 6 = pmol/h/mg
        Activity can be expressed as per mg protein, mill. cells, µg DNA or mg tissue.
    21. For tissue and cell samples, dilution of the sample is generally needed when cpm-values exceed ~6,000.


      Figure 2. SSAT activity assay and preparing the samples for liquid scintillation counting. A. Numbered P81 paper squares for samples, blanks and total counts. B. Washing the paper squares. C. Placing the paper squares to liquid scintillation vials. D. Adding scintillation cocktail with dispenser. E. Capped and uncapped vials and the cassette cover.

Representative data

Table 3. Representative data from an experiment with DU145 cells

Notes

  1. AcCoA is very labile. After thawing the package, aliquot and store at -20 °C.
  2. Using needle-penetrable rubber caps is recommended to avoid any possible release of radioactive CO2 from the tube.
  3. The cells/tissue can be either fresh or frozen at -70 °C.
  4. The amount of buffer used depends on the ODC/SSAT activity in the particular cell/tissue type. The needed amount of cleared lysate is 20 µl for SSAT assay and 100 µl for ODC assay. We recommend using at least 40 mg of tissue and at least 0.5 mill. cells/200 µl buffer.
  5. Use 10 mM L-ornithine for cell samples and 25 mM L-ornithine for tissue samples.
  6. Extra-long pipette tips are convenient when pipetting to the bottom of the tubes. Unexpectedly high cpm-values are usually caused by contaminating reaction mixture on the wall of the glass tube.
  7. SOLVABLE absorbs released [14C]-CO2.
  8. Do not touch the paper with the needle, otherwise bound [14C]-CO2 will be released. If using normal rubber caps, open it just slightly in order to fit the needle into the tube and close the cap immediately after the addition of citric acid.
  9. For optimal results, leave vials o/n at RT before measurement.
  10. Reactions are conveniently done in a thermal cycler, but can also be done on water baths or heat blocks.
  11. Shaking speed depends on the size of the shaker. Use speed that enables the paper squares to gently circle around the flask. Too high speed will lead to loss of paper from the square corners.
  12. High blank values (>500 cpm) indicate insufficient washing or too high amount of samples in the same flask. Either use two flasks to wash the paper squares, or add a couple of additional 5 min washes with H2O.
  13. Triton X-100 is not absolutely necessary for tissue samples, but it enhances cell breakage.
  14. Commercial preparations of [14C]-L-ornithine contain variable amounts of radioactive CO2, which will result in high blank values. Acid treatment removes any residual radioactive CO2 (Jänne and Williams-Ahsman, 1971).

Recipes

  1. Buffer A (Note 13)
    Mix 1.25 ml of Tris-HCl pH 7.4, 250 µl of 200 mM EDTA pH 8.0, 50 µl of Triton X-100 and 500 µl of 100 mM DTT
    Add ddH2O to final volume of 50 ml
    Dissolve one Complete EDTA-free tablet to the solution
    Aliquot and store at -20 °C
  2. 1 M Tris-HCl (pH 7.4 or pH 7.8)
    Dissolve 157.6 g of Tris-HCl to ~800 ml of ddH2O
    Adjust pH to 7.4 or 7.8 with NaOH
    Add ddH2O to final volume of 1,000 ml
    Filter-sterilize (0.2 µm)
    Stored at RT
  3. 200 mM EDTA (pH 8.0)
    Weigh 58.448 g of EDTA to ~800 ml of ddH2O
    Adjust pH slowly to 8.0 with NaOH - EDTA dissolves when pH approaches 8
    Add ddH2O to final volume of 1,000 ml
    Filter-sterilize (0.2 µm)
    Stored at RT
  4. 100 mM DTT
    Dissolve 1.5425 g of DTT to ddH2O in a final volume of 100 ml in the fume hood
    Filter-sterilize (0.2 µm)
    Aliquot and stored at -20 °C
  5. Acid-treated [14C]-L-ornithine (Note 14)
    Mix 0.5 ml of [14C]-L-ornithine with 1 ml of 0.1 M HCl
    Evaporate to dryness in a rotary evaporator in a fume hood
    Dissolve residue into 0.5 ml of 0.01 M HCl
    Aliquot and stored at -20 °C
  6. 10 mM or 25 mM L-ornithine
    Dissolve 168.62 mg or 421.55 mg of L-ornithine monohydrochloride to ddH2O in a final volume of 100 ml to obtain 10 mM or 25 mM concentration, respectively
    Filter-sterilize (0.2 µm)
    Aliquot and stored at -20 °C
  7. 20 mM PLP
    Dissolve 530.32 mg of pyridoxal 5’phosphate monohydrate to ddH2O to a final volume of 100 ml
    Filter-sterilize (0.2 µm)
    Aliquot and stored at -20 °C
  8. 2 M citric acid
    Dissolve 384.24 g of citric acid to ddH2O to a final volume of 1,000 ml
    Filter-sterilize (0.2 µm)
    Stored at +4 °C
  9. 100 mM Spd
    Dissolve 2.546 g of Spd to ddH2O to a final volume of 100 ml
    Filter-sterilize (0.2 µm)
    Aliquot and stored at -20 °C
  10. 1 M hydroxylamine
    Dissolve 6.949 g of hydroxylamine hydrochloride to ddH2O to a final volume of 100 ml
    Filter-sterilize (0.2 µm)
    Stored at RT

Acknowledgments

This work was supported by the strategic funding of the University of Eastern Finland and the Academy of Finland. The protocols were modified from the original versions developed by Jänne and Williams-Ashman (1971) and Libby (1978).

References

  1. Jänne, J. and Williams-Ashman, H. G. (1971). On the purification of L-ornithine decarboxylase from rat prostate and effects of thiol compounds on the enzyme. J Biol Chem 246(6): 1725-1732.
  2. Libby, P. R. (1978). Calf liver nuclear N-acetyltransferases. Purification and properties of two enzymes with both spermidine acetyltransferase and histone acetyltransferase activities. J Biol Chem 253(1): 233-237.

简介

多胺,亚精胺(精)和精胺及其二胺前体腐胺是各种细胞功能(如增殖和分化)的重要调节剂。 多胺稳态在摄取,排泄,生物合成,相互转化和末端分解代谢的水平上受到严格调节。 多胺生物合成和相互转化的速率控制酶分别是鸟氨酸脱羧酶(ODC)和亚精胺/精胺N 1 - 乙酰基转移酶(SSAT)。 在这里,我们描述了测定细胞或组织样品的ODC(Jänne和Williams-Ashman,1971)和SSAT(Libby,1978)活性的方案。

关键字:14C鸟氨酸, 14C乙酰辅酶A, 多胺生物合成, 多胺的乙酰化

材料和试剂

  1. EDTA(Sigma-Aldrich,目录号:E9884)
  2. Tris-HCl(Sigma-Aldrich,目录号:T5941)
  3. Triton X-100(Sigma-Aldrich,目录号:T8787)
  4. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:43815)
  5. 蛋白酶抑制剂混合物,例如完全无EDTA蛋白酶抑制剂片剂(Roche Diagnostics,目录号:11873580001)
  6. L-鸟氨酸单盐酸盐(Sigma-Aldrich,目录号:O2375)
  7. 酸处理的[14 C] -L-鸟氨酸(100μCi/ml,40-60mCi/mmol)(PerkinElmer,目录号:NEC710050UC)
  8. 吡哆醛5'-磷酸一水合物(PLP)(Sigma-Aldrich,目录号:82870)
  9. 柠檬酸(Sigma-Aldrich,目录号:C0759)
  10. SOLVABLE(PerkinElmer,目录号:6NE9100)
  11. [表1] [注释1] [注1] [注释1] [乙酰辅酶A](AcCoA)(20μCi/ml,40-60mCi/>
  12. 亚精胺三盐酸盐(Sigma-Aldrich,目录号:S2501)
  13. 盐酸羟胺(Sigma-Aldrich,目录号:159417)
  14. ddH sub 2 O
  15. 96%乙醇,例如ETAX A(Altia Oyj,目录号:12210143)
  16. 液体闪烁混合物,例如OptiPhase HiSafe 2(PerkinElmer,目录号:1200-436)
  17. 缓冲区A(参见配方)
  18. 1 M Tris-HCl(参见配方)
  19. 200mM EDTA(pH 8.0)(参见配方)
  20. 100 mM DTT(参见配方)
  21. 酸处理的[14 C] -L-鸟氨酸(参见配方)
  22. 10 mM或25 mM L-鸟氨酸(参见配方)
  23. 20 mM PLP(参见配方)
  24. 2 M柠檬酸(见配方)
  25. 100 mM Spd(参见配方)
  26. 1 M羟胺(参见配方)

设备

  1. 10毫升带橡胶帽的玻璃管(注2)
  2. 玻璃管管架
  3. Whatman TM 3MM Chr色谱纸(Thermo Fisher Scientific,目录号:05-713-336)
    注意:切割成3厘米x 2厘米大小,折叠4次(图1A)。
  4. 长18-21G针
  5. 10毫升注射器
  6. 镊子
  7. 组织匀浆器,例如3-ml Potter-Elvehjem玻璃管和杵(Sigma-Aldrich,目录号:P7734)和操作研杵的钻子
  8. 0.5 ml微量离心管
  9. 微量离心机
  10. 带有块的热循环仪适用于0.5 ml管
  11. Whatman?sup?Grade P81离子交换纤维素色谱纸(Thermo Fisher Scientific,目录号:05-171-2A)(切成1.5×1.5cm大小)
  12. Parafilm TM(Sigma-Aldrich,目录号:P7793)
  13. 水平振动器
  14. 一张滤纸
  15. Wallac 4-ml塑料闪烁小瓶和盖(PerkinElmer,目录号:1200-421)
  16. 液体闪烁计数器和适用于4-ml小瓶的平板

程序

  1. 样品制备
    1. 细胞或组织样本
      1. 单元格样本
        重悬细胞沉淀   冰冷的缓冲液A通过吸移上下,直到细胞悬浮液 同质的; 将样品保存在冰上(注3,注4)
      2. 组织样品
        称重组织并在冰上匀浆至2-3x体积的缓冲液A,通过使用 Potter-Elvehjem匀浆器或其他合适的匀浆器(注3-4)。 例如,使用100mg组织和200-300μl缓冲液A.均化 样品直到没有组织块残留。 转移匀浆 干净的微量离心管。
    2. 在冰上孵育管子20分钟。
    3. 在+ 4℃下以15,000×g离心30分钟
    4. 将上清液转移到干净的微量离心管中,弃去沉淀。 将样品保存在冰上。

  2. ODC活性测定
    1. 计算所需的管和反应混合物的量。 每个样本 应重复测定。 还包括两个空白反应和两个   "总计数"(用于确定反应中的总放射性) 混合物)。
    2. 标记所需数量的玻璃管,并将其装入管架。 将机架放在冰水浴上。
    3. 在冰上制备反应混合物(表1)
      表1.用于ODC活性测定的反应混合物(一个反应)
      组件
      金额
      1 M Tris-HCl(pH 7.4)
      25μl
      200 mM EDTA
      5微升
      100 mM DTT
      10微升
      10 mM或25 mM L-鸟氨酸(注5)
      10微升
      100μCi/ml [14 C] -L-鸟氨酸 2微升
      20 mM PLP
      5微升
      ddH sub 2 O
      143μl
      总计
      200μl

    4. 将200μl反应混合物小心地移至试验底部   管,注意不要用移液管尖端接触管壁 (注6)。
    5. 将50μl样品或空白(缓冲液A)加入试管底部
    6. 移取25μl溶于3MM折叠纸(注7),放入 玻璃管的顶部,并用橡胶帽密封(图1B)。
    7. 孵育管(直立)在+37°C水浴温和摇动30分钟(组织样本)或60分钟(细胞样本)。
    8. 将管架放回冰水浴上。
    9. 在通风橱中,加入1ml的2M柠檬酸的壁 管通过使用长的18-21G针连接到10ml注射器(注2, 注8)(图1C,D)。
    10. 继续在+37°C水浴孵育,温和摇动15分钟。
    11. 将折叠的纸放入4毫升闪烁瓶使用镊子(也见图2C)。
    12. 还包括两个"总计"小瓶 - 移液器100μl的反应 混合物倒入折叠的纸中,并将其放入闪烁瓶中
    13. 向每个小瓶中加入3ml闪烁鸡尾酒,并用盖子封闭(参见图2D-E)
    14. 用液体闪烁计数器测量[14 C] - 放射活性(注9)。
    15. 计算结果(另请参见表3):
      1. 检查[ 标记的L-鸟氨酸:
        标记的放射性c(mCi/ml)/比活性(mCi/mmol)
        标记为 = 标记为的标签 n = n 已标记 + n未标记的
      2. 接下来计算多少cpm对应于1pmol的L-鸟氨酸:
        相对比活性(RSA)(pmol/cpm)= n总计/[(平均"总计数"cpm)* 2]
      3. 然后计算结果:
        1. 对于单元格
          [(平均样品cpm-平均空白cpm)* RSA] /(每50μl样品的mg蛋白质)= pmol/h/mg蛋白质
        2. 对于组织
          {[(平均样品cpm-平均空白cpm)* RSA] /(每50μl样品的mg蛋白质)}/0.5 h = pmol/h/mg蛋白质
          活性可以表示为每mg蛋白质,磨。 细胞,μgDNA或mg组织。
    16. 对于组织和细胞样品,当cpm值超过〜15,000时通常需要稀释样品

      图1. ODC活性测定。A.折叠的Whatman 3 MM滤纸。 乙。 ODC反应管,折叠纸就位。 C.停止反应   加入2M柠檬酸,使用可针刺的橡胶 帽和D普通橡胶帽。

  3. SSAT活性测定
    1. 计算所需的管和反应混合物的量。 每个样本 应重复测定。 还包括两个空白反应和两个   "总计数",用于确定反应中的总放射性 混合物
    2. 标记所需量的0.5 ml微量离心管,并将其放在冰上
    3. 在冰上制备反应混合物(表2)。

      表2. SSAT活性测定的反应混合物(一个反应)
      组件
      金额
      1M Tris-HCl(pH7.8)
      10微升
      100 mM Spd
      3微升
      100 mM DTT
      1微升
      (20μCi/ml)[14 C] -AcCoA 6 2.5μl
      ddH sub 2 O
      73.3微升
      总计
      90微升

    4. 吸取90微升反应混合物到每个管。
    5. 吸取10μl样品或空白(缓冲液A)到每个管
    6. 在+ 37℃下在热循环仪中孵育10分钟(注10)。
    7. 冷却至+4℃,加入20μl的1M羟胺
    8. 在+100℃下加热3分钟。
    9. 在RT下以15,000×g离心管5分钟
    10. 将P81纸方格放置在石蜡膜上,并用铅笔将其编号(图2A)
    11. 吸取40μl样品上清液到每个纸方块,并让其干燥 。 还包括两个"总计数" - 移液45μl反应混合物 以过滤纸。
    12. 将"总数"纸张正方形放入 使用镊子的4-ml闪烁小瓶,加入3ml闪烁 鸡尾酒,并用盖子封闭它们(图2C-E)
    13. 将样品和空白纸方格放在1升锥形烧瓶中。
    14. 用dH 2 O洗涤纸张方格3×5分钟和1×10分钟(500ml 每次洗涤),在约200rpm下在水平振荡器上摇动(注释11-12) (图2B)。
    15. 用400 ml 96%乙醇洗涤纸张方格5分钟,在水平振荡器上以约200 rpm振摇(注释11)。
    16. 将纸张正方形用乙醇倒入一个大容器中,用镊子将它们放在一张大张滤纸上,然后让其干燥。
    17. 将纸方格放入4毫升闪烁瓶(图2C)。
    18. 添加3毫升闪烁鸡尾酒到每个小瓶,用盖子关闭它们(图2D-E)
    19. 用液体闪烁计数器测量[14 C] - 放射活性(注9)。
    20. 计算结果(也见表3):
      1. 检查[14 C] -AcCoA批次标记的比活性,并计算其在反应混合物中的摩尔量(90μl):
        c =放射性c(mCi/ml)/比活性(mCi/mmol)
        n = c * V
      2. 接下来计算多少cpm对应于一个pmol的AcCoA:
        相对比活性(RSA)(pmol/cpm)= n/[(平均"总计"cpm)* 2]
      3. 然后计算结果:
        {[(平均样品cpm-平均空白cpm)* RSA] *(120μl/40μl)/(每10μl样品的mg)} * 6 = pmol/h / 活性可以表示为每mg蛋白质,磨。 细胞,μgDNA或mg组织。
    21. 对于组织和细胞样品,当cpm值超过〜6,000时通常需要稀释样品

      图2. SSAT活性测定和制备液体样品 闪烁计数。 A。 编号为P81纸方格的样品, 空白和总计数。 B.洗纸方格。 C.放置 纸方格到液体闪烁瓶。 D.添加闪烁 鸡尾酒与分配器。 E.加盖和未加盖的小瓶和盒 盖。

代表数据

表3.来自使用DU145细胞的实验的代表性数据

笔记

  1. AcCoA非常不稳定。 解冻包装后,分装并储存在-20°C
  2. 建议使用可用针刺穿的橡胶帽,以避免任何可能的放射性CO 2从管中释放。
  3. 细胞/组织可以是新鲜的或冷冻在-70℃
  4. 所用缓冲液的量取决于特定细胞/组织类型中的ODC/SSAT活性。 所需的澄清裂解物的量为SSAT测定为20μl,ODC测定为100μl。 我们建议使用至少40毫克的组织和至少0.5磨。 细胞/200μl缓冲液
  5. 对于细胞样品使用10mM L-鸟氨酸,对组织样品使用25mM L-鸟氨酸
  6. 当移液到管的底部时,超长移液管吸头是方便的。 出乎意料的高cpm值通常是由玻璃管壁上的污染反应混合物引起的
  7. 可溶的吸收释放[ 14 C] -CO 2 。
  8. 请勿用针接触纸张,否则将会释放[ 14 C] -CO 2 。如果使用普通的橡胶帽,只需稍微打开它,以便将针安装到管中,并在添加柠檬酸后立即关闭帽。
  9. 为了获得最佳结果,在测量前将小瓶置于室温下
  10. 反应可以方便地在热循环仪中进行,但也可以在水浴或加热块上进行
  11. 振动速度取决于振动器的尺寸。使用速度,使纸方格轻轻地围绕烧瓶。太高的速度将导致从方角上的纸张丢失。
  12. 高空白值(> 500cpm)表示在同一烧瓶中洗涤不足或样品量太高。或者使用两个烧瓶来洗涤纸方格,或者用H 2 O添加另外两个5分钟的洗涤。
  13. Triton X-100对于组织样品不是绝对必要的,但它增强了细胞破裂
  14. [14 C] -L-鸟氨酸的商业制剂含有可变量的放射性CO 2,这将导致高空白值。 酸处理除去任何残留的放射性CO 2(Jänne和Williams-Ahsman,1971)。

食谱

  1. 缓冲区A(注13)
    将1.25ml Tris-HClpH7.4,250μl200mM EDTA pH8.0,50μlTriton X-100和500μl100mM DTT混合
    将ddH 2 O加到最终体积为50ml的
    中 将一片完全无EDTA的片剂溶解在溶液中
    等分并存储在-20°C
  2. 1 M Tris-HCl(pH 7.4或pH 7.8)
    将157.6g Tris-HCl溶解于〜800ml ddH 2 O中
    用NaOH调节pH至7.4或7.8 将ddH <2> O加入到最终体积为1000ml
    过滤灭菌(0.2μm)
    存储在RT
  3. 200mM EDTA(pH8.0) 称取58.448g EDTA至〜800ml ddH 2 O 2 / 用NaOH缓慢调节pH至8.0 - 当pH接近8时,EDTA溶解。
    将ddH <2> O加入到最终体积为1000ml
    过滤灭菌(0.2μm)
    存储在RT
  4. 100 mM DTT
    在通风橱中将1.5425g DTT溶解于ddH 2 O中,最终体积为100ml。
    过滤灭菌(0.2μm)
    等分并储存在-20°C
  5. 酸处理的[14 C] -L-鸟氨酸(注14)
    将0.5ml [14 C] -L-鸟氨酸与1ml 0.1M HCl混合
    在通风橱中的旋转蒸发器中蒸发至干 将残留物溶于0.5ml 0.01M HCl中 等分并储存在-20°C
  6. 10mM或25mM L-鸟氨酸 在终体积100ml中将168.62mg或421.55mg L-鸟氨酸盐酸盐溶解于ddH 2 O中,分别得到10mM或25mM浓度。
    过滤灭菌(0.2μm)
    等分并储存在-20°C
  7. 20 mM PLP
    将530.32mg吡哆醛5'-磷酸一水合物溶解于ddH 2 O至最终体积为100ml
    过滤灭菌(0.2μm)
    等分并储存在-20°C
  8. 2 M柠檬酸
    将384.24g柠檬酸溶解于ddH 2 O至最终体积为1,000ml
    过滤灭菌(0.2μm)
    储存在+4°C
  9. 100 mM Spd
    将2.546g Spd溶解于ddH 2 O至最终体积为100ml。
    过滤灭菌(0.2μm)
    等分并储存在-20°C
  10. 1 M羟胺
    将6.949g盐酸羟胺溶解于ddH 2 O至最终体积为100ml。
    过滤灭菌(0.2μm)
    存储在RT

致谢

这项工作得到了东芬兰大学和芬兰科学院的战略资助。 协议从Jänne和Williams-Ashman(1971)和Libby(1978)开发的原始版本修改。

参考文献

  1. Jänne,J。和Williams-Ashman,H.G。(1971)。 关于从大鼠前列腺中纯化L-鸟氨酸脱羧酶和硫醇化合物对酶的影响。/b> J Biol Chem 246(6):1725-1732。
  2. Libby,P.R。(1978)。 小牛肝核N-乙酰转移酶。 具有亚精胺乙酰转移酶和组蛋白乙酰转移酶活性的两种酶的纯化和性质.J Biol Chem 253(1):233-237。

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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hyvönen, M. T., Keinänen, T. and Alhonen, L. (2014). Assay of Ornithine Decarboxylase and Spermidine/Spermine N1-acetyltransferase Activities. Bio-protocol 4(22): e1301. DOI: 10.21769/BioProtoc.1301.
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