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Acetyl-coenzyme A Synthetase (Acs) Assay
乙酰辅酶A合成酶(ACS)的活性测定

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Abstract

Acetyl-coenzyme A synthethase (Acs, E.C.6.2.1.1) is an acetate activating enzyme widely represented in nature from bacteria to human. Its function is important for cellular catabolism, especially in order to support microbial growth at low concentrations of acetate (<10 mM) (Castano Cerezo et al., 2011; Castano Cerezo et al., 2009;Renilla et al., 2012). In this protocol, a continuous coupled enzymatic assay for Acs activity is described. Product formation is followed spectrophotometrically by the formation of NADH. The protocol is tailored for E. coli’s Acs, but it can be adapted to assay Acs in any other organism.
The acetyl-coenzyme A synthetase (Acs) assay was first described by Brown et al. (1977). Acs activity is measured using an enzymatic method coupled to malate dehydrogenase (Mdh) and citrate synthase (Cs):
(Acs) acetate + CoASH + ATP -> acetyl-CoA + AMP
(Cs) acetyl-CoA + oxaloacetate -> citrate + CoASH
(Mdh) L-malate + NAD+ -> oxaloacetate + NADH
Net reaction: Acetate + ATP + L-malate + NAD+ -> citrate + AMP + NADH
Under the assay conditions, Mdh and Cs activities are in excess and the rate of NADH formation is limited by Acs activity.

Materials and Reagents

  1. Extraction
    1. Potassium phosphate buffer 65 mM (pH 7.5) (Sigma-Aldrich, catalog number: P5379 )
    2. Cultured cells (approx. 1010 cells) (e.g., for Escherichia coli cells grown in M9 glucose minimal medium, 1 ml of OD600 1 corresponds to approx. 6 x 108 cells)
  2. Enzyme activity
    1. 100 mM Tris-HCl buffer (pH 7.8) (Sigma-Aldrich, catalog number: T1502 )
    2. 20 mM ATP  (Sigma-Aldrich, catalog number: A3377 )
    3. 2 mM Coenzyme A trilithium salt (CoASH) (Sigma-Aldrich, catalog number: C3019 )
    4. 60 mM β-nicotinamide adenine dinucleotide hydrate (NAD+)  (Sigma-Aldrich, catalog number: N7004 )
    5. 50 mM MgCl2  (Panreac Applichem, catalog number:  131396 )
    6. 50 mM L-malate (Sigma-Aldrich, catalog number: 02288 )
    7. 1 M sodium acetate trihydrate (Sigma-Aldrich, catalog number: S8625 )
    8. 50 U/ml malate dehydrogenase (Mdh) from bovine heart (Sigma-Aldrich, catalog number: M9004 )
    9. 25 U/ml citrate synthase from porcine heart (Cs) (Sigma-Aldrich, catalog number: C3260 )
      Reagents 2-4 were prepared in milliQ water and components 5-7 in Tris-HCl buffer (100 mM, pH 7.8). All concentrated stocks (1-7) were prepared in advance and stored at -20 °C. Enzymes (8-9) were stored at 4-6 °C (following the instructions of the manufacturer) and diluted in Tris-HCl buffer (100 mM, pH 7.8) immediately before being used.
    10. 96 well flat bottom, non-treated, non-sterile transpartent plates
    11. Ice-cold phosphate buffer

Equipment

  1. Refrigerated benchtop centrifuge (Eppendorf, catalog number: 5801 R
  2. Ultrasonic homogenizer (Sonics & Materials Inc., Vibra Cell VC 375) equipped with a 3 mm diameter probe
  3. Spectrophotometric plate reader (Bio-Tek)
  4. Novaspec Plus spectrophotometer (Amersham Bioscience GE Healthcare Europe GmbH)
  5. Non-sterile transpartent plates
  6. Water-ice bath

Procedure

  1. Protein extraction procedure
    1. Harvest the cells at the phase of culture of interest, e.g. for E. coli, 50 ml of a culture in exponential growth phase (OD600=0.8) or 20 ml of a culture in stationary phase (OD600=2.0). 
    2. Cool the cells in a water-ice bath for 1-2 min.
    3. Centrifuge the cells for 15 min (10,000 x g, 4 °C).
    4. Discard the supernatant.
    5. Resuspend the cells in ice-cold phosphate buffer (in order to ensure proper washing of cells, use the same initial volume of sample).
    6. Centrifuge the cells for 15 min (10,000 x g, 4 °C). Discard the supernatant.
      Resuspend the cells in ice-cold phosphate buffer. Use an appropriate volume of buffer, calculated according to the following equation; this will result in a concentrated cell suspension of OD600 of 50:
      ml (phosphate buffer)=(OD600 x Vol (ml culture volume))/50
      Where OD600 is considered as readout of the concentration of cells in the sample (Castano-Cerezo et al., 2011).
    7. Disrupt the cells using an ultrasonic homogenizer as follows. Transfer the cell suspension to a 1.5 ml conical tube and place it in an ice bucket. Subject the cell suspension to 3 sonication pulses (20 sec each, 21% amplitude, 3-mm diameter probe, 50% power input). Respect 60 sec intervals between sonication cycles in order to avoid over-heating of samples.
    8. Centrifuge the disrupted cells for 30 min (20,000 x g, 4 °C). 
    9. Collect the supernatant (cell-free extract) and transfer it to a clean 1.5 ml conical tube. Inspect the cell-free extract for suspended particles. If necessary, centrifuge again.
    10. Use the cell- free extract immediately for acetyl-CoA synthetase assay or store the supernatant at -70 °C for long term use.

  2. Acetyl-coenzyme A synthethase assay
    For proper determination of enzyme activity, the optimal dilution of the protein extract has to be determined for each sample, since the activity of extracted enzyme is influenced by the physiological state and the extraction yield of the cells.
    1. Thaw the protein extracts and reagent stocks on ice. Dilute the Mdh and Cs enzyme stocks as explained in the Materials and Methods section and keep them on ice.
    2. Connect the plate reader and allow it to reach 37 °C.
    3. Prepare a "Master Mix" according to the number of samples to be analyzed. The "Master Mix" should contain all the reaction components except for sodium acetate (the substrate of Acs) and the protein extract. Remember to consider sample triplicates. Keep the "Master Mix" on ice. 
      "Master Mix" per reaction:
      100 mM Tris-HCl buffer (pH 7.8): 70 μl
      50 mM L-malate : 10 μl
      20 mM ATP : 10 μl
      50 mM MgCl2 : 10 μl
      2 mM CoASH : 10 μl
      60 mM NAD+ : 10 μl
      50 U/ml Mdh : 10 μl
      25 U/ml Cs : 10 μl
    4. Prepare several dilutions of the protein extract in phosphate buffer. Choose dilutions in a wide enough range (e.g.: 1:1, 1:10, 1:100 and 1:1,000).
    5. In a 96 well flat bottom, transparent plate, add 140 μl of the Master Mix to each well.
    6. Add 40 μl of each of the protein dilutions. Pipet up and down in order to mix the reaction components properly. Tip: Avoid the formation of air bubbles.
    7. Start the reaction by adding 20 ul of 1 M sodium acetate into the sample and mix well by pipetting up and down.
    8. Incubate at 37 °C in the plate reader and record the continuous increase in absorbance at 340 nm (due to the reduction of NAD+) for 5-10 min. Recording should start immediately after starting the reaction. Adjust the reader to get an absorbance value each 10-20 sec. 
    9. Calculate the maximum slope in the OD over time plot (ΔOD/min) for each dilution of the protein extract. For subsequent measurements, select the 2-3 dilutions of the extract to yield a ΔOD/min=10-200. If necessary, assay other dilutions (the typical optimal dilution for Acs activity in cell extracts of glucose-limited stationary phase cultures of E. coli extracts with a protein concentration of approx. 15 mg/ml is 1:20).
    10. Repeat steps 4-10 with the dilutions selected. Perform the assay, at least, in triplicate. To ensure proper determination of enzyme activity in the extract, ΔOD/min should be proportional to protein content.
    11. Calculate the Enzyme Activity Units for the protein extract assayed using the following equation:
      Acs Activity (U/ml) = (ΔOD (/min)*Vr*Df) / (ε *l*Ve)
      Where:
      ΔOD (/min) = maximum slope in the OD vs. t plot (/min)
      Vr = reaction volume (μl)
      Df = dilution factor
      ε NADH 340 nm = 6.22 ml*/μmol*/cm
      l = optical path length (cm)a
      Ve = protein extract volume (μl)b
      aTo determine the optical path length, refer to the specifications of your 96 well plates to get the diameter of the well. Calculate the optical path length from the total liquid volume used in the assay and the diameter of the well.
      bThis volume refers to the volume of diluted extract added to the assay. 
    12. Determine the protein concentration of the extracts assayed [e.g. using the Lowry (Hartree, 1972) or the Bicinchoninic Acid method (Smith et al., 1985)].
    13. Determine the specific Acs activity for each extract using the following equation:
      Acs Specific Activity (U/mg) = Acs Activity (U/ml) / Protein concentration (mg/ml)

Recipes

  1. All the reagents, except for the Mdh and Cs dilution, can be prepared in advance and be stored at -20 °C for up to 1 year.
  2. All the reagents are prepared in 100 mM Tris-HCl buffer (pH 7.8) except for NAD+, CoASH and ATP that should be dissolved in MilliQ water.
  3. Potassium phosphate buffer (65 mM). Adjust pH to 7.5 with 1 M KOH.
  4. Tris-HCl buffer 100 mM. Adjust pH to 7.8 with 1 M KOH.

Acknowledgments

This protocol was first described in and adapted from Brown et al. (1977) and previously used in Castano-Cerezo et al. (2009) and Castano-Cerezo et al. (2011).

References

  1. Brown, T. D., Jones-Mortimer, M. C. and Kornberg, H. L. (1977). The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli. J Gen Microbiol 102(2): 327-336.
  2. Castano-Cerezo, S., Bernal, V., Blanco-Catala, J., Iborra, J. L. and Canovas, M. (2011). cAMP-CRP co-ordinates the expression of the protein acetylation pathway with central metabolism in Escherichia coli. Mol Microbiol 82(5): 1110-1128.
  3. Castano-Cerezo, S., Pastor, J. M., Renilla, S., Bernal, V., Iborra, J. L. and Canovas, M. (2009). An insight into the role of phosphotransacetylase (pta) and the acetate/acetyl-CoA node in Escherichia coli. Microb Cell Fact 8: 54.
  4. Hartree, E. F. (1972). Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem 48(2): 422-427.
  5. Renilla, S., Bernal, V., Fuhrer, T., Castano-Cerezo, S., Pastor, J. M., Iborra, J. L., Sauer, U. and Canovas, M. (2012). Acetate scavenging activity in Escherichia coli: interplay of acetyl-CoA synthetase and the PEP-glyoxylate cycle in chemostat cultures. Appl Microbiol Biotechnol 93(5): 2109-2124.
  6. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. and Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid. Anal Biochem 150(1): 76-85.

简介

乙酰辅酶A合成酶(Acs,E.C.6.2.1.1)是从细菌到人广泛代表的乙酸活化酶。其功能对于细胞分解代谢是重要的,特别是为了支持在低浓度的乙酸盐(<10mM)下的微生物生长(Castano Cerezo等人,2011; Castano Cerezo等人, ,2009; Renilla等人,2012)。在该协议中,描述了Acs活性的连续偶联酶测定。通过NADH的形成,通过分光光度法测定产物形成。该协议是为E定制的。大肠杆菌Acs,但它可以适用于在任何其他生物体中测定Acs。
首先由Brown等人描述了乙酰辅酶A合成酶(Acs)测定法 。 (1977)。使用与苹果酸脱氢酶(Mdh)和柠檬酸合酶(Cs)偶联的酶促方法测量Acs活性:乙酸+ AcASH + ATP→乙酰-CoA + AMP(Cs)乙酰 - CoA +草酰乙酸盐→柠檬酸盐+ Co Wash
(Mdh)L-苹果酸盐+ NAD +→草酰乙酸盐+ NADH
净反应:乙酸盐+ ATP + L-苹果酸盐+ NAD +→柠檬酸盐+ AMP + NADH />在测定条件下,Mdh和Cs活性过量,NADH形成速率受Acs活性限制。

材料和试剂

  1. 萃取
    1. 磷酸钾缓冲液65mM(pH7.5)(Sigma-Aldrich,目录号:P5379)
    2. 对于在M9葡萄糖基本培养基中生长的大肠杆菌细胞,培养的细胞(约10 10个细胞)(例如),1ml OD 600对应于约6×10 8个细胞)
  2. 酶活性
    1. 100mM Tris-HCl缓冲液(pH7.8)(Sigma-Aldrich,目录号:T1502)
    2. 20mM ATP(Sigma-Aldrich,目录号:A3377)
    3. 2mM辅酶A三锂盐(CoASH)(Sigma-Aldrich,目录号:C3019)
    4. 60mMβ-烟酰胺腺嘌呤二核苷酸水合物(NAD +)(Sigma-Aldrich,目录号:N7004)
    5. 50mM MgCl 2(Panreac Applichem,目录号:131396)
    6. 50mM L-苹果酸盐(Sigma-Aldrich,目录号:02288)
    7. 1M的乙酸钠三水合物(Sigma-Aldrich,目录号:S8625)
    8. 来自牛心脏的50U/ml苹果酸脱氢酶(Mdh)(Sigma-Aldrich,目录号:M9004)
    9. 来自猪心脏(Cs)的25U/ml柠檬酸合酶(Sigma-Aldrich,目录号:C3260)
      在milliQ水和组分5-7中在Tris-HCl缓冲液(100mM,pH 7.8)中制备试剂2-4。 预先制备所有浓缩储备物(1-7),并储存在-20℃。 将酶(8-9)储存在4-6℃(按照制造商的说明书),并在即将使用前在Tris-HCl缓冲液(100mM,pH7.8)中稀释。
    10. 96孔平底,未处理的,未灭菌的透明平板
    11. 冰冷的磷酸盐缓冲液

设备

  1. 制冷台式离心机(Eppendorf,目录号:5801R)
  2. 装备有3mm直径探针的超声匀浆器(Sonics& Materials Inc.,Vibra Cell VC 375)
  3. 分光光度计读板器(Bio-Tek)
  4. Novaspec Plus分光光度计(Amersham Bioscience GE Healthcare Europe GmbH)
  5. - 无菌透明板
  6. -ice bath

程序

  1. 蛋白提取程序
    1. 在感兴趣的培养阶段收获细胞,例如用于大肠杆菌,50ml指数生长期的培养物(OD <600> = 0.8)或20ml稳定期培养物(OD <600 = 2.0)。
    2. 冷却细胞在水 - 冰浴1-2分钟。
    3. 将细胞离心15分钟(10,000×g,4℃)。
    4. 弃去上清液。
    5. 将细胞重悬在冰冷的磷酸盐缓冲液中(为了确保正确清洗细胞,使用相同的初始体积的样品)。
    6. 离心细胞15分钟(10,000×g,4℃)。弃去上清液。
      将细胞重悬在冰冷的磷酸盐缓冲液中。使用适当体积的缓冲液,根据以下公式计算:这将导致OD 600的50:
      的浓缩细胞悬浮液 ml(磷酸盐缓冲液)=(OD 600 = 50vol(ml培养物体积))/50
      其中OD <600>被认为是样品中细胞浓度的读出(Castano-Cerezo et al。,2011)。
    7. 使用超声波均化器如下破坏细胞。转移细胞悬浮液到1.5毫升锥形管,并将其放在冰桶。使细胞悬浮液3个超声波脉冲(每个20秒,21%振幅,3毫米直径探头,50%功率输入)。在声波处理周期之间相隔60秒的间隔,以避免样品过热
    8. 将破碎的细胞离心30分钟(20,000×g <4℃,4℃)。
    9. 收集上清液(无细胞提取物),并将其转移到干净的1.5毫升锥形管。 检查无细胞提取物的悬浮颗粒。 如果需要,再次离心
    10. 立即使用无细胞提取物乙酰辅酶A合成酶测定或存储上清液在-70°C长期使用。

  2. 乙酰辅酶A合成酶测定
    为了适当地测定酶活性,必须为每个样品确定蛋白质提取物的最佳稀释度,因为提取的酶的活性受细胞的生理状态和提取产率的影响。
    1. 在冰上解冻蛋白质提取物和试剂储备液。 如材料和方法部分中所述稀释Mdh和Cs酶储备液,并将其保存在冰上。
    2. 连接读板器,使其达到37°C。
    3. 根据要分析的样品数量制备"主混合物"。 "主混合物"应包含除了乙酸钠(Acs的底物)和蛋白质提取物之外的所有反应组分。 记住考虑样品三次。 保持"主混合"在冰上。
      "主混合"每个反应:
      100mM Tris-HCl缓冲液(pH7.8):70μl
      50mM L-苹果酸盐:10μl
      20mM ATP:10μl
      50mM MgCl 2:10μl
      2mM CoASH:10μl
      60mM NAD +:10μl
      50U/ml Mdh:10μl
      25U/ml Cs:10μl
    4. 准备几个稀释的蛋白质提取物在磷酸盐缓冲液。 在足够宽的范围内选择稀释度(例如:1:1,1:10,1:100和1:1,000)。
    5. 在96孔平底,透明板中,向每个孔中加入140μl的主混合物。
    6. 加入40μl每种蛋白质稀释液。上下移动以正确混合反应组分。提示:避免形成气泡。
    7. 通过向样品中加入20ul 1M乙酸钠开始反应,并通过上下吹吸混匀
    8. 在平板读数器中在37℃下孵育,并记录在340nm处的吸光度的持续增加(由于NAD 的减少)5-10分钟。开始反应后应立即开始记录。调整读数器以获得每10-20秒的吸光度值。
    9. 计算每个蛋白质提取物稀释的OD随时间的最大斜率(ΔOD/min)。对于随后的测量,选择提取物的2-3个稀释度以产生ΔOD/min = 10-200。如果必要,测定其它稀释度(蛋白质浓度为约15mg/ml的大肠杆菌提取物的葡萄糖限制性固定相培养物的细胞提取物中Acs活性的典型最佳稀释度为1: 20)。
    10. 重复步骤4-10,选择稀释液。执行测定,至少,一式三份。为了确保提取物中酶活性的适当测定,ΔOD/min应与蛋白质含量成比例
    11. 计算使用下列方程测定的蛋白质提取物的酶活性单位:
      Acs活性(U/ml)=(ΔOD(/min)* Vr * Df)/(ε* l * Ve) 其中:
      ΔOD(/min)= OD相对于t图的最大斜率(/min)
      Vr =反应体积(μl)
      Df =稀释因子
      ε NADH 340nm = 6.22ml * /μmol*/cm l =光程长度(cm) a
      Ve =蛋白提取物体积(μl) b
      a 要确定光程长度,请参考96孔板的规格以获取孔的直径。从测定中使用的总液体体积和孔的直径计算光程长度。
      b 此体积是指添加到测定中的稀释提取物的体积。
    12. 测定所测定的提取物的蛋白质浓度[例如。使用Lowry(Hartree,1972)或二辛可宁酸方法(Smith等人,1985)。
    13. 使用以下公式确定每个提取物的特异性Acs活性:
      Acs比活性(U/mg)= Acs活性(U/ml)/蛋白质浓度(mg/ml)

食谱

  1. 所有试剂,除了Mdh和Cs稀释,可以提前准备,并在-20℃下储存长达1年。
  2. 所有试剂在100mM Tris-HCl缓冲液(pH7.8)中制备,除了应当溶解于MilliQ水中的NAD +,CoASH和ATP。
  3. 磷酸钾缓冲液(65mM)。 用1M KOH调节pH至7.5。
  4. Tris-HCl缓冲液100mM。 用1M KOH调节pH至7.8

致谢

该方案首先在Brown等人(1977)中描述并改编自Brown et al。(1977),并且之前在Castano-Cerezo等人(2009)和Castano-Cerezo et al。(2011)。

参考文献

  1. Brown,T.D.,Jones-Mortimer,M.C.and Kornberg,H.L。(1977)。 大肠杆菌中乙酸盐和乙酰辅酶A的酶相互转化。 J Gen Microbiol 102(2):327-336。
  2. Castano-Cerezo,S.,Bernal,V.,Blanco-Catala,J.,Iborra,J.L.and Canovas,M.(2011)。 cAMP-CRP协调蛋白质乙酰化途径与中枢代谢在大肠杆菌中的表达大肠杆菌。 Mol Microbiol 82(5):1110-1128。
  3. Castano-Cerezo,S.,Pastor,J.M.,Renilla,S.,Bernal,V.,Iborra,J.L.and Canovas,M。(2009)。 深入了解磷酸转乙酰酶(pta)和乙酸/乙酰辅酶A结构在 Escherichia coli 。 Microb Cell Fact 8:54。
  4. Hartree,E.F。(1972)。 蛋白质的测定:对提供线性光度反应的Lowry方法的修改。 Anal Biochem 48(2):422-427
  5. Renilla,S.,Bernal,V.,Fuhrer,T.,Castano-Cerezo,S。,Pastor,J.M.,Iborra,J.L.,Sauer,U.and Canovas,M.(2012)。 大肠杆菌中的乙酸清除活性:乙酰辅酶A合成酶和在chemostat培养物中的PEP-乙醛酸循环。 Appl Microbiol Biotechnol 93(5):2109-2124。
  6. Smith,PK,Krohn,RI,Hermanson,GT,Mallia,AK,Gartner,FH,Provenzano,MD,Fujimoto,EK,Goeke,NM,Olson,BJand Klenk,DC(1985) //www.ncbi.nlm.nih.gov/pubmed/3843705" target ="_ blank">使用二喹啉酸测量蛋白质。 Anal Biochem 150(1):76-85。
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引用:Castaño-Cerezo, S., Bernal, V. and Cánovas, M. (2012). Acetyl-coenzyme A Synthetase (Acs) Assay. Bio-protocol 2(17): e256. DOI: 10.21769/BioProtoc.256.
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shikha srivastava
Jawaharlal Nehru University
hello
I have gone through the above protocol.It was of great help.Can you please let me know as to how to find out Km and Vmax from the above.

regards
7/25/2014 12:34:18 AM Reply
Thanks a lot for giving scientific details
9/13/2012 7:28:52 AM Reply