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Sep 2017

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The ATPase Activity of Escherichia coli Expressed AAA+-ATPase Protein
大肠杆菌表达的AAA + -ATPase蛋白的ATPase活性   

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Abstract

ATPases are the enzymes that breakdown ATP to ADP and release inorganic phosphate (Pi). Here we provide a detailed protocol to determine the ATPase activity of a recombinant AAA+-ATPase protein (GENERAL CONTROL NON-REPRESSIBLE-4 [GCN4]) by spectrophotometric absorption at 360 nm to measure the accumulated inorganic phosphate. In general, the substrate 2-amino-6-mercapto-7-methylpurine riboside (methylthioguanosine, a guanosine analog: MESG) is enzymatically converted in the presence of Pi by purine nucleoside phosphorylase (PNP) to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine. The spectrophotometric shift in maximum absorbance at 330 nm for the MESG substrate and subsequent conversion product at 360 nm due to enzymatic conversion was measured. The GCN4-His-tagged recombinant protein was expressed in Escherichia coli BL21 cells and purified using Ni-NTA column. This purified protein was then used for the quantitation of Pi in solution or the continuous determination of Pi released due to the ATPase activity of GCN4, an AAA+-ATPase protein conserved in many eukaryotes, which in plants regulates stomatal aperture during biotic and abiotic stress in plants.

Keywords: GCN4 (GCN4), ATPase (ATP酶), AAA+-ATPase (AAA+-ATP酶), MESG (MESG), Spectrophotometer (分光光度计)

Background

Adenosine triphosphatases (ATPases) are a class of enzymes that catalyze the breakdown of ATP into ADP and free inorganic phosphate (Pi). This breakdown and release of Pi generates energy used by enzymes to carry out the chemical reactions that require energy. This process is an integral part of all the kingdom of life. ABC transporters are the transmembrane proteins that move solute through the membrane by ATPase activity (Rees et al., 2009). Some ATPases are cytoplasmic or membrane-associated proteins. AAA+ proteins are the ATPases associated with diverse cellular activities such as protein degradation, membrane fusion, disassembly of protein complexes, microtubule dynamics, etc. (Snider et al., 2008). We identified GENERAL CONTROL NON-REPRESSIBLE-4 (GCN4), an AAA+-ATPase, as a novel player in regulating stomatal aperture and thus playing a role in plant innate immunity and drought tolerance (Kaundal et al., 2017). Here we describe the method to prove its ATPase activity. The GCN4 as an ATPase enzyme converts ATP to ADP and Pi, and then the enzyme phosphoribosyl phosphorylase (PNP) converts the MESG substrate into the final substrate. The assay monitors the spectrophotometric shift of the substrate (MESG) in the presence of Pi from 330 to 360 nm. The conversion rate is directly correlated with the amount of Pi present in the solution and, that depends on the conversion of ATP to ADP by GCN4 ATPase activity. We reported the ATPase activity of a recombinant protein (GCN4-His) as absorbance (ABS) at 360 nm vs. protein concentration (Kaundal et al., 2017).

Many techniques have been used to quantify the in vitro ATPase activity of purified proteins. Most of the techniques for quantification are based on the detection of free inorganic phosphate (Pi). The most common method uses the calorimeter substrate malachite green (Carter and Karl, 1982). Besides this, fluorescent and radioactive substrates are also used in various protocols to detect free phosphate in the ATPase reaction (Brune et al., 1994; Shiue et al., 2006). In this protocol, we used 2-amino-6-mercapto-7-methylpurine riboside (methylthioguanosine, a guanosine analog: MESG) as a substrate to detect the presence of free phosphate during ATPase activity (Webb, 1992). The purine nucleoside phosphorylase (PNP) enzyme in the presence of inorganic phosphate, which is released from ATP (an experimental substrate) upon hydrolysis to ADP by ATPase (experimental enzyme to be tested), converts MSEG (assay substrate) to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine. This enzymatic conversion of MSEG results in a spectrophotometric shift in maximum absorbance from 330 nm for the MESG substrate to 360 nm for the product 2-amino-6-mercapto-7-methylpurine (Figure 1). The advantage of this protocol is that it does not require long incubation steps and the reaction can be incubated in the plate reader itself for the required duration. The protocol is based on the ENZcheck Phosphate Assay Kit. Most of the components for enzyme assay come with the kit, including potassium phosphate standard, so the reaction is very convenient to assemble. We optimized the protocol from 1 ml reaction to 300 µl reaction to carry out in a microtiter plate. This protocol describes the detailed assay for calculating the ATPase activity of a recombinant GCN4, an AAA+-ATPase protein in U/ml, and specific activity. This protocol can also be used for the calculation of GTPase activity of a protein by using GTP as a substrate (Webb and Hunter, 1992).



Figure 1. Enzymatic conversion of 2-amino-6-mercapto-7-methylpurine riboside (methylthioguanosine, a guanosine analog: MESG) into ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine by purine nucleoside phosphorylase (PNP) in the presence of inorganic phosphate released from ATP (substrate) by ATPase (enzyme to be tested)

Materials and Reagents

  1. 96-well microtiter plate (BD Biosciences, catalog number: 353075 )
  2. EnzCheck Phosphate Assay Kit (Thermo Fisher, catalog number: E6646 )
  3. ATP (Sigma, catalog number: A2383 )
  4. ATPase (Sigma, catalog number: A7510 )
  5. Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 500-0006 )
  6. Bovine Serum Albumin (BSA) 50 mg/ml (InvitrogenTM, catalog number: 15561020 )
  7. Tris base
  8. Recombinant ATPase protein/enzyme you would like to test (we used GCN4-HisTag Fusion protein and referred to as test protein below)
  9. 1 M Tris-Cl (pH-8.0) (see Recipes)

Equipment

  1. Microtiter plate reader (Tecan, model: Infinite M200 Pro )
  2. Water bath (Thermo Scientific, catalog number: TSGP02 )
  3. Vortex (FisherBrands, catalog number: 14-955-163 )
  4. Autoclave

Procedure

  1. Estimation of protein concentration by Bradford assay (Bradford, 1976)
    1. Prepare a 100 µg/ml stock solution of BSA in water from BSA 50 mg/ml.
    2. Use a 96-well microtiter plate to prepare the reaction mix. Make triplicates for all reactions.
    3. Prepare 2.5, 5, 10, 20, and 50 µg of BSA standard in corresponding well as described in Table 1 below:

      Table 1. Protein estimation reaction mix composition


    4. Prepare a blank by adding 160 µl of water in a blank microtiter plate well.
    5. Prepare test samples in a microtiter plate well by making several dilutions of purified recombinant ATPase protein/enzyme with water to make a total of 160 µl volume.
    6. Add 40 µl of Bradford reagent to each microtiter plate well, mix and incubate for 5 min at room temperature.
    7. Read absorbance (ABS) at 595 nm on a microtiter plate reader.
    8. If the spectrophotometer does not have software to plot the standard curve, then plot the standard curve in Excel by plotting known BSA concentrations on X-axis and ABS595 on Y-axis. Obtain trendline and use the equation for the trendline and the ABS595 of the unknown to resolve the unknown concentration. The representative data are shown in Figure 2.


      Figure 2. BSA standard curve

  2. ATPase assay
    Reagent preparation
    Prepare stock solutions supplied with the EnzCheck Phosphate Assay Kit to perform the assay.
    1. Prepare a 1 mM stock solution of the MESG substrate by adding 20 ml of dH2O directly to the bottle (Component A). Mix extensively to dissolve MESG completely (maybe ~10 min).
      Note: Do not heat to dissolve. Because MESG is near its saturation point, a small amount of solid may remain, even after extensive mixing. Immediately after dissolving the MESG substrate, aliquot the solution into convenient volumes and place immediately at -20 °C.
    2. Thaw an aliquot of MESG substrate before use by placing it in a 37 °C water bath until just melted (not more than 5 min). Vortex vigorously and immediately chill the solution by placing it on ice.
      Note: The solution is stable for at least 4 h on ice at pH 7.5. If left at room temperature, the half-life of MESG is about 4 h at pH 8.0 and 40 h at pH 6.0. Do not refreeze leftover MESG substrate.
    3. Prepare enzyme purine nucleoside phosphorylase (Component B) stock by adding 500 µl of dH2O to a vial to prepare a 100 U/ml stock solution and store at 4 °C.
    4. Prepare recombinant ATPase protein (test protein) stock.
      Note: Use different concentrations of test protein to analyze ATPase activity.
    5. Prepare 1 mM ATP stock. Make 10 mM ATP stock by adding 0.05 g of ATP (disodium salt) in 10 ml of 25 mM Tris-Cl (pH 8.0) buffer (250 µl of 1 M Tris-Cl pH 8.0 in 9.75 ml sterilized water). Dilute to 1 mM ATP stock by adding 1 ml of 10 mM ATP in 9 ml of 25 mM Tris-Cl (pH 8.0).
    6. Dilute a portion of the 50 mM potassium phosphate monobasic (KH2PO4), a phosphate standard, 100-fold with dH2O to generate a 500 µM solution. Further, dilute a portion of 500 µM solution to make 1 µM KH2PO4, working solution.

    Phosphate Standard curve
    1. Prepare 10, 20, 30, 40, and 50 nmol of inorganic phosphate standard with 1 µM KH2PO4 in corresponding well to the final volume of 300 µl reaction mix as per Table 2 below:

      Table 2. Phosphate standard curve reaction mix composition


    2. Use a 96-well microtiter plate to perform the assay.
    3. Start the reaction by the addition of final component purine nucleoside phosphorylase and incubate for 30 min at 22 °C.
    4. Read the absorbance at 360 nm. Correct the absorbance by subtracting blank from each standard.
    5. Plot Phosphate standard curve in Excel by plotting known inorganic phosphate concertation on X-axis and ABS360 on Y-axis and obtain trendline and equation. The representative data is shown in Figure 3.


      Figure 3. Phosphate standard curve

    ATPase reaction
    1. Use a 96-well microtiter plate to perform the assay. If the plate reader is not available, use 1 ml of assay mix to perform the assay in cuvettes.
    2. Make triplicate for all reactions.
    3. Prepare assay mix by mixing the stock solution to make 300 µl assay mix for plate reader in 96-well plates and 1 ml assay mix to use in the cuvette as described in Table 3 below:

      Table 3. ATPase assay reaction mix composition


    4. Prepare blank in a microtiter plate well by adding water instead of recombinant ATPase test protein.
    5. Prepare the sample volume of recombinant ATPase test protein ranging from 0.5 to 2 µg of protein in water to the total volume to 100 µl.
    6. Prepare test samples well by adding 100 µl of recombinant ATPase test protein prepared above.
    7. Prepare positive control microtiter plate well by adding 0.5 units of ATPase (Sigma) instead of recombinant ATPase test protein.
    8. Incubate the reaction at 22 °C for 10 min in the plate reader.
    9. Start the reaction by adding 25 µl of ATP (1 mM) as a substrate in each microtiter plate well and mix in the plate reader. If using 1 ml cuvette the amount of ATP needs to be increased accordingly (83 µl).
    10. Read absorbance in a microtiter plate reader at 360 nm at 0 min and then 10 min intervals for one hour or until saturation point has reached at 22 °C. Incubate the plate in the microtiter plate reader.

Data analysis

  1. Calculate the ΔA360nm/mintest (ABS360 at saturation point (min) - ABS360 at 0 min for test samples).
  2. Calculate the ΔA360nm/minblank (ABS360 at saturation point (Min) - ABS360 at 0 min for blank).
  3. Calculate the ΔΔABS360 = (ΔABS360nm/mintest - ΔABS360nm/minblank). Calculate Average and standard error using all three replicates in each reaction.
  4. Plot ΔΔABS360 on Y-axis vs recombinant protein concertation on X-axis. The representative data for ΔΔABS360 vs recombinant test protein are shown in Figure 4.


    Figure 4. ABS360nm vs. test (GCN4-His) protein concentration

  5. Calculate the amount of inorganic phosphate (Pi) using the phosphate standard curve trendline equation (Figure 3) by substituting y with ΔΔABS360.
  6. Calculate ATPase activity using the following formula:

    ATPase Activity =Pi/(t × V) × D = nmol/min/μl = mU/μl = U/ml

    where,
    Pi is the inorganic phosphate amount (nmol) from the standard curve (obtained in step 5),
    t is the reaction time (min) (time to reach the saturation point),
    V is the sample volume (100 µl of recombinant ATPase test protein) added into the reaction well (μl),
    D is the sample dilution factor (100 µl/300 µl) = 0.34.
    Unit Definition: One unit of ATPase is the amount of enzyme that will generate 1.0 µmol of phosphate per min.
  7. Plot ATPase activity (U/ml) on Y-axis vs recombinant test protein concentration on X-axis.
  8. To calculate specific activity, divide the U/ml by the amount of protein present in the sample (converted to mg/ml).

Recipes

  1. 1 M Tris-Cl (pH-8.0)
    Tris base 12.11 g
    Deionized H2O 80 ml
    Adjust pH to 8.0 with HCl
    Make up volume to 100 ml with deionized water
    Autoclave at 121 °C for 15 min

Acknowledgments

This work was supported by funds from the Noble Research Institute, LLC. This protocol was derived from Kaundal et al., 2017 manuscript.

Competing interests

The authors declare no financial or non-financial competing interests.

References

  1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  2. Brune, M., Hunter, J. L., Corrie, J. E. T. and Webb, M. R. (1994). Direct, Real-Time Measurement of Rapid Inorganic Phosphate Release Using a Novel Fluorescent Probe and Its Application to Actomyosin Subfragment 1 ATPase. Biochemistry 33: 8262-8271.
  3. Carter, S. G. and Karl, D. W. (1982). Inorganic phosphate assay with malachite green: an improvement and evaluation. J Biochem Biophys Methods 7(1): 7-13.
  4. Kaundal, A., Ramu, V. S., Oh, S., Lee, S., Pant, B., Lee, H. K., Rojas, C. M., Senthil-Kumar, M. and Mysore, K. S. (2017). GENERAL CONTROL NONREPRESSIBLE4 degrades 14-3-3 and the RIN4 complex to regulate stomatal aperture with implications on Nonhost disease resistance and drought tolerance. Plant Cell 29(9): 2233-2248.
  5. Rees, D. C., Johnson, E. and Lewinson, O. (2009). ABC transporters: the power to change. Nat Rev Mol Cell Biol 10(3): 218-227.
  6. Shiue, S. J., Kao, K. M., Leu, W. M., Chen, L. Y., Chan, N. L. and Hu, N. T. (2006). XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL. EMBO J 25(7): 1426-1435.
  7. Snider, J., Thibault, G., and Houry, W. A. (2008). The AAA+ superfamily of functionally diverse proteins. Genome Biol 9(4): 216.
  8. Webb, M. R. (1992). A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc Natl Acad Sci U S A 89(11): 4884-4887.
  9. Webb, M. R. and Hunter, J. L. (1992). Interaction of GTPase-activating protein with p21ras, measured using a continuous assay for inorganic phosphate release. Biochem J 287 (Pt 2): 555-559.

简介

[摘要]ATP酶是将ATP分解为ADP并释放无机磷(Pi)的酶。在这里,我们提供了一个详细的方案来测定重组AAA+-ATPase蛋白(一般对照非抑制-4[GCN4])的ATPase活性,在360nm处测定无机磷的累积量。一般而言,底物2-氨基-6-巯基-7-甲基嘌呤核苷(甲基硫代鸟苷,鸟苷类似物:MESG)在Pi存在下通过嘌呤核苷磷酸化酶(PNP)酶转化为核糖1-磷酸和2-氨基-6-巯基-7-甲基嘌呤。测量了由于酶转化而导致的MESG底物和随后的转化产物在330nm处的最大吸收度的分光光度偏移。将gcn4his标记的重组蛋白在大肠杆菌BL21细胞中表达,并用Ni-NTA柱纯化。将纯化后的蛋白质用于溶液中Pi的定量测定或连续测定由于GCN4的atp酶活性而释放的Pi,GCN4是一种在许多真核生物中保存的AAA+-ATPase蛋白,在植物受到生物和非生物胁迫时调节气孔开度。

[背景] 腺苷三磷酸酶(atpase)是催化ATP分解为ADP和游离无机磷(Pi)的一类酶。Pi的分解和释放产生的能量被酶用来进行需要能量的化学反应。这个过程是整个生命王国不可分割的一部分。ABC转运蛋白是通过atp酶的活性使溶质通过膜移动的跨膜蛋白质(Rees等人,2009)。有些atp酶是细胞质或膜相关蛋白。AAA+蛋白是与多种细胞活动相关的atp酶,如蛋白质降解、膜融合、蛋白质复合物的分解、微管动力学等(Snider等人,2008)。我们确定了一般控制非抑制-4(GCN4),一种AAA+-ATP酶,是调节气孔孔径的新参与者,因此在植物先天免疫和耐旱性中发挥作用(Kaundal等人,2017)。这里我们描述了证明其ATPase活性的方法。GCN4作为一种ATP酶将ATP转化为ADP和Pi,然后磷酸核糖磷酸化酶(PNP)将MESG底物转化为最终底物。该方法监测底物(MESG)在Pi存在下从330 nm到360nm的分光光度变化。转化率与溶液中Pi的含量直接相关,这取决于GCN4 ATP酶活性将ATP转化为ADP。我们报告了一种重组蛋白(GCN4-His)的ATP酶活性,即360 nm处的吸光度(ABS)与蛋白质浓度的关系(Kaundal等人,2017年)。

许多技术已经被用来量化纯化蛋白质的体外atp酶活性。大多数定量技术都是基于游离无机磷酸盐(Pi)的检测。最常见的方法是使用量热器基质孔雀石绿(Carter和Karl,1982)。除此之外,荧光和放射性底物也用于各种检测ATP酶反应中的游离磷酸盐的方案(Brune等人,1994年;Shiue等人,2006年)。在该方案中,我们使用2-氨基-6-巯基-7-甲基嘌呤核苷(甲基硫代鸟苷,鸟苷类似物:MESG)作为底物,检测ATP酶活性期间是否存在游离磷酸盐(Webb,1992)。嘌呤核苷磷酸化酶(PNP)在无机磷酸盐存在下,由ATP(实验底物)通过ATP酶(待测实验酶)水解成ADP后释放出来,将MSEG(检测底物)转化为核糖1-磷酸和2-氨基-6-巯基-7-甲基嘌呤。MSEG的这种酶转化导致最大吸光度从MESG底物的330 nm到产物2-氨基-6-巯基-7-甲基嘌呤的360 nm的分光光度变化(图1)。该方案的优点是不需要长时间的孵育步骤,反应可以在读版器中孵育一段时间。该方案基于ENZcheck磷酸盐检测试剂盒。酶分析所用的大部分成分都是试剂盒自带的,包括磷酸钾标准品,所以反应的组装非常方便。我们优化了从1ml反应到300µl反应的方案,以便在微量滴定板中进行。本方案描述了计算重组GCN4、AAA+-ATP酶蛋白(单位:U/ml)的ATP酶活性和比活性的详细分析方法。该协议也可用于计算以GTP为底物的蛋白质的GTPase活性(Webb和Hunter,1992)。





图1。在ATP酶(待测酶)释放的无机磷酸盐存在下,用嘌呤核苷磷酸化酶(PNP)将2-氨基-6-巯基-7-甲基嘌呤核苷(甲基硫代鸟苷,鸟苷类似物:MESG)转化为1-磷酸核糖和2-氨基-6-巯基-7-甲基嘌呤

关键字:GCN4, ATP酶, AAA+-ATP酶, MESG, 分光光度计

材料和试剂


 


1.     96孔微量滴定板(BD Biosciences,目录号:353075)


2.     EnzCheck磷酸盐分析试剂盒(Thermo Fisher,目录号:E6646)


3.     ATP(西格玛,目录号:A2383)


4.     ATP酶(西格玛,目录号:A7510)


5.     Bio-Rad蛋白质测定(Bio-Rad实验室,目录号:500-0006)


6.     牛血清白蛋白(BSA)50 mg/ml(InvitrogenTM,目录号:15561020)


7.     Tris基


8.     重组ATPase蛋白/您想测试的酶(我们使用GCN4 HisTag融合蛋白,以下简称测试蛋白)


9.     1 M Tris Cl(pH-8.0)(见配方)


 


设备


 


1.     微量平板阅读器(帝肯,型号:Infinite M200 Pro)


2.     水浴(Thermo Scientific,目录号:TSGP02)


3.     漩涡(FisherBrands,目录号:14-955-163)


4.     高压灭菌器


 


程序


 


A、 用布拉德福德分析法估计蛋白质浓度(Bradford,1976)


1.     从50 mg/ml牛血清中制备100µg/ml牛血清白蛋白储备溶液。


2.     使用96孔微量滴定板制备反应混合物。所有的反应都要三倍。


3.     制备2.5、5、10、20和50µg BSA标准溶液,如下表1所示:


 


表1。蛋白质估计反应混合物成分





浓度µg/ml


牛血清白蛋白(100微克/毫升)微升


水µl


布拉德福德试剂µl


SD1型


2.5


4


156


40


SD2型


5


8


152


40


SD3型


10


16


144


40


SD4型


20


32


128


40


SD5型


30


64


96


40


SD6型


50


80


80


40


 


4.     通过在空白微量滴定板孔中添加160µl水制备空白。


5.     将纯化后的重组ATP酶蛋白/酶用水稀释几次,使其总体积达到160µl,在微滴度板中制备试验样品。


6.     向每个微量滴定板中加入40µl布拉德福德试剂,搅拌并在室温下培养5分钟。


7.     在微量滴定板阅读器上读取595nm处的吸光度(ABS)。


8.     如果分光光度计没有绘制标准曲线的软件,则通过在X轴上绘制已知BSA浓度,在Y轴上绘制ABS595,在Excel中绘制标准曲线。获得趋势线,并使用趋势线方程和未知物质的ABS595来解析未知浓度。代表性数据如图2所示。


 






图2。BSA标准曲线


 


B、 ATP酶测定


试剂制备


准备与EnzCheck磷酸盐分析试剂盒一起提供的储备溶液,以进行分析。


1.     通过直接向瓶子(a组分)中添加20 ml dH2O,制备1 mM MESG基质储备溶液。充分混合,使MESG完全溶解(大约10分钟)。


注:不要加热溶解。由于MESG接近饱和点,因此即使在大量混合之后,也可能残留少量固体。溶解MESG基质后,立即将溶液等分至合适的体积,并立即置于-20°C下。


2.     使用前,将一小份MESG基质放在37°C水浴中解冻,直到刚好融化(不超过5分钟)。用力旋涡,立即将溶液放在冰上冷却。


注:溶液在pH值为7.5的冰上至少稳定4小时。在室温下,MESG在ph8.0和ph6.0下的半衰期分别为4h和40h。不要重新冻结剩余的MESG基质。


3.     通过向小瓶中添加500µl dH2O来制备酶嘌呤核苷磷酸化酶(B组分)储备,以制备100 U/ml储备溶液,并在4°C下储存。


4.     制备重组ATPase蛋白(试验蛋白)储备。


注:用不同浓度的试验蛋白分析atp酶活性。


5.     准备1毫米ATP储备。将0.05 g ATP(二钠盐)加入10 ml 25 mM Tris-Cl(pH 8.0)缓冲液(9.75 ml灭菌水中250µl 1 M Tris-Cl pH 8.0)中,制成10 mM ATP储备。通过在9 ml 25 mM Tris Cl(pH 8.0)中添加1 ml 10 mM ATP稀释至1 mM ATP储备。


6.     用dH2O稀释一部分50 mM磷酸二氢钾(KH2PO4),磷酸盐标准溶液100倍,生成500µM溶液。此外,稀释一部分500µM溶液,制成1µM KH2PO4工作溶液。


 


磷酸盐标准曲线


1.     用1µM KH2PO4在相应的井中制备10、20、30、40和50 nmol无机磷酸盐标准溶液,至300µl反应混合物的最终体积,如下表2所示:


 


表2。磷酸盐标准曲线反应混合物成分





无机磷酸盐


nmol公司


(KH2PO4 1µM)µl


20x反应缓冲液µl


MESG基板µl


水µl


嘌呤核苷磷酸化酶µl


空白


0


0


15


60


222


3


SD1型


10


3


15


60


219


3


SD2型


20


6


15


60


216


3


SD3型


30


9


15


60


213


3


SD4型


40


12


15


60


210


3


SD5型


50


15


15


60


207


3


 


2.     使用96孔微量滴定板进行分析。


3.     通过添加最终组分嘌呤核苷磷酸化酶开始反应,并在22°C下培养30分钟。


4.     读取360 nm处的吸光度。通过从每个标准品中减去空白来校正吸光度。


5.     通过绘制已知无机磷酸盐在X轴和ABS360在Y轴上的浓度,在Excel中绘制磷酸盐标准曲线,得到趋势线和方程。代表性数据如图3所示。


 






图3。磷酸盐标准曲线


 


atp酶反应


1.     使用96孔微量滴定板进行分析。如果平板阅读器不可用,使用1ml分析混合物在试管中进行分析。


2.     所有反应一式三份。


3.     通过混合储备溶液制备分析混合物,在96孔板中制备300µl供平板阅读器使用的分析混合物,以及在反应杯中使用的1 ml分析混合物,如下表3所示:


 


表3。ATP酶测定反应混合物成分


储备溶液


分析混合物(300µl)


如果1毫升的分析混合物


20x反应缓冲液


15微升


50微升


MESG基质溶液


60微升


200微升


重组试验蛋白/酶(0.5-2µg)


100微升


500微升


嘌呤核苷磷酸化酶(PNP)(1U)


3微升


10微升





补充至300µl


将体积加至1毫升


 


4.     以水代替重组atp酶试验蛋白,在微量平板上制备空白。


5.     制备重组ATP酶试验蛋白样品体积,从0.5到2µg的水中蛋白质到100µl的总体积。


6.     加入100µl上述制备的重组ATP酶试验蛋白,制备好供试品。


6.     用0.5个单位的ATPase(Sigma)代替重组ATPase试验蛋白,制备好阳性对照微量板。


7.     将反应于22℃在平板阅读器中孵育10分钟。摄氏度


8.     通过在每个微量滴定板中添加25µl ATP(1 mM)作为底物,开始反应,并在读版器中混合。如果使用1毫升比色杯,则需要相应增加ATP的量(83微升)。


9.     在360 nm条件下,以0分钟的速度在微量平板阅读器中读取吸光度,然后每隔10分钟读取一小时,或直到在22°C下达到饱和点。将平板培养在微量平板阅读器中。


 


数据分析


 


1.     计算ΔA360nm/min试验(饱和点(min)处的ABS360-试样0 min时的ABS360)。


2.     计算ΔA360nm/Min空白(ABS360在饱和点(Min)-ABS360在0 Min时用于空白)。


3.     计算ΔΔABS360=(ΔABS360nm/min试验-ΔABS360nm/min空白)。使用每个反应中的所有三个复制品计算平均和标准误差。


4.     绘制Y轴上的ΔΔABS360与X轴上重组蛋白的聚集。ΔΔABS360与重组试验蛋白的代表性数据如图4所示。


 






图4。ABS360nm与试验(GCN4 His)蛋白质浓度的比较


 


5.     使用磷酸盐标准曲线趋势线方程(图3)计算无机磷酸盐(Pi)的数量,用ΔΔABS360代替y。


6.     使用以下公式计算ATP酶活性:


 


ATP酶活性=Pi/(t×V)×D=nmol/min/μl=mU/μl=U/ml


 


哪里,


Pi是标准曲线中的无机磷酸盐量(nmol)(在步骤5中获得),


t是反应时间(min)(达到饱和点的时间),


V为加入反应井的样品体积(100µl重组ATP酶试验蛋白),μl,


D是样品稀释系数(100µl/300µl)=0.34。


单位定义:一个uATPase的nit是每分钟产生1.0µmol磷酸盐的酶量。


7.     绘制Y轴上ATPase活性(U/ml)与X轴上重组试验蛋白浓度的关系。


8.     为了计算比活度,用U/ml除以样品中的蛋白质含量(换算成mg/ml)。


 


食谱


 


1.     1 M Tris Cl(pH-8.0)


Tris base 12.11克


去离子水80毫升


用盐酸将pH调至8.0


用去离子水补充至100毫升


在121°C下高压灭菌15分钟


 


致谢


 


这项工作得到了Noble Research Institute,LLC.的资金支持。本协议来源于Kaundal等人,2017年手稿。


 


相互竞争的利益


 


作者声明没有金融或非金融竞争利益。


 


工具书类


 


1.     布拉德福德医学硕士(1976)。一种利用蛋白质染料结合原理快速、灵敏地定量蛋白质的方法。《肛生物化学》72:248-254。


2.     Brune,M.,Hunter,J.L.,Corrie,J.E.T.和Webb,M.R.(1994年)。用新型荧光探针直接、实时测定无机磷的快速释放及其在肌动球蛋白亚组分1atpase中的应用。生物化学33:8262-8271。


3.     Carter,S.G.和Karl,D.W.(1982年)。孔雀绿无机磷酸盐测定方法的改进与评价。生物化学与生物物理方法7(1):7-13。


4.     Kaundal,A.,Ramu,V.S.,Oh,S.,Lee,S.,Pant,B.,Lee,H.K.,Rojas,C.M.,Senthil Kumar,M.和Mysore,K.S.(2017年)。一般控制不可抑制4降解14-3-3和RIN4复合物以调节气孔开度,从而影响非寄主抗病性和耐旱性。植物细胞29(9):2233-2248。


5.     Rees,D.C.,Johnson,E.和Lewinson,O.(2009年)。ABC运输公司:改变的力量。分子细胞生物学10(3):218-227。


6.     施国强、高国明、梁文敏、陈立英、陈新立、胡新泰(2006)。XpsE的寡聚作用是由ATP结合而不是水解引起的,导致XpsE与XpsL的结合。EMBO J 25(7):1426-1435。


7.     Snider,J.,Thibault,G.和Houry,W.A.(2008年)。功能多样性蛋白质的AAA+超家族。基因组生物学9(4):216。


8.     韦伯,M.R.(1992年)。无机磷酸盐的连续分光光度测定法和生物体系中磷酸盐释放动力学的测定。美国科学院学报89(11):4884-4887。


9.     韦伯,M.R.和亨特,J.L.(1992年)。GTPase激活蛋白与p21ras的相互作用,用无机磷酸盐释放连续测定法测定。生物化学杂志287(第2部分):555-559。
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引用:Kaundal, A., Vemanna, R. S. and Mysore, K. S. (2020). The ATPase Activity of Escherichia coli Expressed AAA+-ATPase Protein. Bio-protocol 10(15): e3705. DOI: 10.21769/BioProtoc.3705.
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