搜索

Microplate Assay to Study Carboxypeptidase A Inhibition in Andean Potatoes
采用微孔板分析法研究安第斯土豆中羧基肽酶的抑制作用   

引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Phytochemistry
Dec 2015

Abstract

Metallocarboxypeptidases (MCP) are zinc-dependent exopeptidases that catalyze the hydrolysis of C-terminal amide bonds in proteins and peptides. They are involved in a wide range of physiological processes and have recently emerged as relevant drug targets in biomedicine (Arolas et al., 2007). In this context, the study and discovery of new MCP inhibitors from plants constitute a valuable approach for the development of new therapeutic strategies. Herein we describe a simple and accessible microplate method for the study of the specific and dose-response carboxypeptidase A inhibitory activities present in Andean potato tubers. Our protocol combines an extraction method optimized for small protein inhibitors in plant tissues, with the measurement of enzyme kinetics using a microplate reader. These instruments are capable of reading small sample volumes, for many samples in a very short time-frame, therefore reducing the time and costs of high-throughput screening experiments. Although this protocol describes the study of Andean potatoes, our approach is also applicable to the analysis other plant samples.

Keywords: Metallocarboxypeptidase (金属羧肽酶), Carboxypeptidase A (羧肽酶A), Inhibitor (抑制剂), Inhibitory activity (抑制活性), Microplate assay (微孔板分析), Potatoes (土豆)

Background

In higher plants, small proteinaceous protease inhibitors are wound-induced molecules produced as a part of its defense system against insect attack (Graham et al., 1981; Villanueva et al., 1998). Among the studied inhibitors, only two are specific for MCP, i.e., the potato carboxypeptidase inhibitor (PCI) and its close homolog found in tomato plants (TCI). Over the last few decades, the presence of MCP inhibitors in Solanaceae has been extensively reported, revealing potato (Solanum tuberosum) as one of the most important sources of MCP inhibitors (Hass et al., 1979; Obregón et al., 2012; Lufrano et al., 2015). In humans, MCP action is exquisitely regulated and dysregulation of its function might lead to disease or even to cell death (Arolas et al., 2007). In fact, MCP have been associated with human pathologies such as acute pancreatitis (Appelros et al., 1998), diabetes (Cool et al., 1997), several types of cancer (Ross et al., 2009; Sun et al., 2016; Abdelmagid et al., 2008; Tsakiris et al., 2008), fibrinolysis (Valnickova et al., 2007), inflammation (Deiteren et al., 2009) or neurodegeneration (Rogowski et al., 2010). In this context, there is an interest in the discovery of new MCP inhibitors, and thus we focus our studies in potatoes that are native from the Andean region of South America. In this region, thousands of different potato varieties coexist, constituting a natural reservoir for the discovery of novel MCP inhibitors (Figure 1).


Figure 1. Andean potatoes and potato extract CPA inhibitory activity. A. Picture displaying the large number of potato varieties found within the Andean region. Currently in this region coexist thousands of Andean varieties of Solanum tuberosum (Machida-Hirano, 2015; Clausen et al., 2010). B. Effects of potato extracts on bCPA activity. The activity of bovine CPA (bCPA) was measured using the substrate N-(4-methoxyphenylazoformyl)-Phe-OH determining the decrease in absorbance at 340 nm in function of the time. Due to its high content in MCP inhibitors, the addition of potato extract to the reaction decreases the rate of substrate hydrolysis in a dose-response manner.

Here, we describe a simple protocol to determine the specific and dose-response carboxypeptidase A inhibitory activity present in Andean potatoes and in other biological extracts using microplates. The major advantage of this protocol over other available approaches (Yanes et al., 2007) is that the use of microplates allows multiple enzymatic measurements to be done in a single experiment, therefore reducing the time and costs.

Materials and Reagents

  1. 50 ml tubes
  2. 96-well microplates, clear flat bottom (Corning, catalog number: 3364 )
  3. Syringe filters, 0.45 μm pore size (EMD Millipore, catalog number: SLHP033RS )
  4. Potato tubers
  5. General laboratory materials and instrumentation (e.g., micropipettes, microtubes, tips)
  6. Bradford assay kit, e.g., Coomassie Plus Assay Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23236
  7. Bovine serum albumin (BSA)
  8. N-(4-methoxyphenylazoformyl)-Phe-OH·potassium salt (Bachem, catalog number: M-2245 )
  9. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  10. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  11. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
  12. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D4540 )
  13. Bovine carboxypeptidase A (bCPA) (Sigma-Aldrich, catalog number: C9268 )
  14. Carboxypeptidase A reaction buffer/Extraction buffer (see Recipes)
  15. 2 mg/ml bCPA stock solution (see Recipes)
  16. 10x bCPA working solution (see Recipes)
  17. 1,000x substrate stock solution (see Recipes)
  18. 10x substrate working solution (see Recipes)

Equipment

  1. Laboratory blender or equivalent (Oster, catalog number: 004093-008-NP0 )
  2. Refrigerated centrifuge (suitable for volumes of 50 ml) (Beckman Coulter, model: Avanti J-26 XPI )
  3. UV-Vis microplate spectrophotometer system capable of operating at 340 and 595 nm (e.g., PerkinElmer, model: Victor X 2030-0050 or other equivalent spectrophotometer) 
  4. pH meter (HACH LANGE SPAIN, Crison, model: GLP 21 )
  5. 37 °C oven (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: Heratherm Compact Microbiological Incubator
  6. Multichannel pipette (e.g., Technology Networks, model: CappAero Multichannel Pippete 25-200 μl )

Software

  1. GraphPad Prism 5 software (GraphPad Software, Ing USA)

Procedure

  1. Preparation of Andean potato extracts and protein quantification
    1. Wash two mature potato tubers (stage five, according to Johnson, 2008) with distilled water. Peel, weigh (in our case 11.50 g) and dice them into small pieces of about 2 x 2 cm in size (see Figures 2A and 2B).


      Figure 2. Preparation of potato extracts. A. Workflow followed to obtain the crude potato extract enriched in MCP inhibitors. B. Potato peeling and dicing. C. Mixing of the diced potatoes with the extraction buffer into the blender. D. The appearance of the potato extract before and after two centrifugation steps (from left to right).

    2. Mix the potato pieces with three volumes of ice-cold extraction buffer (in our case 34.50 ml; a mass/volume ratio of 1:3) into the cooled blender and homogenize at gentle speed for 5 min. (Figures 2A and 2C).
      Note: The homogenization should be performed in several sessions of 15-30 sec each, with 60 sec intervals between sessions to prevent excessive heating of the sample.
    3. After homogenization, transfer the potato homogenate to 50 ml tubes and centrifuge the sample at 9,000 x g for 30 min at 4 °C.
    4. Transfer the supernatant to a clean 50 ml centrifuge tube and spin at 25,000 x g for 30 min at 4 °C (Figures 2A and 2D). After the second centrifugation step, collect the resultant supernatant and filter through a 0.45 μm syringe filter to eliminate protein aggregates. The resultant potato extract can be stored at -20 °C until analyzed.
    5. Determine the protein concentration of the samples using the Coomassie Plus Assay Kit, according to the manufacturer’s instructions. In brief, prepare a final volume of 500 μl of each of the six standard solutions containing 0, 2, 5, 10, 15 and 20 μg/ml of BSA and appropriate dilutions of the sample/s. Transfer 150 μl of each standard and potato extract samples into different wells of a 96-well microplate. BSA standards and potato extract samples should be assayed in triplicate. Then, add 150 μl of the Coomassie Plus reagent (see Materials and Reagents section) to each well and mix using the micropipette, by pipetting up and down carefully. After 5 min incubation at room temperature, measure the absorbance at 595 nm using a UV-Vis microplate spectrophotometer. Typically, we obtained 1-2 mg/ml of protein in the extracts.

  2. Enzymatic assays
    1. Determination of bCPA specific inhibitory activity
      1. Prepare triplicates of the reaction mixtures (Table 1) in a microplate without adding the substrate. Calculate the volume of potato extract to be added, in order to obtain 20% to 80% of bCPA inhibition. To obtain these inhibition levels, we typically add around 5-30 μg/ml of final protein concentration to the assay.

        Table 1. Reaction mixture

        Where X is the volume (in μl) of potato extract to be assayed. The substrate should be added immediately before plate reading (see step B1c below)

      2. Cover the microplate with a lid and incubate at 37 °C for 15 min.
      3. Add 25 μl of substrate working solution to each well, mix carefully and thoroughly, by pipetting up and down carefully with a multichannel micropipette (A graphical demonstration of steps B1a, B1b and B1c is shown in Video 1).
        Note: the homogenization should be performed in no longer than 60 sec, to avoid significant consumption of the substrate before absorbance monitoring.
      4. Perform absorbance measurements at 340 nm every 30 sec for 10 min.
      5. One unit of inhibitory activity is defined as the amount of inhibitor able to reduce one unit of bCPA activity, which in turn corresponds to the amount of enzyme that hydrolyzes 1.0 μmol of N-(4-methoxyphenylazoformyl)-Phe-OH per min at 25 °C. Consequently, Equation 1 can be used to calculate the Specific Inhibitory Activity (SIA) found in potato extracts.
        Equation 1:

        Where,
        IA is the Inhibitory Activity in U/ml,
        ΔAbs/Δt is the absorbance variation per unit of time (in min) during the reaction in absence (control), and in presence of the potato extract (Potato ext), respectively,
        ξ is the extinction coefficient for the substrate N-(4-methoxyphenylazoformyl)-Phe-OH (ξ 350 nm = 19 [μmoles/ml]-1 cm-1),
        Vtotal is the assay volume and VPotato ext is the volume of extract added to the reaction,
        D and L are the dilution factor for the extract and the path length (in cm), respectively. Typically, the path length in a microplate for a volume of 250 μl is 0.7 cm. However, for different reaction volumes, or for a more accurate calculation, check your 96-well microplate manufacturer instructions.
        Then, calculate directly the SIA by dividing the resultant IA value by the protein concentration of the sample in mg/ml (see Equation 2 and examples in Table 2).
        Equation 2:
    2. Determination of the IC50: Dose-Response curve assay (see Figure 3)
      1. Prepare the same control reaction as described in step B1a and prepare at least 12 additional reaction mixtures with different final concentrations of the potato extract in the assay, ranging from 0 to 300 μg/ml, (or even with higher concentration to obtain the complete inhibition of bCPA activity).
        Note: We typically assay a total of 15 different extract final concentrations, containing 0, 1, 2, 3, 5, 10, 15, 20, 30, 75, 100, 150, 200, 250 and 300 μg/ml of protein.
        Fit the results obtained to the following Equation 3 and determine the IC50 value.
        Equation 3:

        Where,
        X is the log-transformed protein concentration assayed,
        Y is the normalized bCPA activity (relative to the control condition and expressed as a percentage of the maximal activity).
        Y values can be calculated for each extract concentration, using the following Equation 4.
        Equation 4:

        The IC50 value is the extract concentration necessary to reach a 50% of bCPA inhibition (Copeland, 2005). See representative examples of dose-response bCPA inhibitory plots in Figure 3 and the corresponding IC50 values in Table 2.


        Figure 3. Examples of dose-response inhibitory plots. Representative examples of dose-response inhibitory curves determined for three different varieties of potatoes; Solanum tuberosum subsp. tuberosum var. monalisa (Monalisa, magenta solid line), Solanum tuberosum subsp. andigenum var. churqueña (Churqueña, green solid line), Solanum tuberosum subsp. andigenum var. cuarentilla; (Cuarentilla, blue solid line).

        Table 2. Summary of the inhibitory activities found in different varieties of potatoes

        A: Solanum tuberosum subsp. tuberosum var. monalisa; B: Solanum tuberosum subsp. andigenum var. churqueña; C: Solanum tuberosum subsp. andigenum var. cuarentilla

        The two protocols for the determination of SIA and IC50 are summarized in the scheme of Figure 4 and Video 1.


        Figure 4. Enzymatic assays. General workflow for the measurement of inhibitory activity and determination of the SIA and IC50.

        Video 1. Video demonstration of the protocol to perform the enzymatic measurements

Data analysis

Data fittings and IC50 determination were performed using GraphPad Prism 5 software (GraphPad Software, Ing USA) (Motulsky et. al., 2007). Only fittings with an R2 > 0.97 were considered.

Notes

  1.  Freshly collected samples display higher inhibitory activities; however, frozen samples can also be used.
  2. The protocol described here is suitable for any scale of sample preparation, therefore volumes and reagent quantities should be scaled proportionally.
  3. Here we used a laboratory blender that allows a complete sample blending and homogenization. Alternatively, other appropriate homogenization systems can be used.
  4. The soluble fraction resultant from the step A4 is a crude extract rich in MCP inhibitors. Other proteinaceous and chemical compounds soluble in the homogenization buffer can be also present in the sample. For a higher MCP inhibitor enrichment (e.g., for Ki determination), further purification procedures could be addressed (Pearce and Ryan, 1983).
  5. We used a commercial Bradford assay kit; however, non-commercial Bradford reagents or other different quantification assays can be used (Bradford, 1976; He, 2011a and 2011b).
  6. The microplate reactions can alternatively be performed in conventional spectrophotometer cuvettes by scaling up the reagents proportionately.
  7. Mix the enzyme suspension thoroughly to ensure complete mixing. It is strongly recommended to follow manufacturer’s instructions for an accurate enzyme preparation.
  8. The substrate concentration used is such that the enzyme is at the maximum velocity. The enzyme concentration might be adjusted to consume less than 5-10% of substrate.

Recipes

  1. Carboxypeptidase A reaction buffer/Extraction buffer (1 L, 20 mM Tris-HCl, 500 mM NaCl, pH 7.5)
    2.42 g of Trizma base
    29.22 g of NaCl
    MilliQ water up to 900 ml
    Adjust solution to pH 7.5 by addition of 5 N HCl
    Adjust the final volume with MilliQ water to 1,000 ml
    Store at 4 °C up to two weeks
  2. 2 mg/ml bCPA stock solution
    Prepare 1 ml of a 2 mg/ml bCPA solution (~57 µM) in reaction buffer
    Note: We typically dilute 100 µl of commercial bCPA with 900 µl of carboxypeptidase A reaction buffer. Note that different batches of bCPA may have different enzyme concentrations, therefore the dilutions should be adjusted accordingly.
    Divide into 20 μl aliquots
    For short-term storage, store at 4 °C; for long-term storage, store at -20 °C
  3. 10x bCPA working solution
    Prepare 10 ml of a 50 nM enzyme solution by diluting 8.77 μl of bCPA stock solution in 9.991 ml of carboxypeptidase A reaction buffer
    For short-term storage, store at 4 °C; for long-term storage, store at -20 °C
  4. 1,000x substrate stock solution (100 mM N-[4-methoxyphenylazoformyl]-Phe-OH)
    Dissolve 100 mg of N-(4-methoxyphenylazoformyl)-Phe-OH·potassium salt (Mock et al., 1996) in 2.74 ml of DMSO
    Divide into 100 μl aliquots and store at -20 °C
  5. 10x substrate working solution, 1 mM (N-[4-methoxyphenylazoformyl]-Phe-OH)
    Dilute 100 μl of the substrate stock solution to a final volume of 10 ml with carboxypeptidase A reaction buffer
    Store at -20 °C

Acknowledgments

The protocol described here was adapted from previously published studies (Covaleda et al., 2012; Lufrano et al., 2015; Sanglas et al., 2009). This work was supported by the Spanish Ministry of Innovation and Competitiveness grant BIO2013-44973-R and by UNLP, Argentina grant PPID/X014. The authors acknowledge support of the CONICET (PIP 0120), Universidad Nacional de La Plata (UNLP), PPID X/014, Bilateral Cooperation Program MinCyT-MICINN (project ES/09/24-AR2009/006) and Proyectos Redes Universitarias, PPUA, SPU, Argentina.

References

  1. Abdelmagid, S. A. and Too, C. K. (2008). Prolactin and estrogen up-regulate carboxypeptidase-d to promote nitric oxide production and survival of MCF-7 breast cancer cells. Endocrinology 149(10): 4821-8.
  2. Appelros, S., Thim, L. and Borgstrom, A. (1998). Activation peptide of carboxypeptidase B in serum and urine in acute pancreatitis. Gut 42(1): 97-102.
  3. Arolas, J. L., Vendrell, J., Aviles, F. X. and Fricker, L. D. (2007). Metallocarboxypeptidases: emerging drug targets in biomedicine. Curr Pharm Des 13(4): 349-366.
  4. 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.
  5. Clausen, A. M., Ispizúa, N. and Digilio. (2010). Native andean potato varieties in argentina: conservation and evaluation of an endangered genetic resource contents. AmJPSB 3(1): 72-82.
  6. Cool D. R., Normant E., Shen F., Chen H. C., Pannell L., Zhang Y. and Loh Y. P. (1997). Carboxypeptidase E is a regulated secretory pathway sorting receptor: genetic obliteration leads to endocrine disorders in Cpefat mice. Cell 88(1): 73–83.
  7. Copeland R. (2005). Lead optimization and SAR for reversible inhibitors. In: Copeland R. (Ed). Evaluation of enzyme inhibitors in drug discovery: a guide for medicinal chemists and pharmacologists. John Wiley & Sons, pp: 125-128.
  8. Covaleda, G., del Rivero, M. A., Chavez, M. A., Aviles, F. X. and Reverter, D. (2012). Crystal structure of novel metallocarboxypeptidase inhibitor from marine mollusk Nerita versicolor in complex with human carboxypeptidase A4. J Biol Chem 287(12): 9250-9258.
  9. Deiteren, K., Hendriks, D., Scharpe, S. and Lambeir, A. M. (2009). Carboxypeptidase M: Multiple alliances and unknown partners. Clin Chim Acta 399(1-2): 24-39.
  10. Graham, J. S. and Ryan, C. A. (1981). Accumulation of a metallo-carboxypeptidase inhibitor in leaves of wounded potato plants. Biochem Biophys Res Commun 101(4): 1164-1170.
  11. Hass, G. M. and Derr, J. E. (1979). Purification and characterization of the carboxypeptidase isoinhibitors from potatoes. Plant Physiol 64(6): 1022-1028.
  12. He, F. (2011a). Bradford protein assay. Bio-protocol Bio101: e45.
  13. He, F. (2011b). BCA (bicinchoninic acid) protein assay. Bio-protocol Bio101: e44.
  14. Johnson, D. A. (2008). Potato Health Management (2nd ed.). American Phytopathology Society.
  15. Lufrano, D., Cotabarren, J., Garcia-Pardo, J., Fernandez-Alvarez, R., Tort, O., Tanco, S., Aviles, F. X., Lorenzo, J. and Obregon, W. D. (2015). Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes. Phytochemistry 120: 36-45.
  16. Machida-Hirano, R. (2015). Diversity of potato genetic resources. Breed Sci 65(1): 26-40.
  17. Mock, W. L., Liu,Y. and Stanford, D. J. (1996). Arazoformyl peptide surrogates as spectrophotometric kinetic assay substrates for carboxypeptidase A. Anal Biochem 239(2): 218-222.
  18. Motulsky, H. (2007). Prism 5 statistics guide. GraphPad Software.
  19. Obregón, W. D.; Ghiano, N; Tellechea, M., Cisneros, S., Lazza, C. M.; López, L.M.I. and F. X. Avilés (2012). Detection and characterization of a new metallocarboxypeptidase inhibitor from Solanum tuberosum cv. Desirèe using proteomic techniques. Food Chem 133(4): 1063–1068.
  20. Pearce, G. and Ryan, C. A. (1983). A rapid, large-scale method for purification of the metallo-carboxypeptidase inhibitor from potato tubers. Anal Biochem 130(1): 223-225.
  21. Rogowski, K., van Dijk, J., Magiera, M.M., Bosc, C., Deloulme, J.C., Bosson, A., Peris, L., Gold, N.D., Lacroix, B., Bosch Grau, M., Bec, N., Larroque, C., Desagher, S., Holzer, M., Andrieux, A., Moutin, M.J. and Janke, C. (2010). A family of protein-deglutamylating enzymes associated with neurodegeneration. Cell 143(4): 564–578.
  22. Ross P. L., Cheng I., Liu X., Cicek M. S., Carroll P. R., Casey G. and Witte J. S. (2009). Carboxypeptidase 4 gene variants and early-onset intermediate-to-high risk prostate cancer. BMC Cancer 9: 69.
  23. Sanglas, L., Aviles, F. X., Huber, R., Gomis-Ruth, F. X. and Arolas, J. L. (2009). Mammalian metallopeptidase inhibition at the defense barrier of Ascaris parasite. Proc Natl Acad Sci U S A 106(6): 1743-1747.
  24. Sun, L., Wang, Y., Yuan, H., Burnett, J., Pan, J., Yang, Z., Ran, Y., Myers, I. and Sun, D. (2016). CPA4 is a novel diagnostic and prognostic marker for human Non-Small-Cell lung cancer. J Cancer 7(10):1197-204.
  25. Tsakiris I., Soos G., Nemes Z., Kiss S. S., Andras C., Szanto J. and Dezso B. (2008). The presence of carboxypeptidase-M in tumour cells signifies epidermal growth factor receptor expression in lung adenocarcinomas: the coexistence predicts a poor prognosis regardless of EGFR levels. J Cancer Res Clin Oncol 134(4): 439-451.
  26. Valnickova, Z., Thogersen, I. B., Potempa, J. and Enghild, J. J. (2007). Thrombin-activable fibrinolysis inhibitor (TAFI) zymogen is an active carboxypeptidase. J Biol Chem 282(5): 3066-3076.
  27. Villanueva, J., Canals, F., Prat, S., Ludevid, D., Querol, E. and Aviles, F. X. (1998). Characterization of the wound-induced metallocarboxypeptidase inhibitor from potato cDNA sequence, induction of gene expression, subcellular immunolocalization and potential roles of the C-terminal propeptide. FEBS Lett 440(1-2): 175-182.
  28. Yanes, O., Villanueva, J., Querol, E. and Aviles, F. (2007). Enzymatic measurements for the detection of trypsin and carboxypeptidase A inhibitory activity. Protocol Exchange.

简介

金属羧肽酶(MCP)是锌依赖性外肽酶,其催化蛋白质和肽中的C-末端酰胺键的水解。它们参与多种生理过程,并且最近作为生物医学中的相关药物靶标出现(Arolas等人,2007)。在这种情况下,从植物研究和发现新的MCP抑制剂构成了开发新的治疗策略的有价值的方法。在这里我们描述一种简单和可访问的微孔板方法研究特定和剂量反应羧肽酶A抑制活动存在于安第斯马铃薯块茎。我们的协议结合了针对植物组织中的小蛋白抑制剂优化的提取方法,使用酶标仪测量酶动力学。这些仪器能够在很短的时间内读取小样品体积,因此减少了高通量筛选实验的时间和成本。虽然本协议描述了安第斯土豆的研究,我们的方法也适用于分析其他植物样品。
关键字:金属羧肽酶,羧肽酶A,抑制剂,抑制活性,微孔板测定,土豆

[背景] 蛋白质蛋白酶抑制剂是作为其防御昆虫攻击的防御系统的一部分产生的伤口诱导分子(Graham等人,1981; Villanueva等人,1998)。在研究的抑制剂中,只有两种对MCP具有特异性,即,马铃薯羧肽酶抑制剂(PCI)及其在番茄植物(TCI)中发现的紧密同源物。在过去几十年中,Solanaceae中MCP抑制剂的存在已被广泛报道,揭示马铃薯(马铃薯)作为MCP抑制剂的最重要来源之一(Hass等人, ,1979;Obregón等人,2012; Lufrano等人,2015)。在人类中,MCP作用被精确调节,其功能失调可能导致疾病或甚至导致细胞死亡(Arolas等人,2007)。事实上,MCP已经与人类病理例如急性胰腺炎(Appelros等人,1998),糖尿病(Cool等人,1997)相关联,几种类型的癌症(Ross等人,2009; Sun等人,2016; Abdelmagid等人,2008; Tsakiris等人,2008),纤维蛋白溶解(Valnickova等人,2007),炎症(Deiteren等人,2009)或神经变性(Rogowski等人, al。,2010)。在这种情况下,有兴趣发现新的MCP抑制剂,因此我们的研究集中在来自南美洲安第斯地区的土豆。在这一地区,成千上万种不同的马铃薯品种共存,构成发现新的MCP抑制剂的天然库(图1)。


图1.安地斯土豆和马铃薯提取物CPA抑制活性。 A。图片显示安第斯地区内发现的大量马铃薯品种。目前在该区域中共存数千个安第斯品种的马铃薯(Machida-Hirano,2015; Clausen等人,2010)。 B.土豆提取物对bCPA活性的影响。使用底物N-(4-甲氧基苯基偶氮甲酰基)-Phe-OH测定牛CPA(bCPA)的活性,测定340nm处吸光度随时间的减少。由于其在MCP抑制剂中的高含量,向反应中加入马铃薯提取物以剂量 - 反应方式降低底物水解的速率。

   在这里,我们描述了一个简单的协议,确定存在于安第斯土豆和其他生物提取物使用微孔板的特定和剂量反应羧肽酶A抑制活性。该协议相对于其它可用方法的主要优点(Yanes等人,2007)是使用微板允许在单个实验中进行多个酶测量,因此减少了时间和成本。


关键字:金属羧肽酶, 羧肽酶A, 抑制剂, 抑制活性, 微孔板分析, 土豆

材料和试剂

  1. 50ml管子
  2. 96孔微孔板,透明平底(Corning,目录号:3364)
  3. 0.45μm孔径的注射器过滤器(EMD Millipore,目录号:SLHP033RS)
  4. 马铃薯块茎
  5. 一般实验室材料和仪器(例如,微量移液管,微量管,吸头)
  6. Bradford测定试剂盒,例如,Coomassie Plus测定试剂盒(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:23236)
  7. 牛血清白蛋白(BSA)
  8. N-(4-甲氧基苯基偶氮甲酰基)-Phe-OH·钾盐(Bachem,目录号:M-2245)
  9. (Sigma-Aldrich,目录号:T1503)
  10. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  11. 盐酸(HCl)(Sigma-Aldrich,目录号:258148)
  12. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D4540)
  13. 牛羧肽酶A(bCPA)(Sigma-Aldrich,目录号:C9268)
  14. 羧肽酶反应缓冲液/提取缓冲液(参见配方)
  15. 2 mg/ml bCPA储备溶液(见配方)
  16. 10x bCPA工作解决方案(参见配方)
  17. 1,000x底物储备溶液(见配方)
  18. 10x底物工作溶液(见配方)

设备

  1. 实验室搅拌器或等同物(Oster,目录号:004093-008-NP0)
  2. 冷冻离心机(适用于50ml体积)(Beckman Coulter,型号:Avanti J-26 XPI)
  3. 能够在340和595nm(例如,PerkinElmer,型号:Victor X 2030-0050或其它等效分光光度计)上操作的UV-Vis微板分光光度计系统。
  4. pH计(HACH LANGE SPAIN,Crison,型号:GLP 21)
  5. 37℃烘箱(例如Thermo Fisher Scientific,Thermo Scientific ,型号:Heratherm Compact Microbiological Incubator)
  6. 多通道移液器(例如,Technology Networks,型号:CappAero Multichannel Pippete 25-200μl)

软件

  1. GraphPad Prism 5软件(GraphPad Software,Ing USA)

程序

  1. 安第斯马铃薯提取物的制备和蛋白质定量
    1. 用蒸馏水洗涤两个成熟马铃薯块茎(第五阶段,根据Johnson,2008)。剥离,称重(在我们的例子中为11.50g),并将它们切成约2×2cm大小的小块(见图2A和2B)。


      图2.马铃薯提取物的制备 span style ="color:#666666; font-family:Arial; font-size:13.3333px; text-align:justify; white-space:normal;">  A。工作流程接着获得富含MCP抑制剂的粗土豆提取物。 B.马铃薯剥皮和切片。 C.将切块的马铃薯与提取缓冲液混合到搅拌器中。 D.两次离心分离前后马铃薯提取物的外观(从左到右)。

    2. 将马铃薯块与三体积的冰冷的提取缓冲液(在我们的情况下为34.50ml;质量/体积比为1:3)混合到冷却的混合器中,并以缓慢的速度均化5分钟。 (图2A和2C) 注意:应在每次15-30秒的几个阶段中进行,每次间隔60秒以防止样品过热。
    3. 均化后,将马铃薯匀浆转移至50ml管中,并在4℃下以9,000×g离心样品30分钟。
    4. 将上清液转移到干净的50ml离心管中,并在4℃下以25,000×g离心30分钟(图2A和2D)。在第二次离心步骤后,收集所得上清液并通过0.45μm注射器过滤器过滤以除去蛋白质聚集体。所得到的马铃薯提取物可以在-20℃下储存,直到分析。
    5. 使用Coomassie Plus Assay Kit根据制造商的说明书确定样品的蛋白质浓度。简而言之,制备最终体积为500μl的每种含有0,2,5,10,15和20μg/ml BSA和适当稀释的样品的六种标准溶液。转移150微升的每个标准品和土豆提取物样品到96孔微孔板的不同孔。 BSA标准品和马铃薯提取物样品应一式三份进行测定。然后,向每个孔中加入150μl的Coomassie Plus试剂(参见材料和试剂部分),并使用微量移液器小心地上下吹吸混合。在室温下孵育5分钟后,使用UV-Vis微孔板分光光度计测量595nm处的吸光度。通常,我们在提取物中获得1-2mg/ml的蛋白质
  2. 酶测定
    1. bCPA特异性抑制活性的测定
      1. 在微量培养板中制备反应混合物的三份(表1),不加底物。计算要添加的马铃薯提取物的体积,以获得20%至80%的bCPA抑制。为了获得这些抑制水平,我们通常在测定中加入约5-30μg/ml的最终蛋白质浓度
        表1.反应混合物

        其中X是马铃薯浸膏的体积进行测定。在读板之前(见下面的步骤B1c)
        ,应立即添加底物
      2. 用盖子盖住微孔板,并在37℃孵育15分钟
      3. 加入25微升的底物工作溶液到每个孔,仔细和彻底混合,用多通道微量移液管小心地上下移动(步骤B1a,B1b和B1c的图形演示显示在视频1)。
        注意:匀化应当在不超过60秒内进行,以避免在吸光度监测之前显着消耗底物。
      4. 在340 nm每30秒进行吸光度测量10分钟。
      5. 一个单位的抑制活性定义为能够减少一个单位的bCPA活性的抑制剂的量,其又对应于在25℃下每分钟水解1.0μmol的N-(4-甲氧基苯基偶氮甲酰基)-Phe-OH的酶的量C。因此,方程1可用于计算在马铃薯提取物中发现的比抑制活性(SIA) 公式1:

        在哪里,
        IA是以U/ml为单位的抑制活性,
        ΔAbs/Δt分别是在不存在(对照)和存在马铃薯提取物(Potato ext)的反应过程中每单位时间(分钟)的吸收变化,
        ξ是底物N-(4-甲氧基苯基偶氮甲酰基)-Phe-OH(ξ350nm = 19 [μmoles/ml] -1 cm -1)的消光系数,
        V total是试验体积,V Potato ext是加入到反应中的提取物体积,
        D和L分别是提取物的稀释因子和路径长度(cm)。通常,在250μl体积的微量培养板中的路径长度为0.7cm。然而,对于不同的反应体积或更准确的计算,请检查96孔微量培养板制造商的说明 然后,通过将所得IA值除以样品的蛋白质浓度(mg/ml)直接计算SIA(参见表2中的等式2和实施例)。
        公式2:
    2. IC 50的测定:剂量 - 响应曲线测定(参见图3)
      1. 制备与步骤B1a中所述相同的对照反应,并制备至少12个另外的反应混合物,其中在所述测定中马铃薯提取物的不同终浓度为0至300μg/ml,或甚至具有较高浓度以获得完全抑制的bCPA活动)。
        注意:我们通常测定总共15种不同的提取物最终浓度,包括0,1,2,3,5,10,15,20,30,75,100,150,200,250和300μg/ml的蛋白质。
        将获得的结果乘以下面的等式3,并确定IC 50值。
        公式3:

        在哪里,
        X是测定的对数转化的蛋白质浓度,
        Y是标准化的bCPA活性(相对于对照条件并表示为最大活性的百分比)。
        可以使用以下等式4为每种提取物浓度计算Y值 公式4:

        IC 50值是达到50%bCPA抑制所需的提取物浓度(Copeland,2005)。参见图3中剂量 - 反应bCPA抑制图的代表性实例和表2中相应的IC50值

        图3.对于三种不同品种的马铃薯测定的剂量 - 反应抑制曲线的代表性实例; Solanum tuberosum subsp。 tuberosum var。 monalisa(Monalisa,品红色实线),马铃薯性状变异churqueña(Churqueña,绿色实线), Solanum tuberosum 性状变异; (Cuarentilla,蓝色实线)。

        表2.在不同品种的土豆中发现的抑制活性的总结
        A: Solanum tuberosum subsp。 tuberosum var。莫纳利萨B: Solanum tuberosum subsp。性状变异库尔克C: Solanum tuberosum subsp。性状变异cuarentilla

        用于测定SIA和IC 50的两种方案总结在图4和视频1的方案中

        图4.酶测定。 测量抑制活性和测定SIA和IC50的一般工作流程
        <! - flashid2032v126开始 - >
        视频1.执行酶测量的协议的视频演示
        <! - [if!IE] <! - <![endif] - >

        要播放视频,您需要安装较新版本的Adobe Flash Player。

        获取Adobe Flash Player

        <! - [if!IE]> - >
        <! - <![endif] - >
        <! - flashid2032v126结束 - >

数据分析

使用GraphPad Prism 5软件(GraphPad Software,Ing USA)(Motulsky等人,2007)进行数据拟合和IC50测定。仅具有R 2 > 0.97。

笔记

  1.  新鲜收集的样品显示更高的抑制活性;但是,也可以使用冷冻样品
  2. 这里描述的方案适用于任何规模的样品制备,因此体积和试剂量应按比例缩放。
  3. 在这里我们使用一个实验室搅拌器,允许一个完整的样品混合和均化。或者,可以使用其他合适的均化系统。
  4. 由步骤A4得到的可溶性级分是富含MCP抑制剂的粗提取物。样品中还可以存在可溶于匀浆缓冲液中的其他蛋白质和化学化合物。对于更高的MCP抑制剂富集(例如,用于Ki测定),可以进一步纯化程序(Pearce和Ryan,1983)。
  5. 我们使用商业Bradford测定试剂盒;然而,可以使用非商业Bradford试剂或其他不同的定量测定(Bradford,1976; He,2011a和2011b)。
  6. 或者,可以在常规分光光度计比色杯中通过按比例放大试剂来进行微板反应
  7. 充分混合酶悬浮液以确保完全混合。强烈建议按照制造商的说明准备酶制剂。
  8. 所使用的底物浓度使得酶处于最大速度。可以调节酶浓度以消耗小于5-10%的底物。

食谱

  1. 羧肽酶A反应缓冲液/提取缓冲液(1L,20mM Tris-HCl,500mM NaCl,pH7.5)
    2.42克Trizma碱
    29.22克NaCl MilliQ水至900 ml
    通过加入5N HCl调节溶液至pH7.5 用MilliQ水将最终体积调整到1000ml
    储存在4°C长达两个星期
  2. 2mg/ml bCPA储液
    在反应缓冲液中制备1ml 2mg/ml bCPA溶液(〜57μM) 注意:我们通常用900μl羧肽酶A反应缓冲液稀释100μl的商业bCPA。注意不同批次的bCPA可能具有不同的酶浓度,因此稀释度应相应地调整。
    分成20μl等分
    对于短期储存,储存于4°C;长期储存,-20°C储存
  3. 10x bCPA工作溶液
    通过稀释8.77μlbCPA储备溶液在9.991 ml羧肽酶A反应缓冲液中制备10 ml 50 nM酶溶液
    对于短期储存,储存于4°C;长期储存,-20°C储存
  4. 1,000x底物储备溶液(100mM N- [4-甲氧基苯基偶氮甲酰基] -Phe-OH) 将100mg的N-(4-甲氧基苯基偶氮甲酰基)-Phe-OH·钾盐(Mock等人,1996)溶解在2.74ml的DMSO中
    分成100μl等分试样,存储在-20°C
  5. 10x底物工作溶液,1mM(N- [4-甲氧基苯基偶氮甲酰基] -Phe-OH) 用羧肽酶A反应缓冲液稀释100μl底物储备溶液至终体积为10ml 储存于-20°C

致谢

这里描述的方案改编自以前发表的研究(Covaleda等人,2012; Lufrano等人,2015; Sanglas等人)。 ,2009)。这项工作得到了西班牙创新和竞争力部授予BIO2013-44973-R和阿根廷UNLP授予PPID/X014的支持。作者承认对CONICET(PIP 0120),拉普拉塔国立大学(UNLP),PPID X/014,双边合作计划MinCyT-MICINN(项目ES/09/24-AR2009/006)和Proyectos Redes大学,PPUA ,SPU,阿根廷。

参考文献

  1. Abdelmagid,S.A。和Too,C.K。(2008)。 催乳激素和雌激素上调羧-D,以促进一氧化氮生成和MCF-7乳腺癌细胞的存活。内分泌学149(10):4821-8。
  2. Appelros,S.,Thim,L.和Borgstrom,A。(1998)。  羧肽酶B在急性胰腺炎的血清和尿中的激活肽。 42(1):97-102。
  3. Arolas,J.L.,Vendrell,J.,Aviles,F.X.and Fricker,L.D。(2007)。 Metallocarboxypeptidases:新兴的药物靶标生物医学&NBSP; Curr Pharm Des 13(4):349-366
  4. Bradford,M.M。(1976)。 一种快速灵敏的定量微克数量蛋白质的方法蛋白质 - 染料结合的原理。 72:248-254。
  5. Clausen,A.M.,Ispizúa,N.and Digilio。 (2010)。 在阿根廷安第斯本土马铃薯品种:保护和评估濒危遗传资源的内容  AmJPSB 3(1):72-82
  6. Cool D.R.,Normant E.,Shen F.,Chen H.C.,Pannell L.,Zhang Y.and Loh Y.P。(1997)。 羧肽酶E是一种受控的分泌途径分选受体:遗传性闭塞导致 Cep fat 小鼠的内分泌紊乱。 88(1):73-83。
  7. Copeland R.(2005)。 <一类="KE-的insertFile的"href ="https://books.google.com/books?hl=zh-CN&lr=&id=_5yubYumdqwC&oi=fnd&pg=PT11&dq=Evaluation+of+Enzyme+Inhibitors+in+Drug+发现:+ A +指南+为+药物+化学家+和+药物学家和OTS = HHcOcTWxJX与SIG = W0zHHLucUh2nh5GA28ZZhPMXbrc#v = onepage&q =评估%20of%20Enzyme%20Inhibitors%20英寸%20Drug%20Discovery%3A%20A%20Guide%20for%20Medicinal%20Chemists %20和%20药理学家&f =假"target ="_ blank">可逆性抑制剂的先导优化和SAR。 在:Copeland R. 药物发现中酶抑制剂的评价:药物化学家和药理学家的指南 John Wiley& ,pp:125-128。
  8. Covaleda,G.,del Rivero,M.A.,Chavez,M.A.,Aviles,F.X.and Reverter,D。(2012)。从海洋软体动物小说metallocarboxypeptidase抑制剂晶体结构的杂色蜒 287(12):9250-9258。与人类羧肽酶A4的复合物。
  9. Deiteren,K.,Hendriks,D.,Scharpe,S。和Lambeir,A.M。(2009)。 羧肽酶M:多个联盟和未知合作伙伴。 ; Clin Chim Acta 399(1-2):24-39
  10. Graham,J.S。和Ryan,C.A。(1981)。 在受伤马铃薯植株叶片中积累金属羧肽酶抑制剂。   Biochem Biophys Res Commun 101(4):1164-1170。
  11. Hass,G.M。和Derr,J.E。(1979)。 从土豆中纯化和表征羧肽酶异构酶抑制剂。   植物生理 64(6):1022-1028
  12. 他,F.(2011a)。 Bradford蛋白质测定 生物协议 em> Bio101:e45。
  13. 他,F.(2011b)。 BCA(bicinchoninic acid)protein assay。  Bio -protocol Bio101:e44。
  14. Johnson,DA(2008) Potato Health Management(2 nd ed。) American Phytopathology Society,ISBN 978-0 -89054-353-5。
  15. Lufrano,D.,Cotabarren,J.,Garcia-Pardo,J.,Fernandez-Alvarez,R.,Tort,O.,Tanco,S.,Aviles,F.X.,Lorenzo,J.and Obregon,W.D。 来自各种安第斯土豆的新型羧肽酶抑制剂的生物化学表征。   Phytochemistry 120:36-45。
  16. Machida-Hirano,R。(2015)。 马铃薯遗传资源的多样性  Breed Sci 65(1):26-40。
  17. Mock,W.L.,Liu,Y。和Stanford,D.J。(1996)。 Arazoformyl肽替代作为羧肽酶A的分光光度动力学测定底物。 a>  239(2):218-222。
  18. Motulsky,H。(2007)。  Prism 5统计资料指南  GraphPad软体。
  19. Obregón,W.D。吉亚诺Tellechea,M.,Cisneros,S.,Lazza,C.M。 López,L.M.I。和F.X.Avilés(2012)。 从马铃薯中检测和表征新的金属羧肽酶抑制剂 cv。 Desirèe使用蛋白质组学技术。  Food Chem 133(4):1063-1068。
  20. Pearce,G.and Ryan,C.A。(1983)。 一种快速,大规模的纯化金属羧肽酶的方法 al Biochem 130(1):223-225。
  21. Rogowski,K.,van Dijk,J.,Magiera,MM,Bosc,C.,Deloulme,JC,Bosson,A.,Peris,L.,Gold,ND,Lacroix,B.,Bosch Grau,M.,Bec ,N.,Larroque,C.,Desagher,S.,Holzer,M.,Andrieux,A.,Moutin,MJand Janke,C。(2010)。 与神经变性相关的蛋白质去谷氨酰化酶家族。  143(4):564-578
  22. Ross PL,Cheng I.,Liu X.,Cicek MS,Carroll PR,Casey G. and Witte JS(2009)。羧肽酶4基因变体和早发性中至高危前列腺癌。 9:69.
  23. Sanglas,L.,Aviles,F.X.,Huber,R.,Gomis-Ruth,F.X.and Arolas,J.L。(2009)。 哺乳动物金属肽酶抑制蛔虫的防御屏障em> parasite。  Proc Natl Acad Sci USA 106(6):1743-1747。
  24. Sun,L.,Wang,Y.,Yuan,H.,Burnett,J.,Pan,J.,Yang,Z.,Ran,Y.,Myers,I.and Sun, CPA4是一种新的诊断和预后标记物,用于人类非小细胞肺癌,细胞肺癌  J Cancer 7(10):1197-204。
  25. Tsakiris I.,Soos G.,Nemes Z.,Kiss S.S.,Andras C.,Szanto J.and Dezso B.(2008)。 肿瘤细胞中羧肽酶-M的存在表示表皮生长因子受体在肺腺癌中的表达:共存预测不考虑EGFR水平的不良预后。  J Cancer Res Clin Oncol 134(4):439-451。
  26. Valnickova,Z.,Thogersen,I.B.,Potempa,J。和Enghild,J.J。(2007)。 凝血酶可活化纤维蛋白溶解抑制剂(TAFI)酶原是一种活性羧肽酶。   J Biol Chem 282(5):3066-3076
  27. Villanueva,J.,Canals,F.,Prat,S.,Ludevid,D.,Querol,E。和Aviles,F.X。(1998)。 从马铃薯cDNA序列表征伤口诱导的金属羧肽酶抑制剂,诱导基因表达,亚细胞免疫定位和C-末端前肽的潜在作用。 FEBS Lett 440(1-2):175-182。
  28. Yanes,O.,Villanueva,J.,Querol,E。和Aviles,F。(2007)。 用于检测胰蛋白酶和羧肽酶A抑制活性的酶测量。 协议交换 br />
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tellechea, M. E., Garcia-Pardo, J., Cotabarren, J., Lufrano, D., Bakas, L., Avilés, F. X., Obregon, W. D., Lorenzo, J. and Tanco, S. (2016). Microplate Assay to Study Carboxypeptidase A Inhibition in Andean Potatoes. Bio-protocol 6(23): e2032. DOI: 10.21769/BioProtoc.2032.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。