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Jun 2018

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Quantification of Protein Kinase A (PKA) Activity by An in vitro Radioactive Assay Using the Mouse Sperm Derived Enzyme
用小鼠精子衍生酶体外放射性测定蛋白激酶A(PKA)活性   

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Abstract

In order to acquire fertilizing potential, mammalian sperm must undergo a process known as capacitation, which relies on the early activation of Protein Kinase A (PKA). Frequently, PKA activity is assessed in whole-cell experiments by analyzing the phosphorylation status of its substrates in a western-blot. This technique faces two main disadvantages: it is not a direct measure of the kinase activity and it is a time-consuming approach. However, since PKA can be readily obtained from sperm extracts, in vitro assays such as the “radioactive assay” can be performed using the native enzyme. Unlike western-blot, the radioactive assay is a straightforward technique to evaluate PKA activity by quantification of incorporated 32P into a peptidic substrate. This approach easily allows the analysis of different agonists or antagonists of PKA. Since mouse sperm is a rich source of soluble PKA, this assay allows a simple fractionation that renders PKA usable both for in vitro testing of drugs on PKA activity and for following changes of PKA activity during the onset of capacitation.

Keywords: Protein Kinase A (PKA) (蛋白激酶A(PKA)), Capacitation (精子获能), Sperm (精子), Kinase activity assay (激酶活性测定), Fertilization (受精)

Background

Mammalian sperm are not able to fertilize an oocyte immediately after ejaculation. In order to acquire fertilization competence, they must undergo a series of cellular changes collectively known as capacitation (Stival et al., 2016). This process takes place within the female reproductive tract but can be emulated in the laboratory by incubating spermatozoa in a defined medium containing Ca2+, albumin and HCO3-, i.e., the “capacitation medium” (Visconti et al., 1995). Both HCO3- and Ca2+ act synergistically on a soluble adenylyl cyclase (sAC), producing an elevation of intracellular cAMP levels. Among different targets, cAMP directly activates the Ser/Thr Protein Kinase A (PKA), which acts as a central player orchestrating capacitation signaling events (Buffone et al., 2014). Usually, two different approaches can be used to analyze its activity. One of them relies on western-blots, using commercially available antibodies that detect a consensus phosphorylation sequence of PKA (Krapf et al., 2010). However, the steady-state phosphorylation status of any protein depends on the relative activities of both kinases and phosphatases acting on it. Thus, this approach fails to directly analyze actual PKA activity. A second approach involves measuring in vitro PKA activity by direct quantification of 32P incorporated into a peptidic substrate, in a controlled reaction mixture containing phosphatase inhibitors (Stival et al., 2018). This allows analysis of PKA independently of other factors that could modulate the phosphorylated state of the substrate. The chemical nature of the peptidic substrate named Kemptide, named after Dr Kemp, who first synthesized it in 1977 (Kemp et al., 1977), includes a phosphorylable serine residue and two arginine on positions -3 and -2, allowing high PKA specificity. In addition, the Kemptide possesses high relative positive charge which accounts for its strong binding to the negatively charged Whatman P81 cellulose paper, simplifying washing of excess radioactive material (Kemp et al., 1977). The enzyme PKA is composed by two regulatory and two catalytic subunits (Akamine et al., 2003; Zhang et al., 2012). Both types of subunits can be found in Triton X-100-soluble and –insoluble fractions of mouse sperm (Visconti et al., 1997). The activity of PKA within the soluble fraction increases during sperm capacitation. However, the activity of PKA that remains in the insoluble fraction does not change during the course of capacitation, and thus, acts as an excellent source of PKA for enzymatic studies (Visconti et al., 1997).

The protocol described herein is used to analyze the effect of agonists or antagonists on PKA activity, using mouse sperm as the source for the kinase, lowering costs while keeping high efficiency. However, this protocol can be easily adapted to analyze variations of PKA activity during the course of sperm capacitation, by using total unfractionated sperm extracts. In this regard, phosphorylation of the Kemptide reflects the given activity of PKA at any stage of capacitation.

Finally, other sources of PKA can also be used, such as purified PKA from plasmid expression.

Materials and Reagents

Note: Unless specified, all reagents are stored at room temperature (RT, 15-25 °C).

  1. P81 Whatman cellulose chromatography paper (Sigma-Aldrich, catalog number: Z753645 ), in 2 x 2 cm pieces
    Note: There should be as many squares as conditions, plus 9 extra squares for controls, all in triplicates. All pieces should be labeled with a pencil to differentiate them after washing.
  2. 7 ml Copolymer Plastic Vial, Unlined White Poly Screw Cap (RPI, catalog number: 125509 )
  3. Eppendorf tube (1.5 ml and 2 ml)
  4. 0.45 µm filter
  5. Male mouse (8-20 weeks old)
  6. 70% ethanol
  7. Tris(hydroxymethyl)aminomethane (Tris base) (Cicarelli, catalog number: 1131214 )
  8. Triton X-100 (Neo Lab, catalog number: 0 1685 )
  9. NaCl (Cicarelli, catalog number: 750 )
  10. MgCl2 (Sigma-Aldrich, catalog number: M8266 )
  11. Adenosine 5′-triphosphate disodium salt hydrate (ATP) (Sigma-Aldrich, catalog number: A7699 ), store at -20 °C
  12. Sodium orthovanadate (Na3VO4) (Sigma-Aldrich, catalog number: 450243 ), store at -20 °C
  13. β-Glycerophosphate disodium salt hydrate (G3P) (Sigma-Aldrich, catalog number: G6251 ), store at -20 °C
  14. Para-Nitrophenyl Phosphate (NPP) (Cayman chemicals, catalog number: 400090 )
  15. cOmpleteTM, EDTA-free Protease Inhibitor Cocktail (Protease Cocktail) (Roche, catalog number: 4693132001 ), store at 4 °C
  16. Bovine Serum Albumin fatty-acid free (BSA) (Sigma-Aldrich, catalog number: A7906 ), store at 4 °C
  17. Adenosine 3′,5′-cyclic monophosphate sodium salt monohydrate (cAMP) (Sigma-Aldrich, catalog number: 3A6885 ), alternatively, 8-BrcAMP or db-cAMP can be used, store at -20 °C
  18. 3-Isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich, catalog number: I5879 ), store at -20 °C
  19. Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) (Sigma-Aldrich, catalog number: K1127 ), store at -20 °C
  20. HEPES (Sigma-Aldrich, catalog number: H3375 )
  21. Trichloroacetic acid (TCA) (BioChemica, catalog number: PAA1431 )
  22. ortho-Phosphoric acid 85% (Merck, catalog number: 100573 )
  23. ATP (ATP-32Pγ) 3000 Ci/mmol; 10 mCi/ml (Perkin Elmer, catalog number: BLU502A )
  24. Specific drugs to be tested in the experiment (inhibitors, agonists, etc.)
  25. KCl (Sigma-Aldrich, catalog number: S-7653 )
  26. Na-Pyruvate (Sigma-Aldrich, catalog number: P-4562 )
  27. CaCl2·2H2O (Sigma-Aldrich, catalog number: C-7902 )
  28. KH2PO4 (Sigma-Aldrich, catalog number: P-5655 )
  29. MgSO4·7H2O (Sigma-Aldrich, catalog number: 63138 )
  30. Glucose (Sigma-Aldrich, catalog number: G-6152 )
  31. DMSO (Sigma-Aldrich, catalog number: D2650 )

Solutions
For detailed instructions on how to prepare the following solutions, refer to the Recipes heading:
  1. 1 M Tris pH 7.4
  2. 3 M NaCl
  3. 1 M MgCl2
  4. 1 mM ATP
  5. 100 mM Na3VO4
  6. 800 mM Glyceraldehyde 3-phosphate (G3P)
  7. 500 mM p-Nitrophenyl Phosphate (NPP)
  8. 50 mg/ml BSA
  9. 5 mM Kemptide
  10. 200 mM HEPES pH 7.3
  11. 40% TCA
  12. 5 mM Ortho phosphoric acid
  13. H-TYH medium (see Recipes)
  14. Lysis buffer (see Recipes)
  15. 100 mM IBMX (see Recipes)
  16. Kinase buffer (see Recipes)

Equipment

  1. Scintillation Counter (LKB Wallac Rackbeta 1209, catalog number: 0 90161 )
  2. Neubauer chamber
  3. Surgery scissors (WPI, catalog number: 14393 )
  4. Tweezers
  5. Orbital shake
  6. Incubator
  7. Centrifuge

Software

  1. Microsoft® Office Excel (Microsoft Office 365), or similar

Procedure

  1. Prepare Lysis buffer (Recipe 2) on the day of use. Leave the protease inhibitor cocktail to be added just before usage. Keep buffer on ice.

  2. Calculate the volume of Kinase buffer that will be needed, considering 10 µl per reaction tube, and taking into account that each condition (and controls) will be performed in triplicates. While prepare Kinase buffer (Recipe 4) adding all components but Kemptide, protease inhibitors, and ATP-32Pγ, which will be added before use.

  3. Isolate the insoluble PKA fraction from mouse sperm extracts
    Note: This section describes how to obtain PKA from the Triton-insoluble fraction of mouse sperm extracts, to be used as source of PKA in the activity assay.
    1. Euthanize 1 male mouse (8-20 weeks old) and perform surgery to extract the cauda regions from both epididymides (Figure 1). To do this:
      1. Lay the euthanized mouse dorsally on a dissection board.
      2. Spray the ventral area with 70% ethanol to prevent dry hair from detaching
      3. Using scissors, incise the skin and muscle tissue from the abdominal wall, exposing the lower abdominal viscera (Figure 1A).
      4. Using forceps, remove the fat pads that cover the testis to get the epididymis exposed (Figure 1A).
      5. Identify the epididymis attached to the testis, excise it, and localize the cauda region (Figure 1B, red area). Remove it from the caput and corpus regions (Figure 1B grey area), and adipose tissue (Figure 1B blue area).
      6. While holding the cauda with forceps, perform 3 to 4 incisions, using surgery scissors (Figure 1C).
    2. Place both epididymides in a round-bottom 2 ml Eppendorf containing 500 µl of H-TYH medium (Figure 1D) and incubate for 15 min at 37 °C.


      Figure 1. Sperm preparation. A. Ventral incision to excise the epididymis. Note the tweezers holding the cauda epididymis, attached to the testicle (red circled). B. Whole epididymis excised. Red, cauda; grey, caput and corpus; blue, adipose tissue. The cauda region is used for sperm swim out. C. Three to four incisions should be performed in each cauda, before allowing the sperm to swim out. D. Place both epididymides in a round-bottom Eppendorf tube containing 500 µl of H-TYH medium.

    3. Transfer the sperm suspension (500 µl) to a new round-bottom 2 ml Eppendorf tube, leaving the epididymides behind.
    4. Check sperm concentration by counting in a Neubauer chamber. Adjust concentration to 25 x 106 sperm/ml in H-TYH medium.
    5. Transfer the swim-out to a 1.5 ml Eppendorf tube.
    6. Centrifuge the sperm suspension at 10,000 x g for 3 min at RT
    7. Discard supernatant. If the amount of swim out was of 25 x 106 sperm in 1 ml, resuspend the pellet in 330 µl of ice-cooled Lysis buffer supplemented with protease inhibitor cocktail before use. If a different amount of sperm is used, modify buffer addition accordingly.
    8. Incubate cells in Lysis buffer on ice for 30 min
    9. Centrifuge cells at 10,000 x g for 10 min at 4 °C.
    10. Discard supernatant and resuspend the pellet in 330 µl of Lysis buffer (or same amount used in Step C7), with freshly added protease inhibitor cocktail. This fraction contains PKA. Each condition with 10 µl of fraction will have the soluble PKA fraction extracted from 7.5 x 105 sperm. Consider that a minimum of 1.5 x 105 sperm are needed for each condition, which should be tested in triplicate.
    11. Keep on ice for PKA activity assays. From now on this fraction is called PKA fraction.

  4. Kinase reaction assay
    1. Consider as many tubes as needed, keeping in mind that each condition should be performed in experimental triplicates, plus the following controls:
      1. Negative control: it contains all the reaction reagents except for PKA fraction, which is replaced by Lysis buffer. It is used to determine the amount of Kemptide that is marked by 32P in the absence of PKA.
      2. Positive control: it is a control of maximum PKA activity, i.e., in the presence of 1 mM cAMP and 100 µM IBMX.
    2. Add 10 µl of Lysis buffer containing any drug to be analyzed. Consider that it will be diluted 3x at the time of the reaction. Whatever drug to be tested must be considered within the 10 µl corresponding to the addition of this 10 µl of Lysis buffer.
    3. Add to each tube 10 µl of PKA fraction, except for the Negative control, in which PKA fraction should be replaced by 10 µl of Lysis buffer.
    4. Keep all tubes on ice until used.

    From Step D5 onwards the procedures must be performed in the radioisotope room. Please note that security measures must be taken when working with radioactive material. Proper shielding for the personnel is required, as well as adequate waste disposal according to institutional regulations.
    1. Complete preparation of the Kinase buffer by adding the proper amount of ATP-32Pγ (see Table 1), taking into consideration of the decay time and manufacture date, then add 1 µCi/assay.

      Table 1. Stock solutions and concentration for the preparation of Kinase buffer

      Notes:
      1. All reagents are dissolved in distilled water, unless specified otherwise.
      2. The protease inhibitor cocktail is added as to prepare a 2x concentrated buffer, since the PKA fraction already contains cocktail.
      3. Addition of BSA is done to help precipitate all proteins after TCA addition, but keeping the Kemptide soluble.

    2. Sequentially add 10 µl of Kinase buffer to all tubes and place them at 37 °C. Start a new reaction every 30 s by adding 10 µl of Kinase buffer. Keep track of the sequential order of all tubes. This is crucial in order to stop the reactions exactly at the specified time.
    3. Incubate tubes at 37 °C for 30 min.
    4. Stop the reaction after 30 min by addition of 10 µl 40% TCA. Note that the total volume in each tube now is 40 µl.
    5. Incubate tubes on ice for 20 min.
    6. Centrifuge at 10,000 x g for 3 min, at RT.
    7. For each tube, take 10 µl of the supernatant, and spot onto the center of a P81 piece of paper, while holding it with tweezers (Figures 2A and 2B). Each piece of P81 paper should be labeled with pencil in order to identify them later. Wait 5 s for the drop to be absorbed, and release it in a 5 L beaker containing 1 L of 5 mM ortho-phosphoric acid (Figure 2C).
    8. Wash together all papers 6 x 5 min using 1 L of 5 mM ortho-phosphoric acid each time, in a beaker placed on an orbital shaker.
    9. While the washing in Step D12 proceeds, spot 1 µl of Kinase buffer on a P81 paper. Do this in triplicate using 3 different papers, and then directly insert each one in a different plastic vial, without washing. This step is necessary for data analysis to calculate the total amount of total ATP-32Pγ, initially present in the reaction media.
    10. After washing, place the P81 papers on a plastic tray, without contact between each other, and let them air dry for at least 2 h (Figure 2D). At this step, you can discontinue the protocol until the next day. Papers are identified according to the pencil mark written on them before spotting the reaction mixture.
    11. Once dried, place papers individually in a plastic vial, containing 1 ml of scintillation counter liquid, and register counts per minute (CPM) a vial counter.


      Figure 2. P81 paper spotting. A. Prepare 2 x 2 cm P81 papers labeled with pencil marks. B. Spot each paper with 10 µl the supernatant of the reaction mixture after centrifugation (Step D11). Notice that the washing is being performed behind the protecting shield in a radioisotopes room. C. Place one by one all the papers in the same beaker with 1 L of 5 mM ortho-phosphoric acid (Step D11). D. Let all pieces to air dry without touching each other.

Data analysis

On an Excel file, write down counts per minute (CPM) values of each condition, obtained in step D15 (Table 2). These measures correspond to the 10 µl of the supernatant of the reaction mixture spotted on P81 paper.

Table 2. In vitro PKA kinase assay data analysis. Schematic view of steps A-H for data processing.

  1. Calculate the mean value (mean) for each condition considering the experimental triplicates.
  2. Obtain the corrected mean by subtracting the negative control mean from all means, except for the Kinase buffer mean.
  3. Adjust the mean CPM value of each condition to the total volume of the assay (40 µl) by multiplying the corrected mean by 4, since you put 10 µl on the P81 papers out of 40 µl.
  4. Use the Kinase buffer mean CPM to determine the total amount of ATP-32Pγ present in each reaction tube. This will be used to address the amount of 32P incorporated into the Kemptide. To do this, first multiply the mean CPM value of the Kinase buffer by the dilution factor (in this case it is a factor of 10 since you spotted 1 µl of Kinase buffer out of 10 µl present in the reaction).
  5. To determine how many [CPM/ρmol ATP], noted as “b” in Table 2, are in the mixture, divide by the total ATP moles added (in this case the reaction mixture contained 800 pmol of ATP, noted as “a” in Table 2).
  6. To determine how many ρmoles of ATP were incorporated in each condition, divide total CPM of each condition by [CPM/ρmol ATP] of the mixture.
  7. To determine how many ρmoles of ATP were incorporated in each condition per minute, divide this last value by the time of the reaction. In this case divide by 30, since the reaction lasted 30 minutes.
  8. Report this value considering the amount of sperm used for each condition. Please see Table 3 for an example of results.

    Table 3. Example of results. The table exemplifies results obtained using the PKA inhibitor H-89 (Sigma #B1427, diluted in DMSO), at the concentrations specified.
    Note: In this example, all tubes contained cAMP and IBMX (see Table 1).

Recipes

  1. H-TYH medium
    General composition:
    119.3 mM NaCl
    4.7 mM KCl
    0.8 mM Na-Pyruvate
    1.71 mM CaCl2
    1.2 mM KH2PO4
    1.2 mM MgSO4
    5.4 mM glucose
    20 mM HEPES
    pH = 7.2-7.4
    To prepare 25 ml of buffer, weight:
    NaCl                  174.3 mg
    KCl                     8.76 mg
    CaCl2·2H2O      6.28 mg
    KH2PO4            4.08 mg
    MgSO4·7H2O   7.39 mg
    HEPES               119.15 mg
    1. Dissolve all reagents in Mili-Q water to a final volume of 25 ml
    2. Sterilize by filtration using a 0.45 µm filter and store at 4 °C up to 1 month
    3. On the day of use, add 2.20 mg Sodium Pyrutave and 24.78 mg Glucose
    4. Bring to a final pH of 7.2-7.4 with freshly prepared 5 N NaOH
  2. Lysis buffer (prepare day of use)
    General composition:
    25 mM Tris pH 7.4
    150 mM NaCl
    1x Cocktail protease inhibitor (EDTA-free, SIGMA)
    1% Triton X-100
    To prepare 900 µl of buffer:
    1 M Tris, pH 7.4                                  22.5 µl
    3 M NaCl                                              45 µl
    25x Cocktail protease inhibitor        36 µl
    Triton X-100                                         9 µl
    Distilled water                                    787.5 µl
  3. 100 mM IBMX
    Dissolve 22.22 mg in 1 ml of DMSO
  4. Kinase buffer
    Prepare 3x Kinase buffer as it will be diluted 3 times by addition of 1 volume (10 µl) of PKA fraction plus 1 volume of buffer solution (10 µl), accounting for 30 µl of reaction tube (see Table 1). The following volumes are calculated to prepare the amount of buffer needed for 30 reaction tubes (10 different conditions in triplicates), considering that 10 µl of Kinase buffer is added to each tube.

Acknowledgments

The work was funded by Agencia Nacional de Promoción Científica y Tecnológica, PICT 2015-3164 and 2017-3217 awarded to DK. The heptapeptide known as Kemptide was synthesized for the first time by Kemp et al, to study the specificity of the PKA towards its substrate (Kemp et al., 1977). The adapted protocol described in that manuscript has used for the first time by Visconti et al. (1997)

Competing interests

The authors declare no competing interests.

Ethics

Cauda epididymides were collected from C57BL/6 young adult male mice (8-20 weeks old) and sacrificed under supervision of the Animal Care and Use Committee of the Facultad de Ciencias Bioquímicas y Farmacéuticas de Rosario (UNR) (approved protocol numbers 7298/532).

References

  1. Akamine, P., Madhusudan, Wu, J., Xuong, N. H., Ten Eyck, L. F. and Taylor, S. S. (2003). Dynamic features of cAMP-dependent protein kinase revealed by apoenzyme crystal structure. J Mol Biol 327(1): 159-171.
  2. Buffone, M. G., Wertheimer, E. V., Visconti, P. E. and Krapf, D. (2014). Central role of soluble adenylyl cyclase and cAMP in sperm physiology. Biochim Biophys Acta 1842(12 Pt B): 2610-2620.
  3. Kemp, B. E., Graves, D. J., Benjamini, E. and Krebs, E. G. (1977). Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase. J Biol Chem 252(14): 4888-4894.
  4. Krapf, D., Arcelay, E., Wertheimer, E. V., Sanjay, A., Pilder, S. H., Salicioni, A. M. and Visconti, P. E. (2010). Inhibition of Ser/Thr phosphatases induces capacitation-associated signaling in the presence of Src kinase inhibitors. J Biol Chem 285(11): 7977-7985.
  5. Stival, C., Puga Molina Ldel, C., Paudel, B., Buffone, M. G., Visconti, P. E. and Krapf, D. (2016). Sperm capacitation and acrosome reaction in mammalian sperm. Adv Anat Embryol Cell Biol 220: 93-106.
  6. Stival, C., Ritagliati, C., Xu, X., Gervasi, M. G., Luque, G. M., Baro Graf, C., De la Vega-Beltran, J. L., Torres, N., Darszon, A., Krapf, D., Buffone, M. G., Visconti, P. E. and Krapf, D. (2018). Disruption of protein kinase A localization induces acrosomal exocytosis in capacitated mouse sperm. J Biol Chem 293(24): 9435-9447.
  7. Visconti, P. E., Moore, G. D., Bailey, J. L., Leclerc, P., Connors, S. A., Pan, D., Olds-Clarke, P. and Kopf, G. S. (1995). Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Development 121(4): 1139-1150.
  8. Visconti, P. E., Johnson, L. R., Oyaski, M., Fornes, M., Moss, S. B., Gerton, G. L. and Kopf, G. S. (1997). Regulation, localization, and anchoring of protein kinase A subunits during mouse sperm capacitation. Dev Biol 192(2): 351-363.
  9. Zhang, P., Smith-Nguyen, E. V., Keshwani, M. M., Deal, M. S., Kornev, A. P. and Taylor, S. S. (2012). Structure and allostery of the PKA RIIbeta tetrameric holoenzyme. Science 335(6069): 712-716.

简介

[Abstract]为了获得受精潜能,哺乳动物的精子必须经历一个被称为电容的过程,这个过程依赖于蛋白激酶A(PKA)的早期激活。通常,在全细胞实验中,通过分析其底物在western-blot中的磷酸化状态来评估PKA的活性。这种技术面临着两个主要的缺点:它不是对激酶活性的直接测量,而且是一种耗时的方法。然而,由于PKA可以很容易地从精子提取物中获得,体外检测如"放射性检测"可以使用原生酶进行。与western-blot不同的是,放射性测定法是一种直接的技术,通过将整合的32P定量到肽类底物中来评价PKA的活性。这种方法很容易允许分析PKA的不同激动剂或拮抗剂。由于小鼠精子是可溶性PKA的丰富来源,这种测定法可以进行简单的分馏,使PKA既可用于体外测试药物对PKA活性的影响,也可用于跟踪PKA活性在增容开始时的变化。

[Background] 哺乳动物的精子不能在射精后立即使卵母细胞受精。为了获得受精能力,它们必须经历一系列的细胞变化,统称为增容(Stival等,2016)。这一过程发生在女性生殖道内,但可以在实验室中通过将精子在含有Ca2+、白蛋白和HCO3-的规定培养基中进行模拟,即"增容培养基"(Visconti等,1995)。HCO3-和Ca2+协同作用于可溶性腺苷酸环化酶(sAC),产生细胞内cAMP水平的升高。在不同的靶点中,cAMP直接激活Ser/Thr蛋白激酶A(PKA),它作为协调电容信号事件的核心参与者(Buffone等,2014)。通常,可以使用两种不同的方法来分析其活性。其中一种依赖于western-blots,使用市售抗体检测PKA的共识磷酸化序列(Krapf等,2010)。然而,任何蛋白质的稳态磷酸化状态都取决于作用于它的激酶和磷酸酶的相对活性。因此,这种方法不能直接分析实际的PKA活性。第二种方法涉及通过直接定量测量体外PKA活性,在含有磷酸酶抑制剂的受控反应混合物中,将32P纳入肽类底物中(Stival等,2018)。这允许分析PKA独立于可能调节底物磷酸化状态的其他因素。命名为Kemptide的多肽底物的化学性质,以1977年首次合成它的Kemp博士的名字命名(Kemp等,1977),包括一个可磷酸化的丝氨酸残基和在-3和-2位置上的两个精氨酸,允许高PKA特异性。此外,Kemptide具有高的相对正电荷,这说明它与带负电荷的Whatman P81纤维素纸有很强的结合力,简化了多余放射性物质的洗涤(Kemp et al. , 1977)。酶PKA由两个调节亚单位和两个催化亚单位组成(Akamine等,2003;Zhang等,2012)。这两种亚基都可以在小鼠精子的Triton X-100可溶性和-不溶性部分中找到(Visconti et al. , 1997)。在精子增容过程中,可溶性部分的PKA活性增加。然而,留在不溶性部分的PKA的活性在增容过程中不会发生变化,因此,它是酶学研究中PKA的极好来源(Visconti等,1997)。
这里描述的协议是用来分析激动剂或拮抗剂对PKA活性的影响,使用小鼠精子作为激酶的来源,降低成本,同时保持高效率。然而,这个协议可以很容易地调整,以分析PKA活性的变化在精子增容的过程中,通过使用总的未分馏精子提取物。在这方面,Kemptide的磷酸化反映了PKA在增容的任何阶段的给定活性。
最后,PKA的其他来源也可以使用,例如从质粒表达中纯化的PKA。

关键字:蛋白激酶A(PKA), 精子获能, 精子, 激酶活性测定, 受精

材料和试剂


注:除非特别说明,所有试剂均在室温下保存(RT,15-25℃)。
1. P81 Whatman纤维素层析纸(Sigma-Aldrich,目录号:Z753645),2 x 2厘米一张。
注:应该有尽可能多的方块条件,加上9个额外的方块控制,所有的一式三份。所有的碎片都应该用铅笔标明,以便在清洗后将它们区分开来。
2. 7 毫升共聚物塑料瓶,无衬里白色聚乙烯螺旋帽(RPI,目录号:125509)。
3. Eppendorf管(1.5毫升和2毫升)
4. 0.45 µm 过滤器
5. 雄性小鼠(8-20周)
6. 70%乙醇
7. 三(羟甲基)氨基甲烷(Tris 碱)(Cicarelli,目录号:1131214)
8. Triton X-100 (Neo Lab,目录号:01685)
9. 氯化钠(Cicarelli,目录号:750)
10. 氯化镁(Sigma-Aldrich,目录号:M8266)
11. 腺苷5′-三磷酸二钠水合物(ATP)(Sigma-Aldrich,目录号:A7699),-20℃保存。
12. 正钒酸钠(Na3VO4)(Sigma-Aldrich,目录号:450243),储存于-20℃。
13. β-甘油磷酸二钠水合物(G3P)(Sigma-Aldrich,目录号:G6251),在-20℃保存。
14. 对硝基苯磷酸酯(NPP)(开曼化学品,目录号:400090)
15. cOmpleteTM,不含EDTA的蛋白酶抑制剂鸡尾酒(蛋白酶鸡尾酒)(罗氏,目录号:4693132001),4℃保存。
16. 不含脂肪酸的牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906),4℃保存。
17. 腺苷3′,5′-环一磷酸钠盐一水合物(cAMP)(Sigma-Aldrich,目录号:3A6885),也可使用8-BrcAMP或db-cAMP,储存于-20℃。
18. 3-异丁基-1-甲基黄嘌呤(IBMX)(Sigma-Aldrich,目录号:I5879),储存于-20℃。
19. Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly)(Sigma-Aldrich,目录号:K1127),储存于-20℃。
20. HEPES(Sigma-Aldrich,目录号:H3375)
21. 三氯乙酸(TCA)(BioChemica,目录号:PAA1431)。
22. 正磷酸(默克公司,目录号:100573)
23. ATP(ATP-32Pγ)3000 Ci/mmol;10 mCi/ml(Perkin Elmer,目录号:BLU502A)。
24. 实验中要测试的具体药物(抑制剂、激动剂等)。
25. KCl(Sigma-Aldrich,目录号:S-7653)
26. 丙酮酸钠(Sigma-Aldrich,目录号:P-4562)
27. CaCl2-2H2O(Sigma-Aldrich,目录号:C-7902)。
28. KH2PO4(Sigma-Aldrich,目录号:P-5655)
29. MgSO4-7H2O(Sigma-Aldrich,目录号:63138)
30. 葡萄糖(Sigma-Aldrich,目录号:G-6152)
31. DMSO(Sigma-Aldrich,目录号:D2650)


解决办法
关于如何制备以下溶液的详细说明,请参见配方标题。
1. 1 M Tris pH 7.4
2. 3 M氯化钠
3. 1 MgCl2
4. 1 mM ATP
5. 100 mM Na3VO4
6. 800 mM 甘油醛-3-磷酸(G3P)
7. 500mM对硝基苯磷酸酯(NPP)
8. 50 mg/ml BSA
9. 5mM Kemptide
10. 200mM HEPES,pH 7.3。
11. 40%三氯乙酸
12. 5mM正磷酸
13. H-TYH中号(见食谱)
14. 裂解缓冲液(见配方)
15. 100 mM IBMX (见配方)
16. 激酶缓冲液(见配方


装备


1. 闪烁计数器(LKB Wallac Rackbeta 1209,目录号:090161)
2. 诺伊鲍尔室
3. 手术剪刀(WPI,目录号:14393)
4. 镊子
5. 轨道摇动
6. 孵化器
7. 离心机


軟件


1. Microsoft® Office Excel (Microsoft Office 365),或类似的软件。


程序e


A. 准备裂解缓冲液(配方2)在使用当天。留下的蛋白酶抑制剂鸡尾酒,在使用前加入。保持缓冲液在冰上。


B. 计算将需要的Kinase缓冲液的体积,考虑到每个反应管10微升,并考虑到每个条件(和控制)将在一式三份进行。虽然准备Kinase缓冲液(配方4)添加所有组件,但Kemptide,蛋白酶抑制剂,和ATP-32Pγ,这将是使用前添加。


C. 从小鼠精子提取物中分离出不溶性的PKA部分。
注:本节介绍了如何从小鼠精子提取物的Triton-不溶性部分中获得PKA,以作为PKA在活性测定中的来源。
1. 安乐死1雄性小鼠(8-20周龄),并进行手术,从两个表皮(图1)提取尾巴区域。要做到这一点。
a. 躺在解剖板上的安乐死鼠标背部。
b. 用70%的乙醇喷洒腹腔,防止干发脱落。
c. 使用剪刀,从腹壁切开皮肤和肌肉组织,露出下腹内脏(图1A)。
d. 使用镊子,去除覆盖睾丸的脂肪垫,使附睾暴露(图1A)。
e. 识别附睾连接到睾丸,切除它,并定位尾部区域( 图1B,红色区域)。从caput和corpus区域(图1B灰色区域),和脂肪组织(图1B蓝色区域)删除它。
f. 虽然用镊子夹住尾巴,执行3至4个切口,使用手术剪刀(图1C)。
2. 将这两个表皮在一个圆底2毫升Eppendorf含有500微升的H-TYH培养基( 图1D),并在37℃下孵育15分钟。




图1.精子制备。精子准备。A.腹侧切口切除附睾。注意镊子持有尾部附睾,连接到睾丸(红圈)。B.整个附睾切除。红色,尾巴;灰色,caput和语料库;蓝色,脂肪组织。尾部区域用于精子游出。C.在让精子游出之前,应在每条尾巴上做三到四个切口。D.将两个附睾放在一个含有500微升H-TYH培养基的圆底Eppendorf管中。


3. 将精子悬浮液(500微升)转移到一个新的圆底2毫升Eppendorf管中,留下附睾。
4. 检查精子浓度通过计数在Neubauer室。在H-TYH培养基中调整浓度至25×106精子/ml。
5. 转移游出到1.5毫升Eppendorf管。
6. 将精子悬浮液在10,000 xg离心,RT下3分钟
7. 丢弃上清液。如果游出的精子量为1毫升中的25×106个,则在使用前将颗粒重悬于330微升补充有蛋白酶抑制剂鸡尾酒的冰冷裂解缓冲液中。如果使用不同量的精子,相应修改缓冲液的添加量。
8. 在冰上裂解缓冲液中孵育细胞30分钟。
9. 离心机细胞在10,000 xg离心10分钟,在4℃。
10. 弃去上清液,将颗粒重悬于330微升的裂解缓冲液中(或步骤C7中使用的相同量),并加入新的蛋白酶抑制剂鸡尾酒。该部分含有PKA。每个条件与10微升的馏分将有可溶性PKA馏分从7.5×105精子提取。考虑到每个条件至少需要1.5×105个精子,应一式三份进行测试。
11. 保存在冰上,用于PKA活性检测。从现在起这部分称为PKA馏分。


D. 激酶反应检测
1. 考虑尽可能多的管,牢记每个条件应在实验三倍,加上以下控制进行。
a. 阴性对照:含有除PKA部分以外的所有反应试剂,PKA部分由裂解缓冲液代替。它用于确定在没有PKA的情况下被32P标记的Kemptide的量。
b. 阳性对照:它是一个最大PKA活性的控制,即 ,在1mM cAMP和100μM IBMX的存在。
2. 加入10微升含有任何待分析药物的裂解缓冲液。考虑到在反应时它将被稀释3倍。不管是什么药物,都必须在加入这10微升裂解缓冲液对应的10微升内考虑。
3. 向每个试管中加入10微升PKA馏分,但阴性对照除外,在阴性对照中,PKA馏分应以10微升裂解缓冲液代替。
4. 在使用之前,所有的管子都要放在冰上。


从步骤D5开始,这些程序必须在放射性同位素室进行。请注意,在处理放射性材料时必须采取安全措施。必须对人员进行适当的防护,并根据机构条例进行适当的废物处理。
5. 考虑到衰变时间和生产日期,加入适量的ATP-32Pγ(见表1),完成激酶缓冲液的制备,然后加入1μCi/检测。

表1.制备激酶缓冲液的原液和浓度。
试剂 反应管中的最终浓度(30微升)。 库存集中度 在3倍激酶缓冲液中的浓度。 3x激酶缓冲液的库存量(µl) 笔记
蒸馏水 - - 24.85 -
HEPES 25 mM 200mM,pH值7.3 75毫微米 131.25 -
G3P 40毫微米 800毫微米 120毫微米 52.5 -
BSA 1毫克/毫升 50毫克/毫升 3毫克/毫升 21 -
氯化镁 10 mM 1 M 30毫微米 10.5 -
氯化钠 100 µM 100 mM 300 µM 1.05 -
NPP 5 mM 500毫微米 15 mM 10.5 -
IBMX 0.1mM 100mM(在DMSO中) 0.3mM 1.05 -
cAMP 1 mM 1 M 3 mM 1.05 -
三磷酸腺苷 40 µM 1 mM 120 µM 42 *使用前添加
* 蛋白酶抑制剂 1x 25x 2x 28
*Kemptide 100 µM 5 mM 300 µM 21
**ATP-32Pγ 1 µCi/检测器 10 µCi/µl 3 µCi/检测器 5.25 **加在放射性同位素室。
总容量 350 -
注:
a. 除非另有规定,所有试剂均溶解于蒸馏水中。
b. 蛋白酶抑制剂鸡尾酒的加入是为了制备2倍浓缩缓冲液,因为PKA部分已经包含鸡尾酒。
c. 添加BSA是为了帮助沉淀TCA添加后的所有蛋白质,但保持Kemptide可溶性。


6. 依次加入10微升的Kinase缓冲液到所有管中,并将其放置在37℃。开始一个新的反应,每30秒加入10微升的Kinase缓冲液。追踪所有试管的顺序。这对于准确地在指定时间停止反应至关重要。
7. 孵育管在37℃下30分钟。
8. 30分钟后,加入10微升40%的TCA停止反应。请注意,现在每管中的总体积为40 µl。
9. 在冰上孵育管20分钟。
10. 在10,000 xg离心3分钟,在RT。
11. 对于每个管子,取10微升的上清液,并点到P81纸的中心,同时用镊子夹住它(图2A和2B)。每张P81纸都应该用铅笔标记,以便以后识别它们。等待5秒的下降被吸收,并将其释放在含有1L的5mM正磷酸( 图2C)的5L烧杯中。
12. 一起洗所有的纸6×5分钟,每次使用1L的5mM正磷酸,在烧杯中放置在一个轨道振动器上。
13. 当步骤D12中的洗涤进行时,在P81纸上点1微升的激酶缓冲液。使用3张不同的纸,一式三份,然后直接将每张纸插入不同的塑料瓶中,无需洗涤。这一步是必要的数据分析,以计算总ATP-32Pγ的总量,最初存在于反应介质中。
14. 洗涤后,将P81纸放在塑料托盘上,彼此之间不接触,让它们至少晾晒2小时(图2D)。在这一步,你可以停止协议,直到第二天。纸张的识别是根据铅笔标记写在他们身上,然后再点上反应混合物。
15. 干燥后,将纸单独放在塑料小瓶中,含有1毫升的闪烁计数器液体,并登记每分钟计数(CPM)一个小瓶计数器。




图2.P81纸斑。P81纸的斑纹。A.准备2×2厘米的P81纸,用铅笔标记。B.用10微升离心后的反应混合物的上清液斑点每张纸(步骤D11)。注意,洗涤是在放射性同位素室的保护罩后面进行。C.将所有的纸片逐一放入同一个烧杯中,加入1 L的5 mM正磷酸(步骤D11)。D.让所有的纸片晾干,不要相互接触。


数据分析


在Excel文件上,写下每个条件的每分钟计数(CPM)值,在步骤D15中获得(表2)。这些测量值对应于P81纸上斑点的10微升反应混合物上清液。


表2.体外PKA激酶检测数据分析。体外PKA激酶检测数据分析。数据处理步骤A-H的示意图。
1/10 激酶缓冲液控制 负面控制 积极控制 测试样本1 测试样本n
A B C D E
CPM改为1 1 A1 B1 C1 D1 E1
CPM改为2(重复) 2 A2 B2 C2 D2 E2
CPM读数3(一式三份) 3 A3 B3 C3 D3 E3
平均值 4 (A1+A2+A3)/3. (B1+B2+B3)/3。 (C1+C2+C3)/3。 (D1+D2+D3)/3。 (E1+E2+E3)/3。
更正后的平均值 5 - - C4-B4 D4-B4 E4-B4
稀释因子(df)=反应管中的总体积/P81上的斑点体积。 
筒子里的总CPM 6 A4 x 10 - C5 x df D5 x df E5 x df
A6的CPM代表加入反应介质中的ρmol ATP("a")。
混合物中含有"b"CPM/ρmol ATP,相当于(A4/"a")/1000。
反应过程中加入的ATP(ρmol) 7 - - C6/"b" D6/"b" E6/"b
每分钟反应中加入的ATP(ρmol/min) 8 - - C6/30 D6/30 E6/30


1. 考虑到实验三联,计算各条件的平均值(平均值)。
2. 通过减去阴性对照平均值,从所有的平均值,除了Kinase缓冲平均值,获得修正的平均值。
3. 将每个条件的平均CPM值调整到检测的总体积(40微升),将校正后的平均值乘以4,因为您将40微升中的10微升放在P81纸上。
4. 使用激酶缓冲液平均CPM来确定每个反应管中存在的ATP-32Pγ的总量。这将用于解决32P纳入Kemptide的量。要做到这一点,首先将Kinase缓冲液的平均CPM值乘以稀释因子(在这种情况下,它是一个10的因素,因为你发现1微升的Kinase缓冲液中的10微升存在于反应中)。
5. 要确定混合物中的[CPM/ρmol ATP]有多少,在表2中记为"b",除以加入的ATP总摩尔(在本例中反应混合物含有800pmol ATP,在表2中记为"a")。
6. 要确定每个条件中加入了多少ρ摩尔ATP,将每个条件的总CPM除以混合物的[CPM/ρmol ATP]。
7. 要确定每分钟在每个条件下加入多少ρ摩尔ATP,将最后一个值除以反应时间。在这种情况下,除以30,因为反应持续了30分钟。
8. 考虑到每种情况下的精子使用量,报告这个值。结果的例子请见表3。


表3.结果的例子。该表举例说明了使用PKA抑制剂H-89(Sigma #B1427,在DMSO中稀释),在指定浓度下获得的结果。
注:在这个例子中,所有管子都含有cAMP和IBMX(见表1)。
1/10 激酶缓冲液控制 负面控制 H89 (μM)
  0(阳性对照) 0.1 1 10 50 100
CPM改为: 复制1 275374 10172 303106 321092 239832 44063 12470 11207
复制2 285443 11604 333105 327384 209389 48771 10125 12164
复制3 239039 10563 313784 32639 219980 43795 12634 10993
卑鄙 266618.67 10779.67 316665.00 227038.33 223067.00 45543.00 11743.00 11454.67
平均值----消极控制 0.67 305886.00 216259.33 212288.00 34764.00 964.00 675.67
中央方案管理费总额(40微升中的10微升) 1223544.00 865037.33 849152.00 139056.00 3856.00 2702.67
800摩尔的ATP代表2666190CPM。 
因此,混合物含有3332.7 CPM/pmol ATP。
反应中加入的ATP的量(pmol) 367.13 259.56 254.79 41.72 1.16 0.81
每分钟反应中加入的ATP的摩尔数(pmol/min) 12.24 8.65 8.49 1.39 0.04 0.03
由于每管含有2.40E5个精子,那么反应中加入的ATP(pmol/min/5E6sperm)。 254.91 180.22 176.91 28.97 0.80 0.56




食谱


1. H-TYH中号
一般组成:
119.3mM氯化钠
4.7mM KCl
0.8mM丙酮酸钠。
1.71mM CaCl2
1.2mM KH2PO4
1.2mM MgSO4
5.4mM葡萄糖
20mM HEPES
pH值=7.2-7.4
制备25毫升的缓冲液,重量。
氯化钠 174.3毫克
氯化钾 8.76毫克
CaCl2- 2H2O6.28毫克
KH2PO44.08毫克
MgSO4- 7H2O7.39毫克
HEPES119.15毫克
a. 将所有试剂溶解在Mili-Q水中,最终体积为25毫升。
b. 使用 0.45 µm 过滤器过滤消毒,在 4 °C 下保存 1 个月。
c. 用药当天,加入2.20毫克吡鲁特韦钠和24.78毫克葡萄糖。
d. 用新制备的5N NaOH使最终pH值达到7.2-7.4。
2. 裂解缓冲液(使用当天准备)
一般组成:
25mM Tris pH 7.4
150mM氯化钠
1x 鸡尾酒蛋白酶抑制剂(不含EDTA, SIGMA)
1% Triton X-100
准备900微升的缓冲液。
1 M Tris, pH 7. 422.5 µl
3 M氯化钠 45 µl
25x 鸡尾酒蛋白酶抑制剂 36 µl
Triton X- 1009 µl
蒸馏水 787.5微升
3. 100 mM IBMX
将22.22毫克溶解在1毫升的DMSO中。
4. 激酶缓冲液
准备3倍的Kinase缓冲液,因为它将被稀释3倍,加入1体积(10微升)的PKA部分加上1体积的缓冲液(10微升),占30微升的反应管(见表1)。考虑到10微升的Kinase缓冲液加入到每个管中,计算出以下体积,以准备30个反应管所需的缓冲液量(10个不同条件的三联)。


鸣谢


这项工作由Agencia Nacional de Promoción Científica y Tecnológica资助,PICT 2015-3164和2017-3217授予DK。被称为Kemptide的七肽由Kemp等人首次合成,以研究PKA对其底物的特异性(Kemp et al., 1977)。在该手稿中描述的改编协议已由Visconti等人(1997年)首次使用。


公司利益


提交人声明没有竞争利益。


职业道德


从C57BL/6年轻成年雄性小鼠(8-20周龄)收集尾状表皮,并在罗萨里奥生物科学和药物学院(UNR)动物护理和使用委员会的监督下进行牺牲(批准协议号7298/532)。


参考文献


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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Stival, C., Baro Graf, C., Visconti, P. E. and Krapf, D. (2020). Quantification of Protein Kinase A (PKA) Activity by An in vitro Radioactive Assay Using the Mouse Sperm Derived Enzyme. Bio-protocol 10(12): e3658. DOI: 10.21769/BioProtoc.3658.
  2. Stival, C., Ritagliati, C., Xu, X., Gervasi, M. G., Luque, G. M., Baro Graf, C., De la Vega-Beltran, J. L., Torres, N., Darszon, A., Krapf, D., Buffone, M. G., Visconti, P. E. and Krapf, D. (2018). Disruption of protein kinase A localization induces acrosomal exocytosis in capacitated mouse sperm. J Biol Chem 293(24): 9435-9447.
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