JP2008079610A - Measuring method of phosphorylation enzyme activity and dephosphorylation enzyme activity of protein kinase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a measuring method capable of accurately, quickly and simply measuring phosphorylation or dephosphorylation enzyme activity of protein kinase. <P>SOLUTION: The enzyme activity measuring method of protein kinase comprises reacting a substrate peptide with protein kinase in the presence of metal colloid, or reacting the substrate peptide with protein kinase and then adding the metal colloid, and measuring the enzyme activity due to phosphorylation of protein kinase based on color tone change of the obtained reaction liquid. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for measuring phosphorylase activity and phosphatase activity of protein kinases. The present invention further relates to a method for screening a compound that promotes or inhibits the kinase activity of protein kinase.

All the cells of the organism take in information from the outside, and in response to the information, the cells start to proliferate and differentiate. Information from the outside is transmitted through humoral factors secreted by other cells and contact between cells. The information transmitted in this way is sensed by a receptor possessed by the cell, and the signal is transmitted through the cell to cause various responses with gene expression. It is known that phosphorylation of many proteins is involved in the process of information transmission in cells.
Such phosphorylation of proteins is performed by the action of protein phosphorylase (hereinafter also referred to as “protein kinase” or simply “kinase”). Protein phosphorylation by this protein kinase plays an extremely important role not only in cell signal transduction but also in most metabolic pathways (see, for example, Non-Patent Document 1). In other words, protein kinases are involved in the regulation of cell proliferation and differentiation.
For example, if DNA is damaged by UV irradiation or drugs, causing abnormalities in protein kinase expression and other functions, it is a matter of course that protein phosphorylation by protein kinases is abnormal, and cell communication alone However, abnormalities will occur in most metabolic pathways, resulting in diseases such as cancer. Thus, if this kinase dysfunction can be detected accurately and simply, it is highly expected to help elucidate such diseases.

  As a method for detecting protein kinase dysfunction, a method for measuring enzyme activity of protein kinase is employed, and for example, radioimmunoassay (RIA), enzyme immunoassay (ELISA), etc. are used. (For example, refer nonpatent literature 1, patent documents 1 and 2).

  However, since the radioimmunoassay method handles radioactive substances, it is necessary to pay close attention to the handling thereof, and since a special facility is required, it is difficult to measure easily. On the other hand, the enzyme immunoassay method is usually a method in which a synthetic peptide is used as a substrate and the enzyme phosphorylation is detected mainly using a fluorescent dye for an antibody that recognizes the phosphorylated peptide. There are disadvantages in that the reaction efficiency between the substrate peptide to be bound to the phase and the protein kinase is not good, and phosphorylation may not be performed sufficiently (for example, see Non-patent Document 1 and Patent Document 2). Therefore, there is a need for a measurement method that further improves the conventional methods such as the radioimmunoassay method and the enzyme immunoassay method.

Therefore, as such an improved method, a method using colloidal particles such as a metal such as gold has been proposed (for example, see Non-Patent Documents 1 and 2). Metal colloidal particles have useful material properties derived from their nanoparticle size and have been proposed for application to chemical sensors, microimaging, and the like. At present, control techniques for metal colloidal particles and substances have been developed, and a method for reversibly forming colloidal particles and substances into useful aggregates and substances has been developed (for example, non-patented). Reference 2). As these methods, a method of immobilizing a substrate peptide of protein kinase to a metal colloid is also known (see, for example, Non-Patent Document 1 and Patent Document 3). However, since this method uses an extremely expensive ATP derivative such as biotin-ATP, the measurement cost becomes abnormally high, and the reaction between the substrate peptide-metal particle conjugate and the protein kinase causes the reaction. There is a problem of low efficiency.
Wang, Z., etal .: J. Am. Chem. Soc. 2006, 128, 2214-2215 Mirkin, CA, et al .: Nature 1996,382, 607-609 Japanese Patent Laid-Open No. 10-48217 JP 2000-325086 A JP 08-278306 A

  As a result of various studies to improve the prior art, the present inventors have improved the reactivity of protein kinase by reacting a substrate peptide and protein kinase in the presence of a metal colloid such as gold nanoparticles. In addition, the present inventors have found that the protein kinase enzyme activity can be measured based on the change in color of the metal colloid, and the present invention has been completed.

  Therefore, the present invention, as one aspect thereof, reacts a substrate peptide and a protein kinase in the presence of a metal colloid such as a gold nanoparticle, or after reacting a substrate peptide and a protein kinase. A protein kinase consisting of measuring the enzyme activity of a protein kinase based on the color change depending on the degree of aggregation of the substrate peptide and the metal colloid particles by adding metal colloid and phosphorylating or dephosphorylating protein kinase in the sample It aims at providing the enzyme activity measuring method of this.

  The present invention relates to a method for measuring phosphatase activity comprising measuring a phosphatase activity of a phosphatase by dephosphorylating a phosphatase by dephosphorylating a substrate peptide phosphorylated by phosphorylation of a protein kinase Another purpose is to provide.

  Furthermore, another object of the present invention is to provide a method of use consisting of screening for compounds that promote or inhibit phosphorylation or dephosphorylation of a substrate peptide.

  Another object of the present invention is to provide a measurement kit that can be used in a method for measuring the phosphorylation or dephosphorylation enzyme activity of protein kinase as another aspect thereof.

  In order to achieve the above object, the present invention is directed to reacting a substrate peptide with a protein kinase in the presence of a metal colloid such as a gold nanoparticle or after reacting a substrate peptide with a protein kinase. Provided is a method for measuring the enzyme activity of a protein kinase, which comprises adding a colloid and measuring the enzyme activity due to phosphorylation of the protein kinase based on the color change of the metal colloid.

  The present invention also provides a method for measuring phosphatase activity by dephosphorylating a substrate peptide phosphorylated by a protein kinase.

  Furthermore, the present invention provides a method of use consisting of screening for compounds that promote or inhibit phosphorylation or dephosphorylation of a protein kinase substrate peptide.

  The present invention also provides a measurement kit that can be used in a method for measuring protein kinase phosphorylation or phosphatase activity.

In the present invention, phosphorylation reaction is achieved by using only ATP which is generally used, compared to conventional methods such as a measurement method using an expensive reagent called biotin-ATP and a measurement method based on an antigen-antibody reaction. In addition, since it is not necessary to use an antibody by using a metal colloid, the cost is low and the kinase reactivity with the substrate peptide can be improved.
The present invention also has an effect that the dephosphorylation of the substrate peptide phosphorylated by the protein kinase can be easily performed.
Furthermore, this invention also has the effect that screening of compounds that promote or inhibit phosphorylation by protein kinases can be performed easily and simply.
Furthermore, the present invention provides a measurement kit that can measure phosphorylation or dephosphorylation of a substrate peptide by protein kinase, thereby making it possible to easily and easily measure phosphorylation or dephosphorylation by protein kinase. There is an effect that can be performed.

  According to this invention, the kinase activity of protein kinase can be measured by detecting the phosphorylation level of a substrate peptide phosphorylated by a protein kinase contained in a sample. More specifically, the present invention relates to the phosphorylation of a substrate peptide by a protein kinase in the presence of metal colloidal particles when the substrate peptide is phosphorylated by a protein kinase present in a sample, or After reacting with protein kinase, the phosphorylase activity of protein kinase is measured by detecting the change in its phosphorylation level by adding metal colloid.

  That is, in the present invention, first, a measurement sample containing a protein kinase is prepared in a reaction medium by a substrate peptide, a phosphate group-donating substrate that donates a phosphate group as a component necessary for the phosphorylation reaction, and a gold Incubate with metal colloids. Examples of the reaction medium include a buffer made of a mixture of HEPES and magnesium chloride. As the phosphate group-donating substrate, for example, adenosine triphosphate (ATP) can be used.

  The presence of the metal colloid in the phosphorylation or dephosphorylation reaction of the substrate peptide by the protein kinase causes aggregation of the phosphorylated substrate peptide due to the intervention of the metal colloid particles, and this aggregation changes the color of the metal colloid. The color tone of the metal colloid varies depending on the degree of aggregation substantially proportional to the phosphorylation rate of the phosphorylated substrate peptide.

Therefore, the phosphorylase activity or the dephosphorase activity can be measured substantially quantitatively by comparing the change in the color tone with the change in the color tone by the control. More specifically, as a control, various protein kinases with known phosphorylation ability were used to phosphorylate the substrate peptide in the presence of metal colloid, and the degree of phosphorylation of the substrate peptide, that is, aggregation of the substrate peptide The result of measuring the change in the color tone of the metal colloid depending on the ratio can be used. Compared with this control result, the enzyme activity of protein kinase can be measured substantially quantitatively by detecting the degree of color tone change of the metal colloid due to phosphorylation of protein kinase in the sample.
In addition, the dephosphorylation activity of the substrate peptide phosphorylated by protein kinase is substantially reduced by comparing the color of the metal colloid based on the aggregation ratio of the dephosphorylated substrate peptide with the color of the control result. It can be measured similarly.

  Although it does not specifically limit as a protein kinase which can be used for this invention, For example, PKC (protein kinase C), PKA (cyclic AMP dependence protein kinase), cdc2 kinase, raf-1 kinase, Examples include protein kinases such as MAP kinase, CaMK, Rho kinase, and MAPKAP.

  The substrate peptide that can be used in the present invention depends on the protein kinase to be measured, but is not particularly limited. For example, when the protein kinase is PKA, it is kemptide (SEQ ID NO: 1) or PKC. In the case of PKC64 (SEQ ID NO: 2), in the case of CaMK, the peptide (SEQ ID NO: 3) may be used, and in the case of MAPKAP, the peptide (SEQ ID NO: 4) may be used.

  On the other hand, examples of the metal colloid include gold colloid, silver colloid, platinum colloid, iron colloid, iron hydroxide colloid, aluminum hydroxide colloid, palladium colloid, and rhodium colloid. Of these metal colloids, gold colloid and silver colloid are preferred, and gold colloid is more preferred.

  These metal colloids can be produced by methods described in the literature, and commercially available products can also be used. For example, gold nanoparticles can generally be produced by a liquid phase reduction method in which a reducing solution is added to a solution containing gold ions to reduce the gold ions (see, for example, JP-A-2003-253310, JP No. 2006-152438).

  The particle size of these metal colloids is not particularly limited, but may be, for example, about 1 nm to 500 nm, preferably 3 nm to 200 nm, more preferably 5 nm to 100 nm. Moreover, although a metal colloid is based also on the particle size, a gold colloid shows red and a silver colloid shows yellow.

  According to the present invention, for example, the substrate peptide is phosphorylated by a protein kinase, and the substrate peptide aggregates due to the addition of the gold colloid, so that the color of the gold colloid changes from red to blue. Based on this change in color tone, the enzyme activity of protein kinase can be measured.

  The change in color of the metal colloid is generally examined by measuring the absorbance in the range of about 600 nm to 800 nm, preferably about 600 nm to 750 nm, more preferably about 700 nm.

  According to another aspect of the present invention, the enzyme activity of a dephosphorylation enzyme for a phosphorylated substrate peptide phosphorylated by a protein kinase can also be measured. That is, in the dephosphorylating enzyme activity measurement method according to the present invention, when a phosphorylated substrate peptide that is phosphorylated by a protein kinase in the presence of a metal colloid, the substrate peptide is dephosphorylated by a dephosphorylating enzyme, According to this dephosphorylation rate, the aggregation rate of the substrate peptide changes, and at the same time, the color tone of the metal colloid also changes, and the dephosphorylating enzyme activity is measured based on this change in color tone.

  In the present invention, the measurement of phosphatase activity based on the change in the color tone of the metal colloid is performed by comparing the change in the color tone of the metal colloid according to the measurement result of the control, similarly to the measurement of the kinase activity of the protein kinase. be able to.

  Examples of dephosphorylating enzymes that can be used in the present invention include Escherichia coli alkaline phosphatase, bovine intestinal alkaline phosphatase, and protein phosphatases such as human Cdc25 and Cdc14.

  By using the measurement method according to the present invention, it is possible to screen for compounds that promote or inhibit phosphorylation of protein kinase. In other words, a compound that promotes or inhibits phosphorylation of protein kinase promotes or inhibits phosphorylation of the substrate peptide by protein kinase compared to a phosphorylation reaction system consisting of protein kinase, substrate peptide, and metal colloid alone. In turn, this makes it possible to screen for such promoting or inhibiting compounds.

  On the other hand, the presence of a phosphorylation-inhibiting compound naturally inhibits phosphorylation by protein kinase as compared to a phosphorylation reaction system consisting only of protein kinase, substrate peptide, and metal colloid. The degree of phosphorylation promotion due to the presence of the phosphorylation-inhibiting compound can be measured by comparing with the change in the color tone of the phosphorylation reaction system in the absence of the phosphorylation-inhibiting compound. Examples of such phosphorylation-inhibiting compounds include H-89 as a PKA inhibitor, Ro31-8220 as a PKC inhibitor, and KN62 as a CaMK II inhibitor.

  Conversely, compounds that promote or inhibit dephosphorylation can be screened in a substantially similar manner. That is, the presence of a dephosphorylation promoting compound naturally promotes dephosphorylation of a phosphorylated substrate peptide phosphorylated by a protein kinase, a substrate peptide, and a metal colloid. The degree of promotion of dephosphorylation by the presence of this dephosphorylation promoting compound can be measured by comparing with the change in the color tone of the dephosphorylation reaction system in the absence of the dephosphorylation promoting compound. On the other hand, the presence of a dephosphorylation inhibiting compound naturally inhibits the dephosphorylation of the phosphorylated substrate peptide phosphorylated by the protein kinase, the substrate peptide, and the metal colloid. The degree of inhibition of dephosphorylation due to the presence of this dephosphorylation inhibiting compound can be measured by comparing with the change in color tone of the dephosphorylation reaction system in the absence of the dephosphorylation inhibiting compound.

  The measurement kit according to the present invention can quickly and easily measure the phosphorylation or dephosphorylation enzyme activity of a protein kinase. When such a kit is used, for example, to measure the kinase activity of protein kinase, it is preferable that the kit comprises at least a substrate peptide, a metal colloid, and a solvent such as a buffer as a reaction medium. . On the other hand, when such a kit is used, for example, for measuring the dephosphorylating enzyme activity of protein kinase, it comprises at least a solvent such as a buffer as a reaction medium for performing the dephosphorylation reaction. It is good. In any case, the components of the measurement kit can be appropriately changed, added, or reduced so that the measurement can be performed quickly and easily.

(Experimental Example 1-1: Citrate-reduced gold colloid / peptide titration)
475 μL of phosphorylation buffer (10 mM HEPES, 1 mM magnesium chloride, 0.2 mM ATP) and 5 μL of 500 mM EDTA were mixed, and then 20 μL of 25.3 mM citrate-reduced gold colloid was added. 1 μL of 0.5 mM peptide (kemptide: SEQ ID NO: 1) was added to the resulting solution, and the UV spectrum after mixing was measured (final concentration 1 μM, 0.1 μM interval). All solutions were stable within 1 minute after mixing.
As a result of the measurement, detection at a level of several hundreds nM was possible, and high linearity was observed in absorption near 650 nm (FIGS. 1 and 2). However, no color change due to aggregation was observed even 24 hours after mixing.

(Experimental Example 1-2: Detection by citrate-reduced gold colloid and phosphorylation ratio)
In order to confirm the correlation between the phosphorylation rate and the color change of the colloidal gold by a microplate reader, the following experiment was conducted.

  A 50 μM peptide (kemptide: SEQ ID NO: 1) solution was mixed in the amount shown in Table 1 (unit: μL), diluted with 485 μL of phosphorylation buffer (10 mM HEPES, 1 mM magnesium chloride, 0.2 mM ATP) and vortexed. After 5 μL of 500 mM EDTA was added to the resulting solution and vortexed, 20 L of 25.3 mM citrate-reduced gold colloid was added, vortexed, and 200 μL was added to each well of a 96-well plate. The absorption at 650 nm and the spectrum at 400-800 nm were measured without shaking the plate. The results of absorption measurement at 650 nm are shown in FIG. 3 and FIG. Note that only a buffer was used as a blank.

In addition, (P)% means a phosphorylation rate (%), Sub (P) means a phosphorylated peptide, and Sub means a non-phosphorylated peptide.

(Experimental example 2-1: phosphorylation time course with purified enzyme)
Add PKA 16U / mL and 120 μL of 50 μM peptide (kemptide) to 5.8 mL of phosphorylation buffer (10 mM HEPES, 1 mM magnesium chloride, 0.2 mM ATP) 485 μL, pipette up to 6 mL with phosphorylation buffer, and 37 ° C. Reacted. 500 μL was sampled every 5 minutes, and the reaction was stopped by boiling for 5 minutes with a block heater set to 100 ° C. in advance. Then, after allowing to stand at 4 ° C. for 1 hour, 20 μL of gold colloid was added to the reaction solution to which 5 μL of EDTA was added, and absorption or absorption spectrum at 650 nm was confirmed with a microreader.

(Experimental example 2-2: IC50 measurement of phosphorylation inhibitor H89 by colloidal gold)
A solution having the composition shown in Table 4 was prepared, and phosphorylation was performed at 37 ° C. The peptide was added last. After 20 minutes, a phosphorylation buffer containing gold colloid and EDTA (10 mM HEPES, 1 mM magnesium chloride; 20 μL, 5 μL, 500 μL) was added to the reaction solution and vortexed. Thereafter, the absorption and spectrum at 700 nm were measured with a microplate reader, and as a result, it was confirmed that phosphorylation was not achieved in all samples. In Table 2, the peptide is kemptide SEQ ID NO: 1).

(Experimental example 2-3: EC50 measurement with gold colloid)
10 mM ATP 20 μL, 10 M kemptide (SEQ ID NO: 1) 100 μL, phosphorylated buffer (10 mM
880 μL of HEPES, 1 mM magnesium chloride) was mixed. 1 μL or 5 μL of 0.01 U / L, 0.01 U / L, 0.1 U / L and 1 U / LPKA, respectively, was added to this mixture, and incubated at 30 ° C. for 0.5 or 2.0 hours. 10 μL of the obtained reaction solution was added to 500 μL of gold colloid solution (phosphorylated buffer, ATP, EDTA), and the absorbance at 700 nm was measured. As a result of the measurement, a decrease in absorbance was confirmed as the enzyme concentration increased (FIG. 4).

(Experimental example 2-4: Inhibition experiment of phosphorylation inhibitor H89 by colloidal gold)
Reagents, reaction conditions, etc .:
Sample 1: Contains neither PKA (cyclic AMP-dependent protein kinase) nor H89.
Sample 2: Contains H89.
Sample 3: Contains PKA.
Sample 4: Contains PKA (cyclic AMP-dependent protein kinase) and H89.
Phosphorylation buffer: 10 mM HEPES, 1 mM magnesium chloride Peptide: kemptide (SEQ ID NO: 1)
Kinase: 50M PKA (50 units / ml); ATP 0.1 mM
Inhibitor: H89 30 μM
Phosphorylation reaction: 37 ° C, 45 minutes

The experiment was performed according to the following procedure. The peptide, PKA, and inhibitor H89 were added to the phosphorylation buffer and incubated for 10 minutes, then ATP was added and incubated at 37 ° C. for 45 minutes. 10 μL of the resulting reaction solution was diluted with 490 μL of buffer (ATP / EDTA) and vortexed, and then 20 μL (final concentration 1 mM) of citrate-reduced gold colloid was added and vortexed. The spectrum of each processed sample was measured with a microreader (FIG. 5). At the same time, the progress of phosphorylation was confirmed with a microchip (MALDI-TOF-MS).
As a result of the above experiment, it has been found that the inhibitory effect of the inhibitor H89 can be detected with a gold colloid, and that the inhibitor H89 does not affect the aggregation of the gold colloid.

  In the same manner as in Experimental Example 2-1, PKA and kemptide (SEQ ID NO: 1) (peptide concentrations: 100, 10, 5, 1, 0.5, 0.1, 0.01, 0.001 μM) were phosphorylated and then reacted. 500 μL of 1 mM gold colloid was added and incubated. The reaction solution was measured for absorption at 700 nm. FIG. 6 shows the results obtained by plotting the substrate peptide concentration logarithmically and drawing a sigmoid curve. As shown in FIG. 6, it was found that quantitative aggregation occurred when the substrate peptide concentration was approximately 0.1 μM to 1 μM.

(Application to IC50 measurement)
16.7 μL of the reaction solution obtained by reacting each sample having a composition (10 mM HEPES (1 mM magnesium chloride, 0.1 mM ATP, 10 U / mL PKA, 30 M kemptide)) for 30 minutes with a phosphorylation buffer (EDTA / ATP) Dilute to 470 μL and add 20 μL of gold colloid. The absorbance at 650 nm and 700 nm was measured for each sample thus treated. The results are shown in FIGS. 7 and 8, respectively.

Cell disruption solution (lysate) of forskolin (Fsk) that activates intracellular PKA, cells co-stimulated with IBMX, and unstimulated cells was prepared using breast cancer cells MCF-7 as cells. After phosphorylating this under the following phosphorylation conditions, a HEPES buffer containing 5 mM EDTA (10 mM HEPES (1 mM magnesium chloride, 0.1 mM)
Gold colloid was added after dilution to a peptide concentration of 1 μM with ATP, 100 μg / mL lysate, 30 μM kemptide)). The colloidal gold concentration is the same as in Example 4. The results are shown in FIG. 9 (in the figure, GNP means gold colloid).

  Since it is also important to observe the effect of the foreign gene on the kinase activity, the same experiment as in Example 5 was performed on cells transfected with a plasmid encoding PKA. Liver cancer cell HepG2 was used as a cell, and a plasmid encoding PKA was transfected with lipofectamine 2000. Thereafter, as in Example 5, a lysate was prepared and assayed with gold colloid under the same conditions. The absorption spectrum at 400-800 nm was measured. The results are shown in FIG.

In this example, the PKC activity of the cultured cell lysate was evaluated.
After culturing each cell of A431, A549, B16, CHO, HeLa, HepG2, HuH-7, Neuro-2a, NIH3T3, uE, WiDr, MCF-7, 70% to 80% confluence The lysate was prepared by the following procedure, and the phosphorylation reaction of the peptide (Ac-FKKQSFAKKK-NH2) was performed to evaluate the activity with gold nanoparticles. Moreover, phosphorylation was also confirmed by MALDI-TOF-MS, and the presence of PKCα was confirmed by Western blotting.

(Create lysate)
The above cells were collected with a cell scraper, centrifuged at 1,000 rpm for 3 minutes, and the supernatant was removed. The obtained precipitate was suspended by adding 100 μL of a buffer (containing 10 mM HEPES, 10% sucrose, protease inhibitor), and sonicated for 15 seconds. 5,000 g of this suspension was centrifuged at 4 ° C. for 15 minutes, and this supernatant was used for the reaction.

(Western blotting)
Solutions were prepared so that the protein concentration was 200 μg / mL or 400 μg / mL, Western blotting was performed, and the activity of phospho PKC was examined (FIG. 11).

(Kinase assay)
A final concentration of 30 μM peptide, 200 μM ATP, 1 mM MgCl ₂ , and a lysate protein concentration of 100 μg / mL or 200 μg / mL, 10 mM HEPES were prepared, and phosphorylation was performed at 37 ° C. for 1 hour.
Next, the final concentration was adjusted to 0.3 μM peptide, 5 mM EDTA, 3.5 × 10 ¹¹ particles / mL gold nanoparticles, and the difference in color tone was confirmed (FIG. 13).
Furthermore, phosphorylation was confirmed by MALDI-TOF-MS (FIG. 12).

  The phosphorylation rate calculated from MALDI-TOF-MS, the abundance of PKC confirmed by Western blotting, and the results of the assay with gold nanoparticles were almost the same. A431 (human epithelial cancer), A549 (human lung cancer), B16 (mouse skin cancer), CHO (Chinese hamster ovary), HeLa (human cervical cancer), HepG2 (human liver cancer) ), Neour-2a (mouse neuroblastoma), NIH3T3 (mouse skin cancer) and NuE. Therefore, it was found that this assay can be applied to the diagnosis of cancer tissue with PKCa activation.

Using the tissue lysate of the tumor-bearing mouse obtained above, PKCa activity was evaluated as follows.
Normal skin tissue and tumor tissue were taken out from B16 (melanoma) cancer-bearing mouse, and the peptide (Ac-FKKQGSFAKKK-NH ₂ ) was used for phosphorylation reaction, and detection with gold nanoparticles was performed.
First, B16 cells (10 ⁷ cells) were injected into the right lower abdomen of Balb / c mice and left to grow to 7-8 mm. Next, tumor tissues and corresponding normal subcutaneous tissues of the left lower abdomen were collected from the mice.
Tissue treated in this way was buffered (10 mM
HEPES, 10% sucrose, including protease inhibitor (1 mL) was homogenized. 800 g of the obtained tissue was centrifuged at 4 ° C. for 5 minutes to remove the supernatant, and then washed twice with a buffer. The obtained tissue was again suspended in 1 mL of buffer and sonicated for 30 seconds. 5,000 g of this suspension was centrifuged at 4 ° C. for 15 minutes, and this supernatant was used for the reaction.

The supernatant obtained above was prepared such that the final concentration was 30 μM peptide, 200 μM ATP, 1 mM MgCl ₂ , and the protein concentration of lysate was 100 μg / mL or 200 μg / mL, and the volume was increased to 50 μL with buffer.
The obtained solution was phosphorylated at 37 ° C. for 1 hour, and phosphorylation was confirmed by MALDI-TOF-MS.

In addition, the final concentration was 0.4 μM peptide, 5 mM EDTA, and 3.5 × 10 ¹¹ particles / mL of gold nanoparticles, and the difference in color was confirmed (sample number / protein concentration of lysate) (FIG. 14).
Normal cells were hardly phosphorylated, but tumor tissue was phosphorylated. Moreover, this difference can be confirmed by the difference in color of the gold nanoparticles, and the reaction solution exhibited blue in normal tissues and purple to red in tumor tissues.

Phosphorylation assay using B16 cancer-bearing mouse tissue (in the presence of inhibitor)
The phosphorylation assay procedure was the same as described above, and a PKC inhibitor (Ro-31-7549) was added during phosphorylation.
It was shown by MALDI-TOF-MS that the phosphorylation rate decreases with the addition of an inhibitor. Moreover, it turned out that the change of a color tone reflects this (FIG. 15).

Phosphorylation assay using HepG2-bearing mouse tissue
A tumor tissue was taken out from a HepG2-bearing mouse, and PKC64 (Ac-FKKQGSFAKKK-NH ₂ ) was phosphorylated by using the tumor tissue, and detection with gold nanoparticles was performed. It was distinguishable from normal tissue (FIG. 16).

A kinase assay using human breast cancer real samples was performed as follows.
First, the tissue of a breast cancer patient was lysed, and phosphorylation of PKC64 (Ac-FKKQGSFAKKK-NH2) was performed, and phosphorylation was confirmed with gold nanoparticles and MALDI-TOF-MS.

  A part (5 mm square) of a breast cancer sample was placed in 500 μL of a buffer (containing 10 mM HEPES, 10% sucrose, protease inhibitor) and homogenized. The homogenized sample 800-900 g was centrifuged at 4 ° C. for 5 minutes, and the supernatant was removed. The resulting precipitate was suspended in 100 μL of buffer and sonicated for 15 seconds. 5,000 g of the obtained suspension was centrifuged at 4 ° C. for 15 minutes, and this supernatant was used for the reaction.

The final concentrations were 30 μM peptide, 200 μM ATP, 1 mM MgCl ₂ , lysate protein 25, 50, 100, 200 μg / mL, and 10 mM HEPES, and phosphorylation was performed at 37 ° C. for 1 hour.
Next, the final concentration was adjusted to 0.3 μM peptide, 5 mM EDTA, 3.5 × 10 ¹¹ particles / mL gold nanoparticles, and 10 mM HEPES, and the difference in color tone was confirmed (FIG. 17).
Furthermore, phosphorylation was confirmed by MALDI-TOF-MS.

  Since this invention can easily and easily perform phosphorylation and dephosphorylation of protein kinases, it is possible to accurately and simply detect functional abnormalities of this protein kinase and to elucidate the causes of diseases, etc. To help.

Ultraviolet absorption spectrum by citrate-reduced gold colloid / peptide titration. The graph which shows the linearity in 650 nm absorption by acid reduction gold colloid peptide titration. The ultraviolet absorption spectrum figure by a citrate reduction gold colloid and the phosphorylation rate. The linear diagram which shows the relationship between a phosphorylation rate and 650 nm absorption. The sigmoid curve figure which shows EC50 measurement result by a gold colloid. The ultraviolet absorption spectrum figure which shows the inhibition result of the phosphorylation inhibitor H89 by a gold colloid. The sigmoid curve figure which shows the examination result of a detection limit. The sigmoid curve figure which shows IC50 measurement result by a gold colloid. The ultraviolet absorption spectrum figure which shows the result of having performed the phosphorylation reaction using chemical | medical agent stimulation lysate. The ultraviolet absorption spectrum figure which shows the result of having performed the phosphorylation reaction using PKA transfection cell lysate. The figure (Example 7) which shows the result (lysate density | concentration: 200 g / mL) of a western blotting. A graph (Example 7) which shows the phosphorylation rate (lysate concentration: 100 g / mL) calculated | required by MALDI-TOF-MS. The figure (Example 7) which shows the result of the gold | metal | money nanoparticle assay of a lysate (lysate concentration: 100 g / mL). The figure which shows the assay result of a B16 cancer bearing mouse | mouth (Example 8). The figure which shows the result of the phosphorylation assay of a B16 cancer bearing mouse | mouth (Example 9). Bottom: phosphorylation rate determined by MALDI-TOF-MS. The figure which shows the result of the phosphorylation assay of a HepG2 cancer bearing mouse | mouth (Example 10). Bottom: phosphorylation rate determined by MALDI-TOF-MS. The figure which shows the result of the gold nanoparticle assay of a lysate (Example 11).

Claims (12)

After reacting the substrate peptide and protein kinase in the presence of a metal colloid, or after reacting the substrate peptide and protein kinase, the metal colloid is added, and the protein kinase phosphorous is determined based on the color change of the resulting reaction solution. A method for measuring an enzyme activity of a protein kinase, which comprises measuring an enzyme activity by oxidation. The enzyme activity measurement method according to claim 1, wherein the kinase is from PKC (protein kinase C), PKA (cyclic AMP-dependent protein kinase), MAPK, Rho kinase, MAPKAP, cdc2 kinase, raf-1 kinase, or the like. A method for measuring an enzyme activity of a protein kinase, which comprises using a selected protein as a substrate.   3. The protein kinase enzyme activity measuring method according to claim 1 or 2, wherein the substrate peptide is a cationic substrate peptide comprising kemptide (SEQ ID NO: 1) or PKC64 (SEQ ID NO: 2). Method for measuring enzyme activity of kinase. The enzyme activity measurement method for protein kinase according to any one of claims 1 to 3, wherein the metal colloid is gold colloid, silver colloid, platinum colloid, iron colloid, iron hydroxide colloid, aluminum hydroxide colloid, palladium. A method for measuring an enzyme activity of a protein kinase, which is a colloid or a rhodium colloid. By dephosphorylating the substrate peptide phosphorylated by protein kinase through the metal colloid, dephosphorylating enzyme activity of dephosphorylating enzyme based on the color change of the reaction mixture obtained A method for measuring phosphatase activity, comprising measuring. 5. The method for measuring phosphatase activity according to claim 4, wherein the phosphatase is a protein phosphatase such as Escherichia coli alkaline phosphatase, bovine intestinal alkaline phosphatase, human Cdc25, Cdc14 or the like. Activity measurement method. In the presence of a compound that inhibits phosphorylation of protein kinase, the substrate peptide and protein kinase are reacted in the presence of metal colloid, or the substrate peptide and protein kinase are reacted, and then metal colloid is added. Protein kinase phosphorylation inhibition characterized by screening protein kinase phosphorylation inhibition compounds by measuring the enzyme activity of the compound that inhibits protein kinase phosphorylation based on the color change of the reaction mixture obtained Compound screening method. The method for screening a protein kinase phosphorylation inhibiting compound according to claim 7, wherein the protein kinase is PKC (protein kinase C), PKA (cyclic AMP-dependent protein kinase), MAPK, Rho kinase, MAPKAP, CaMK, A method for screening a protein kinase phosphorylation-inhibiting compound, wherein a protein selected from cdc2 kinase or raf-1 kinase is used as a substrate. 9. The screening method for a protein kinase phosphorylation inhibiting compound according to claim 7 or 8, wherein the metal colloid is a gold colloid, a silver colloid, a platinum colloid, an iron colloid, an iron hydroxide colloid, an aluminum hydroxide colloid, a palladium colloid or A screening method for a protein kinase phosphorylation-inhibiting compound, which is a rhodium colloid. 10. The method for screening a protein kinase phosphorylation inhibitor compound according to any one of claims 7 to 9, wherein the protein kinase phosphorylation inhibitor compound is H-89 as a PKA inhibitor and Ro31- as a PKC inhibitor. 8220, a method for screening a protein kinase inhibitor compound comprising KN62 or the like as an inhibitor of CaMK.   A kit for measuring the kinase activity of a protein kinase, comprising at least a substrate peptide that can be phosphorylated by a protein kinase, a metal colloid, and a solvent such as a buffer as a reaction medium. A protein kinase dephosphorylating enzyme activity measurement kit comprising at least a solvent such as a buffer as a reaction medium for performing a dephosphorylation reaction.