JPH07227281A - Peptidase and its production - Google Patents

Abstract

PURPOSE:To obtain a new peptidase having a specific molecular weight and optimum pH, capable of hydrolyzing a proline-rich protein and useful for diagnosis, prevention, treatment, etc., of diseases caused by abnormality of metabolism of an intracranial physiologically active peptide containing proline. CONSTITUTION:This new peptidase has the following physicochemical properties and physiologically active properties: Estimated molecular weight, about 140kDa based on a gel permeation method; optimum pH, about 7.5-8.0; the peptidase hydrolyses the formulas Pro-Pro-Pro-Pro, Pro-Pro-Pro and Pro-Pro, and it recognizes proline existing in the second residue from the N-terminal of the peptide and isolates an amino acid in the N-terminal. The enzymatic activity is activated by manganese chloride and inhibited by o-phenanthroline and 2mercaptoethanol and hardly inhibited by p-chloromercury benzoate, iodoacetamide, pepstatin and bestatin. The peptidase is obtained by adding acetone to a supernatant obtained by homogenizing and centrifuging a bovine brain to precipitate a active fraction.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel peptidase and a method for producing the same. More specifically, a proline-containing bioactive peptide present in the living body of mammals such as the brain,
The present invention relates to a peptidase that hydrolyzes oligoproline, proline-rich protein and the like, and a method for producing the same.

[0002]

2. Description of the Related Art Proline having an imino group in its molecule is
It forms an acid imide bond with other amino acids during peptide formation. Generally, the peptide chain bends at the site where proline exists, and when a large number of prolines are connected, it takes a unique helix structure.
Proteins) are less likely to be degraded by ordinary proteases. Therefore, proline is contained in many physiologically active peptides, and proline-rich protein is present in large amounts between plant cell walls and animal cells and plays an important function in maintaining the structure of living tissues.
Particularly in mammals, collagen involved in tissue structure maintenance and cell adhesion has been known for a long time, but recently, the acidic domain of the human transcription promoting factor CTF1 (J.
Biol. Chem., 268, 20866, 1993), a synapsin (Scienc) that regulates neurotransmitter release in the nervous system.
e, 259, 780, 1993), NDPP-1 (Bioche) whose expression is specifically regulated during formation of the central nervous system of mice.
m. Biophys. Acta, 1132, 240, 1992), and other proline-rich proteins with important functions are being discovered one after another.

However, since such proline-containing peptides and proline-rich proteins are also hydrolyzed to individual amino acids in vivo, the presence of an enzyme specific to proline is predicted, and so far prolyl is Various proline-specific enzymes such as endopeptidase have been reported (Mol. Cell. Biochem., 30: 111, 1980, F.
EBS Letters, 234, 251 pages, 1988). Of these, Pro
As an enzyme capable of degrading -Pro binding, proline iminopeptidase (EC 3.4.11.
5) and aminopeptidase P (EC 3.4.11.9) are known, but the proteolytic activity of aminopeptidase P is very low (Eur. J. Biochem., 198, 45).
1 page, 1991, Eur. J. biochem., 210, 93 pages, 1992
Year). In addition, when the properties of proline iminopeptidase purified from rat brain, rat kidney, and human liver were examined, it was revealed by Turzynski and Matsushima that it was an enzyme of the same type as leucine aminopeptidase (EC 3.4.11.1). (Eur. J. Biochem., 190, 509
Page, 1990, Biochem. Biophys. Res. Commun., 178
Volume, 1459, 1991, Biomed. Res., 12: 323, 199
1 year). Therefore, it is currently unknown whether or not proline iminopeptidase is present in mammals. Leucine aminopeptidase has a wide substrate specificity and
Can release the terminal proline, but Pro-Pro
Does not affect binding. An enzyme that cleaves the Pro-Pro bond from the C-terminal side is a proline-specific dipeptidyl carboxypeptidase (J. Biochem., 112) isolated from actinomycetes.
Vol., 253, 1992), but not confirmed in mammals.

As described above, the elucidation of the existence and function of proline-rich proteins in the living body of mammals has progressed rapidly in recent years, and the control of the neurotransmission system and the formation of the central nervous system, especially in the brain, is particularly proline-rich.・ The importance of proteins is increasing. Therefore, the present inventors have found that an enzyme that cleaves the periphery of proline (especially proline having a proteolytic activity for Pro-Pro bond) that is useful for inhibiting the action of such a proline-rich protein and elucidating its function. The present invention has been completed by discovering the enzyme in bovine brain and purifying and isolating the enzyme, as a result of repeated studies on specific enzyme). That is, the present invention is Pro-
The purpose of the present invention is to provide a novel peptidase having a proteolytic activity and a method for producing the same, and the use of the peptidase as a metabolic improver, a reagent and the like is expected.

[0005]

The peptidase of the present invention has the following physicochemical properties and physiological activities. 1) Estimated molecular weight: Approximately 140 kDa by gel filtration method. 2) Optimum pH: about 7.5-8.0 3) Pro-Pro-Pro-Pro, Pro-Pro-Pro and Pro-Pro are hydrolyzed, and are present at the second residue from the N-terminal of the peptide. It recognizes proline and releases the N-terminal amino acid. 4) Enzyme activity is activated with manganese chloride. 5) Enzymatic activity was inhibited by o-phenanthroline and 2-mercaptoethanol, while PCBM (p-chlo)
There is little inhibition with romercuribenzoate), iodoacetamide, pepstatin A and bestatin. Further, the method for producing peptidase of the present invention comprises obtaining the peptidase having the above-mentioned physicochemical properties and physiological activity from bovine brain.

The peptidase of the present invention can be produced, for example, as follows. The raw material is not particularly limited as long as it is a material containing the peptidase of the present invention, but preferably mammalian tissues, organs, etc., for example,
The brain, lungs, kidneys, spleen, liver, etc. are used. The production of the peptidase of the present invention from such a biomaterial is basically a general method for isolating and purifying proteins from a biomaterial, for example, gel filtration chromatography, ion exchange chromatography, affinity chromatography, Adsorption chromatography, salting out, dialysis, centrifugation, ultrafiltration, freeze-drying, electrophoresis and the like can be appropriately combined and carried out.

A concrete example of a particularly preferable operation method will be described in detail. First, the raw material, for example, bovine brain, is homogenized while adding physiological saline, and then centrifuged at 4 ° C. and 20,000 × g for 20 minutes. Acetone was added to the supernatant to precipitate, and ammonium sulfate fractionation, Q-Sepharo
se anion exchange chromatography, Superde
x200 gel filtration chromatography, Mono Q anion exchange chromatography, TSK-GEL G3
The peptidase of the present invention can be obtained by purification by sequentially performing 000SW gel filtration chromatography. The peptidase of the present invention can also be obtained by using commercially available bovine brain acetone powder (commercially available from Sigma, etc.) as a material and purifying it according to the above method.

The peptidase of the present invention is obtained by the above method, and the peptidase is specified by the above-mentioned characteristics,
Such characteristics are supported by the examples described below. That is, the peptidase of the present invention is Pro-Pro-Pro-Pro, Pro-Pro-Pro, Pr.
In addition to hydrolyzing oligoproline such as o-Pro to proline, it recognizes proline existing at the second residue from the N-terminal of the peptide and releases any amino acid at the N-terminal. Also, the N-terminal amino acids of the physiologically active peptides bradykinin and neuropeptide Y in the brain are released. The estimated molecular weight of the peptidase of the present invention is about 140 kDa by gel filtration method.
Is. The optimum pH is about 7.5-8.0, and the activity drops sharply at 9.0 or more and 7.0 or less. Further, the peptidase of the present invention is activated by manganese chloride and is inhibited by 2-mercaptoethanol in addition to o-phenanthroline which is a metal enzyme inhibitor. On the other hand, cysteine enzyme inhibitors (P
CMB, iodoacetamide), aspartic enzyme inhibitor (pepstatin A), and aminopeptidase inhibitor (bestatin) show almost no inhibition.

Two enzymes, aminopeptidase P (EC 3.4.11.9) and dipeptidyl aminopeptidase IV (EC 3.4.14.5), which are specific to proline existing at the second residue from the N-terminal of the peptide, are used. Is known, but the latter is an enzyme distinct from the peptidase of the present invention because it cleaves the carboxyl group side of proline to release the N-terminal dipeptide and has no ability to decompose Pro-Pro bond. . On the other hand, the former releases the N-terminal amino acid similarly to the peptidase of the present invention and has some Pro-Pro binding resolution, so the peptidase of the present invention is presumed to be an aminopeptidase P-like enzyme. Aminopeptidase P has been confirmed to exist in bacteria, yeast, fish, mammals, etc., and in mammals, purification examples from rats (brain), cows (lungs), pigs (kidneys), humans (white blood cells), etc. have been reported. ing.

The molecular weight of the peptidase of the present invention is TSK-
It was estimated to be about 140 kDa in GEL G3000SW, but the molecular weight estimated by gel filtration was close to that of aminopeptidase P derived from rat brain and human leukocytes (about 140 kDa under non-denaturing conditions), and the optimum pH was about 7 as well. .5
The value is -8.0, which is close to the value of aminopeptidase P derived from other mammals (pH 6.0-8.0). Furthermore, the aminopeptidase P reported so far has a common property of being activated by Mn ²⁺ and being inhibited by a metalloenzyme inhibitor and a cysteine enzyme inhibitor, and the peptidase of the present invention also has a metalloenzyme. It was confirmed that it was inhibited by the inhibitor o-phenanthroline and activated by Mn ²⁺ . However, the peptidase of the present invention is
It was found that there was no inhibition by a cysteine enzyme inhibitor such as iodoacetamide, and that it was inhibited by the SH group protecting agent 2-mercaptoethanol. Further, in rat brain-derived aminopeptidase P, the relative activity of Pro-Pro-Pro with respect to the degrading activity of bradykinin is 0.6%, whereas in the peptidase of the present invention, it is 8.4%, which is about a 14-fold difference. Admitted. In addition, Pro-Pro-Pro-Pro and Pro-Pro
, The degradation activity was 2 to 7 times that of Pro-Pro-Pro. From these, it can be judged that the peptidase of the present invention has a significantly high activity of degrading oligoproline. From the above results, it was shown that the peptidase of the present invention is different from other mammalian aminopeptidases P.

[0011]

INDUSTRIAL APPLICABILITY As described above, it is speculated that certain diseases are caused by proline-containing proteins such as proline-rich protein in the living body of mammals, and the peptidase of the present invention is proline-rich protein. Since it has a function of hydrolyzing a protein, it is used as a drug for preventing or treating a disease involving proline-rich protein, as a clinical diagnostic reagent for testing such a disease, or for elucidating the function of the protein. It is useful as a chemical reagent. In particular, the proline-rich protein in the brain is involved in the control of the neurotransmission system, the formation of the central nervous system, etc., and the peptidase of the present invention decomposes physiologically active peptides in the brain such as bradykinin and neuropeptide Y into an insoluble form. Since it has an activating effect, it can be used as a drug for preventing or treating diseases caused by abnormal metabolism of physiologically active peptides in the brain or a reagent for elucidating brain function.

[0012]

The present invention will be described in more detail based on the following examples, but the invention is not intended to be limited to these examples. Example 1 Measurement of peptidase activity The enzyme activity was measured using Leu-Pro-Pro-Pro as a substrate. More specifically, diluted enzyme solution 20 μl, 50 m
M Tris-HCl buffer (pH 7.5) 50 μl, 10 m
10 μl of a mixed solution of M manganese chloride / 1 mM bestatin was mixed, ice-cooled for 20 minutes, and then preheated at 37 ° C. for 10 minutes. 20 μl of 1 mM Leu-Pro-Pro-Pro was mixed, and 37
After reacting for 20 minutes at 0 ° C., 100 μl of 1N hydrochloric acid was added to stop the reaction, and 10 μl of the supernatant was loaded on a reverse-phase HPLC column to detect the peak of Leu-Pro-Pro-Pro remaining in the reaction solution. . The activity intensity was determined by comparing the peak heights of the reaction solution and the control. The conditions of HPLC are shown below. Column: μ Bondasphere C ₈ -300Å (3.9 x 150 mm, Japan Millipore) Eluent: Linear concentration gradient of acetonitrile / distilled water (7 / 93-63 / 37, v / v) containing 0.1% trifluoroacetic acid Flow rate: 1 ml / min Detection: 210 nm This system was used as a standard activity measurement system. In the following purification step, the enzyme activity of the column eluate was measured using this measurement system, and the activity peak was detected.

Example 2 Purification of Peptidase of the Present Invention To 400 g of crushed bovine brain was added sufficiently cooled 20 mM Tris-hydrochloric acid buffer (pH 7.5, 4 ° C.), and the container was homogenized with a Waring blender while cooling with ice. The same buffer was added to the homogenate, and the mixture was homogenized more finely using a polytron, and then at 4 ° C., 30,000 ×.
After centrifugation at g for 20 minutes, the resulting supernatant was used as a crude enzyme solution. Acetone (4 ℃) sufficiently cooled in crude enzyme solution
Was added dropwise with stirring with a stirrer so that the final concentration was 50% (v / v). After allowing the precipitate to stand overnight in a low greenhouse for aging, centrifugation was performed at 4 ° C. and 20,000 × g for 20 minutes to obtain an active fraction precipitate.

Next, the above precipitate was dissolved in 20 mM Tris-hydrochloric acid buffer (pH 7.5), ammonium sulfate was added to 30% saturation, and the mixture was allowed to stand in a low temperature room overnight.
The precipitate was removed by centrifugation at 000 xg for 20 minutes. Ammonium sulfate was further added to the obtained 30% saturated solution to 50% saturation, and the same operation was performed to obtain a precipitate of an active fraction. 10 mM of the obtained precipitate containing 1 mM manganese chloride
After dissolving in Tris-HCl buffer (pH 7.5), YM
Ammonium sulfate was removed by ultrafiltration using a -30 membrane (exclusion limit: 30,000 Da, Amicon) to obtain 60 ml of an active fraction. This sample was subjected to the next step.

Q-Sepha suspended in 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM manganese chloride.
Rose (Pharmacia) column (1.6 × 60c)
m), and the same buffer was flowed at least 3 times the bed volume for equilibration, the sample was loaded, and then the same buffer was again flowed at least 3 times the bed volume of the column. Flow rate 1 ml
/ Min the adsorbed protein was eluted with a linear concentration gradient of 0.2-0.8 M sodium chloride and fractionated in 9 ml portions, and the active fraction measured by the method of Example 1 was collected and subjected to the following gel filtration chromatography. It was That is, Supe equilibrated with 50 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing 1 mM manganese chloride and 200 mM sodium chloride.
An rdex 200 column (1.6 × 60 cm, Pharmacia) was loaded with the sample obtained in the previous step, eluted with the same buffer and fractionated in 5 ml fractions to obtain the active fraction (Frc.22-2
4) was recovered.

Next, the active fraction obtained in the previous step was desalted using Centricon 30 (Amicon),
MonoQ equilibrated with 10 mM citric acid-sodium hydroxide buffer (pH 6.0) containing 1 mM manganese chloride.
It was loaded onto an HR5 / 5 column (0.5 x 5 cm, Pharmacia). Elution was performed with a linear concentration gradient of 0-0.6 M NaCl at a flow rate of 1 ml / min, fractionation was performed in 1 ml increments, and the active fraction (Frc.41-43) was collected. Finally, the active fraction obtained in the previous step was concentrated using Centricon 30 (Amicon), and then 5 mM containing 1 mM manganese chloride was added.
TSK-GEL G3000SW column (0.5 × 30) equilibrated with 0 mM Tris-HCl buffer (pH 7.5).
cm: Tosoh Corporation) and eluted with the same buffer.
The flow rate was 0.5 ml / min, fractionated by 0.5 ml,
The enzyme activity of each fraction was measured and the active fraction was separated. The active fraction thus obtained was used as a purified preparation of the peptidase of the present invention.

Example 3 Purification of peptidase of the present invention from bovine brain acetone powder 50 g of bovine brain acetone powder (purchased from Sigma)
Dissolve in 10 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing 1 mM manganese chloride, and use a magnetic stirrer for 1
It was extracted by stirring for a time. 22,000 × g insoluble matter 2
Centrifugation was carried out for 0 minutes, a buffer solution was added to the precipitate, extraction was carried out for another 1 hour, and then the same operation was performed. 765 ml of the supernatant obtained by the above operation was used as a crude enzyme solution, and purified by the following ammonium sulfate fractionation and various chromatographies as in Example 2,
A purified standard was obtained.

Example 4 Investigation of various properties of peptidase of the present invention Measurement of molecular weight by gel filtration method In gel filtration of TSK-GEL G3000SW,
From the relationship between the molecular weight marker (Gel Filtration Standards, Bio-Rad) and the relative retention time of the peptidase of the present invention on the column, the molecular weight of the peptidase of the present invention was about 14
It was estimated to be 0 kDa.

Optimum pH measurement In a standard activity measuring system, the activity was measured by replacing the buffer solution with that of each pH. To adjust the pH, 100 mM Tris-HCl buffer (pH 7.0-
9.0), 100 mM citric acid-sodium hydroxide buffer (pH 4.0-6.5), 100 mM sodium borate-hydrochloric acid (pH 8.5-9.0), 100 mM sodium borate-sodium hydroxide (pH 9). .5-10.0)
Various buffer solutions of The result is shown in FIG. As shown in FIG. 1, the optimum pH was about 7.5-8.0, and the activity drastically declined between 9.0 and 7.0.

[0020]Effects of metal ions and inhibitors  Effects of metal ions and inhibitors on the peptidase of the present invention
Hibiki is 10 in a standard activity measurement system (see Example 1).
Various solutions of mM manganese chloride / 1 mM bestatin
Measure activity by replacing with metal salt solution or inhibitor solution
Was obtained by doing. The results are shown in Table 1. Metal salt
Alternatively, 50 mM Tris-HCl buffer instead of the inhibitor
The one to which (pH 7.5) was added was used as a control.
The value was expressed as relative activity with the value being 100%. In metal salt, salt
Inhibited by about 16% of control with nickel chloride, sulfuric acid
Completely inhibited by zinc. Also, about 14 with manganese chloride
It was confirmed to be activated to 0%. Influence of chemical reagents
Was examined to find that the metalloenzyme inhibitor o-phenanthroly
Of about 85% with 2-mercaptoethanol and about 67% with 2-mercaptoethanol
I was hindered. Serine enzyme inhibitors (DFP, PMS
F), cysteine enzyme inhibitor (PCMB, iodoacetate
Amide), aspartic enzyme inhibitor (pepstatin
A), aminopeptidase inhibitor (bestatin)
I was not hindered. From the above results, the present invention
Peptidase is Mn²⁺Is a dependent metalloprotease
Was estimated. So next, Mn²⁺Over the concentration of activity
I investigated the influence. 10 mM salt in standard activity assay system
Chlorination of manganese oxide / 1 mM bestatin mixed solution at each concentration
The activity was measured by replacing with a manganese solution.
I have As a result, the addition of 1-10 mM manganese chloride
Most activated.

[0021]

[Table 1]

Examination of Substrate Specificity A hydrolysis reaction was carried out using various synthetic peptides containing proline and physiologically active peptides as substrates to examine the substrate specificity of the peptidase of the present invention. The synthetic peptides were synthesized by the solid phase method, and as the physiologically active peptides (neuropeptide Y, substance P, bradykinin, brain diuretic peptide-32, gastrin releasing peptide), the standard products purchased from Peptide Institute Inc. were used. The enzymatic reaction was carried out by substituting various peptides for the substrate in a standard activity measurement system (see Example 1). Decomposition rate of the synthetic peptide was determined by comparing the peak heights of the reaction solution and control in reverse phase HPLC, and the degradation rate of the physiologically active peptide was determined by amino acid analysis by the ninhydrin method (Hitachi 835 high-speed amino acid analyzer). It was The results are shown in Table 2. As shown in Table 2,
The peptidase of the present invention acted on a peptide containing proline at the second residue from the N-terminal to release the N-terminal amino acid. Leu-Pro-Pr is the degradation activity for oligoproline.
Relative ratio with o-Pro, Pro-Pro-Pro-Pro 27%, Pro-Pro
-Pro 11% and Pro-Pro 66%. In the decomposition of the physiologically active peptide, the decomposition activity against bradykinin was remarkably high, and the decomposition activity against neuropeptide Y was also relatively high.

[0023]

[Table 2]

[Brief description of drawings]

FIG. 1 shows the relative activity of the peptidase of the present invention at each pH.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hideoki Tanaka 1-3-1, Higashi Tsukuba-shi, Ibaraki Industrial Technology Institute of Industrial Science and Technology (72) Inventor Hidekatsu Maeda 3-7-1, Higashi Toyota, Hino City, Tokyo (72) Inventor Tsutomu Kobayashi 1727 Higashino, Omiya-cho, Naka-gun, Ibaraki Prefecture (72) Inventor Omori Oka 3-3 Midorigahara, Tsukuba-shi, Ibaraki Central Research Laboratories, Nippon Ham Co., Ltd.

Claims (2)

[Claims] 1. A peptidase having the following physicochemical properties and physiological activity. 1) Estimated molecular weight: Approximately 140 kDa by gel filtration method. 2) Optimum pH: about 7.5-8.0 3) Pro-Pro-Pro-Pro, Pro-Pro-Pro and Pro-Pro are hydrolyzed, and are present at the second residue from the N-terminal of the peptide. It recognizes proline and releases the N-terminal amino acid. 4) Enzyme activity is activated with manganese chloride. 5) Enzymatic activity was inhibited by o-phenanthroline and 2-mercaptoethanol, while PCBM (p-chlo)
There is little inhibition with romercuribenzoate), iodoacetamide, pepstatin A and bestatin. 2. A method for producing a peptidase, which comprises obtaining from the bovine brain the peptidase having the physicochemical properties and physiological activity according to claim 1.