JPH0678357B2 - New peptide - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to a novel peptide, and more specifically to a novel peptide having a physiological activity of decreasing blood Ca ²⁺ concentration derived from the chick gill posterior body. .

[Prior art]

Calcitonin (hereinafter referred to as “CT”) is used for birds, fish,
It is a peptide hormone that is present in the gill body such as round mouths and in the thyroid gland of mammals. This hormone has an action of antagonizing parathyroid hormone, and is known to act on bones and the like to lower the blood Ca ²⁺ concentration. Conventionally known
CT is a single-chain peptide consisting of 32 amino acids. The 1st and 7th cysteines from the amino group terminal (hereinafter referred to as "N terminal") form a disulfide bond, and the carboxyl group terminal (hereinafter referred to as "C terminal"). In common) in that they are proline amides. To date, CT has been extracted and purified from pig, cow, sheep, human, rat, salmon, and eel, revealing that the peptide structures differ slightly depending on the animal species, while having the above-mentioned structural commonalities. Has been done.

Moreover, when the 1st and 7th disulphide bonds from the N-terminal of CT were cleaved, their biological activity almost disappeared (HBBr
ewer, Jr., et al, Federation Proc., 27 , 690 (1968), 2
8, 383 (1969)), this disulfide bond to O connexion bioactivity to ethylene combined using aminosuberic acid is retained (JP-B 53-41677), the 1,7-position of the cyclic conformation CT It has been reported to be essential for biological activity.

Recently, the present inventors extracted and purified CT from the hindgill of chicken, and the obtained chicken CT has the following formula (I): (In the formula, Cys is cysteine, Ala is alanine, Ser is serine, Leu is leucine, Thr is threonine, and Val is
Is valine, Gly is glycine, Lys is lysine, Gln is glutamine, Glu is glutamic acid, His is histidine, Tyr is tyrosine, Pro is proline, Arg is arginine, and Asp is aspartic acid. ), A new structure shown in
The application has already been filed as 58-230593 (JP-A-60-123500).

Furthermore, as a result of various examinations on the extraction and purification conditions in the process of extraction and purification of chicken gill posterior bodies, the present inventors extracted the chicken gill posterior bodies with an acidic aqueous solution and then subjected to a specific treatment to obtain the above formula (I). CT indicated by (hereinafter "chicken CT I")
Another new CT-like peptide (hereinafter referred to as "chicken CT II"), which is different from that described above), has been isolated. This chicken CT II is different from the conventionally known CT in which the disulfide bond is cleaved. The inventors have found that they have high CT activity in spite of their completely different structures, and completed the present invention.

[Structure of Invention]

The chicken CT II of the present invention has the following formula (II): (In the formula, γ-Glu represents γ-glutamic acid, and Cys, Al
a, Ser, Leu, Thr, Val, Gly, Lys, Gln, Glu, His, Ty
r, Pro, Arg and Asp are as defined above).

The chemical properties of chicken CT II of the present invention are as follows.

Property of substance: White powder Solubility in solvent: Soluble in water, especially acidic aqueous solution, insoluble in chloroform, carbon tetrachloride, benzene, hexane, etc. Molecular weight: 3,985 Basicity: Chicken CT II of the present invention <Chick CT I Primary structural formula: Formula (II) Immunological properties: An organism that can be detected by utilizing the crossing of chicken CT II of the present invention with the antiserum using anti-eel CT serum and anti-salmon CT I serum Activity: 3,000 to 7,000 MRCU / mg (Bioactivity measurement method) After appropriately diluting the test substance with a 1% sodium acetate (pH 4.0; containing 0.1% bovine serum albumin) aqueous solution, several diluted liquids were used as males. 0.1 ml per juvenile rat (around 100 g) was injected into the tail vein, and 1 hour after the injection, blood was collected from the heart, centrifuged to collect serum, and the Ca ²⁺ concentration in serum was spectroscopically determined. method; measured at (reagent Wako calcium measurement Kitsuto), Ca ²⁺ conc. 10 to reduce the degree by 10%
It is defined as mMRC (Medical Research Council) U.

Efficacy of the effect: Chicken CT of the present invention II> Pig CT (Measurement method) A CT aqueous solution equivalent to 6 pmol CT was intravenously injected into the rat, and serum Ca ²⁺
The change in concentration over time is measured.

The chicken CT II of the present invention can be collected from the posterior gill of an avian as follows.

That is, after extraction of the post-gill tribe with an acidic aqueous solution, the following treatments (a) and (b): (a) The extract is brought into contact with a cation exchange resin, and the adsorbed basic fraction is eluted with the aqueous solution. b) Molecular weight of 3500-6000 by gel chromatography
It can be collected by subjecting at least one kind of the collected fractions.

As a raw material for extraction, the gill rear body of chicken is used. Existence of postgillary body was observed before and after hatching, adjacent to the common carotid artery below the thyroid gland and parathyroid body, and 1 mm in diameter in adult chickens.
It is a spherical organ of a degree. The weight and CT content of the posterior gill body change depending on the age of the chicken. Considering the availability of a large amount of homogeneous chickens, the posterior gill body having an appropriate size, and the ease of extraction, it is preferable to use chickens of broiler species 6 to 80 weeks old.

Extraction of CT from the starting material can also be performed as follows.

For example, the starting material is crushed and mixed in about 5 to 10 times the amount (weight ratio) of an acidic aqueous solution, preferably a volatile acidic aqueous solution (eg, an aqueous solution of acetic acid, formic acid, etc.), and chicken CT is used.
A suspension containing I and II is obtained. In this case, the acidic aqueous solution used preferably has a pH of 1-5. Then, this solution is centrifuged to form a slightly transparent CT extract in the intermediate layer. Here, the precipitate is mainly a tissue piece and insoluble matter of the posterior gill body, and the upper layer part is mainly a white pool of fat. The intermediate layer is collected and subjected to the next processing step.

The extract thus obtained can be purified by the treatment (a) or (b) described above, but it is preferable to combine the treatments (a) and (b), and the treatment (b) is applied. After that, it is more preferable to carry out the treatment (a).

In the treatment (a), the extract or its treated liquid is subjected to chromatography using a cation exchange resin such as SP-Sephadex or carboxymethyl cellulose to recover the basic fraction. The chromatograph is
It is preferably carried out in a water solvent, but can also be carried out in an aqueous solution to which a hydrophilic organic solvent such as lower alcohol or acetone is added.

When SP-Sefadex is used as the cation exchange resin, the eluent is a basic aqueous solution having a pH of 8 to 11, usually,
An aqueous pyridine solution, an aqueous ammonia solution or the like is used.

When carboxymethyl cellulose is used as the cation exchange resin, the eluent is an acidic aqueous solution of pH 3 to 6,
Usually, an aqueous solution of formic acid, an aqueous solution of ammonium formate, an aqueous solution of ammonium acetate or the like is used.

The gel chromatography of the treatment (b) is, for example, acetic acid,
It is preferably carried out in a volatile buffer such as ammonium hydrogen carbonate or ammonium formate. An example of a column is
Bio-gel P10 and Sephadex G50.

In the collection method of the present invention, in the above treatment, the following treatments (c) and / or (d): (c) A hydrophilic organic solvent is added to the aqueous solution containing the target substance to precipitate and remove the high molecular weight polymer. It is further preferable to combine d) collecting a fraction having CT-like physiological activity by high performance liquid chromatography.

In the treatment (c), a sufficient amount of a hydrophilic organic solvent (for example, lower alcohol, acetone, etc.) which does not precipitate chicken CT II (molecular weight 3,985) of the present invention but is sufficient to precipitate higher molecular weight is added. Add to the extract to remove high molecular weight material.

High-performance liquid chromatography of treatment (d) (hereinafter "HPL
C ") can be carried out according to a conventional method, and ammonium formate (10 mM to 1.0 M) -acetonitrile, water-acetonitrile-10% trifluoroacetic acid (TFA) (90: 10: 1: 1).
~ 40: 60: 1) (volume ratio) and the like, and elute with a linear concentration gradient to recover a fraction having calcitonin-like physiological activity.

In the collecting method of the present invention, it is most preferable to combine all the above-mentioned treatments and carry out in the order of (c), (a), (b) and (d).

The assay of each fraction in each of the above purification steps was performed using anti-eel CT.
Utilizing the fact that serum cross-reacts with chicken CT II of the present invention, a radioimmunoassay method was used, and a biological activity measurement method was appropriately used in combination.

In the radioimmunoassay method, using the eel CT which is an antigen,
Rabbits are immunized several times, and after immunization, blood is collected to obtain antiserum.
Here, the present radioimmunoassay is a method for detecting the amount of chicken CT II of the present invention by a competitive reaction between an antibody (antiserum) of eel CT and chicken CT II of the present invention and a radiolabeled antigen. . On the other hand, the labeled antigen is chloramine T that binds radioactive iodine ( ¹²⁵ I) to the tyrosine part of eel CT.
Method or peroxidase method. Next, the chicken CT II extract of the present invention or several solutions obtained by appropriately diluting each fraction (test sample) of each step and the anti-eel CT serum obtained above are allowed to react well (4 ° C, 1 night), by measuring after separation of the ¹²⁵ I- eel CT combined with ¹²⁵ I- eel CT and antibodies not bound to the antibody, either an amount of at γ- counter relative contained in test samples CT activity is required. According to this radioimmunoassay, the amount of CT consumed by the assay is about 1/100 of that of the bioactivity assay.
The radioimmunoassay method of the present invention has the advantages of not only being reduced but also easy procedure, excellent quantification and reproducibility, and short test time.
Can be easily and quickly separated and purified.

〔The invention's effect〕

According to the present invention, a novel peptide can be provided,
This novel peptide is considered to be useful as a drug because it has a high CT activity, although it has a structure in which the disulfide bond is cleaved, which is completely different from that of conventionally known CT. That is, CT has a strong bone resorption inhibitory action, hypercalcemia, bone page et's disease, treatment of osteoporosis, since it has a gastric acid secretion and pancreatic juice secretion inhibitory action, peptic ulcer, an effect on acute pancreatitis, Since it has an anti-inflammatory effect, it is expected to have a wide range of drug effects such as an effect on rheumatoid arthritis.

Example of Invention

Next, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

Example 1 A) Starting material: 3,384 chickens for 10-week-old meat were exsanguinated and immediately their posterior gills were removed. The fat was removed as much as possible and frozen on dry ice. The obtained gill body was about 179.11 g.

B) Extraction step The posterior gill bodies obtained by the above method are subdivided (for approximately 500 animals),
Each was immersed in 1 N acetic acid (5 times the weight of the gill body (weight ratio)), homogenized with a Polytron, and centrifuged (9,000 rpm for 30 minutes). Next, the intermediate layer was collected so as not to contain fat, and a part of this extract was freeze-dried.

A part of this lyophilized powder was dissolved in 0.1N formic acid and the biological activity was measured, and it was found to be 1.0 to 1.2 MRC U / animal (chicken).

C) Purification step 1800 ml of acetone was gradually added to 900 ml of an acetic acid aqueous solution containing the target physiologically active substance obtained as described above, and the mixture was allowed to stand overnight to precipitate a high molecular weight substance. Centrifuge (9,000 rpm, 30
After that, the supernatant was collected.

This supernatant was filled with SP-Sephadex C-25 and
Pour onto a column (3.2 cm diameter x 22 cm height) equilibrated with acetic acid. At this time, the acidic fraction not adsorbed on SP-Sephadex is removed, and the basic fraction containing the target physiologically active substance is ionically adsorbed on SP-Sephadex C-25. Then, from the basic fraction adsorbed on SP-Sephadex with 300 ml of 2M pyridine aqueous solution (pH 10), chicken CT II was used.
The fraction containing was eluted. This eluate was confirmed to have physiological activity on CT.

In the subsequent experiments, the eluate was neutralized with concentrated acetic acid, lyophilized and used. 500 mg of this freeze-dried powder was added to 1 m of 1N acetic acid
A column (3.0 cm in diameter x 150 c in height) that was dissolved in 1 l and pre-packed with Sephadex G-50 (Fine) and equilibrated with 1N acetic acid.
m) was poured over this solution. At this time, the elution conditions were 1N acetic acid as an eluent and an elution rate of 27 ml / h. 15 ml of eluate
/ Fractions were collected for each and the active fraction (fraction No. 48 to 53) by radioimmunoassay was collected. Lyophilize this and powder
17.4 mg was obtained.

The amount of the immunoreactive chicken CT II of the present invention calculated from the above assay was 219 μg (corresponding to 3384 animals).

17.4 mg of this powder was dissolved in 1.5 ml of solution A (described later), and this solution was subjected to HPLC according to the following measurement conditions.

Measurement conditions: Column; TSK GEL CM 2 SW (4.6mm, manufactured by Toyo Soda Industry Co., Ltd.)
φ × 250mm) Flow rate; 2.0ml / min Differential pressure; 35Kg / cm ² Eluent; Solution A (10mM ammonium formate: acetonitrile =
9: 1) (pH4.0) Solution B (1.0M ammonium formate: acetonitrile = 9: 1)
One minute after injection of (pH4.0) sample, linear concentration gradient elution from solution A to solution B (140 minutes) The results obtained show that the activity derived from the radioimmunoassay and the peptide derived at 280 nm from the absorbance measurement method The absorbance was in agreement. Further, similarly, the sample was divided into three under the following conditions and HPLC was repeated.

Measurement conditions: Column: Chemcosorb 5 ODS-H (4.6 mmφ x 2 manufactured by Chemco)
50mm) Eluent; 0.5ml / main flow velocity; 1.0ml / min Differential pressure; 140Kg / cm ² Eluent; Solution A (water: acetonitrile: 10% TFA = 90: 10: 1) Solution B (water: acetonitrile: 10% TFA = 40: 60: 1) 8 minutes after the sample injection, a linear concentration gradient elution from solution A to solution B (160 minutes) resulted in a strong peptide-based absorbance in the active fraction (holding time 84 minutes). (A ₂₈₀ , A ₂₁₀ ) was recognized. This active fraction was freeze-dried to obtain 36.1 μg of active powder. The bioactivity of this powder was 5300 MRCU / mg.

Table 1 shows the amino acid analysis values of the active powders obtained as described above.

As the measuring method, 3.5 μg of active powder was hydrolyzed with 6N hydrochloric acid at 110 ° C. for 22 hours, dehydrochlorinated, dissolved in 50 μ of 0.2N citrate buffer (pH 3.25), and the whole amount was subjected to analysis.

Furthermore, the amino acid sequence of the novel peptide represented by the above formula (II) was determined by a known protein determination method mainly utilizing the similarity with chicken CT I.

Example 2 In the same manner as in Example 1, 625 ml of an aqueous solution of extracted acetic acid containing the chicken CT II of the present invention was obtained from a chicken gill rear body (500).

This solution was injected into a SP-Sephadex C-25 column (diameter 2.0 cm × height 17 cm) that had been equilibrated with 1N acetic acid in advance.
Then, it was eluted with a 2M pyridine solution. This eluate contained the chicken CT II of the present invention.

Acetone (240 ml) was added to this eluate (120 ml), and the mixture was allowed to stand overnight and then centrifuged (9,000 rpm, 30 minutes). The supernatant was collected and freeze-dried to obtain 100 mg of powder.

This lyophilized powder was dissolved in 1N acetic acid to make a solution of about 1 ml, and a Sephadex G-50 (Fine) column (diameter 1.2 cm
× height 103 cm). At this time, the elution conditions were 1N acetic acid as an eluent and an elution rate of 5.7 ml / h. The active fraction by radioimmunoassay was collected and freeze-dried to obtain 2.0 mg of freeze-dried powder. The amount of immunoreactive chicken CT II of the present invention calculated from the above-mentioned assay was 55 μg. This powder was subjected to high performance liquid chromatography operation in the same manner as in Example 1 to obtain 5 μg of active powder of chicken CT II. The biological activity of this powder was 4,800 MRCU / mg.

Furthermore, amino acid analysis was performed, and the amino acid sequence was determined based on a conventional amino acid sequencing method, and it was confirmed that this active powder was the chicken CT II of the present invention.

Example 3 In the same manner as in Example 1, 625 ml of an extracted acetic acid solution containing the chicken CT II of the present invention was obtained from a chicken gill rear body (500).
And lyophilized to obtain 1.5 g of powder.

This powder was dissolved in 15 ml of 1N acetic acid and then centrifuged (3,000 r
pm, 20 minutes), and add the supernatant to Sephadex G-50.
It was injected into a (Fine) column (1.2 cm in diameter x 103 cm in height).

This active fraction was injected onto a SP-Sephadex C-25 column, and in the same manner as in Example 1, 1.0 mg of a powder having CT activity was injected.
Got

This powder was subjected to high performance liquid chromatography operation in the same manner as in Example 1 to obtain 4 μg of CT active powder.
The bioactivity of this powder was 5,100 MRCU / mg.

Further, amino acid analysis was performed, and the amino acid sequence was determined in the same manner as in Example 1, and it was confirmed that this active powder was the chicken CT II of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Homma Homma 1315 Wakaguri, Ami-cho, Inashiki-gun, Ibaraki Sanryo Petrochemical Co., Ltd. Central Research Institute (72) Inventor Eiji Adachi 5 Shinmori, Asahi-ku, Osaka-shi, Osaka Chome 16-6

Claims (1)

[Claims] 1. The following formula: (In the formula, Cys is cysteine, Ala is alanine, Ser is serine, Leu is leucine, Thr is threonine, and Val is
Is valine, Gly is glycine, Lys is lysine, Gln is glutamine, Glu is glutamic acid, His is histidine, Tyr is tyrosine, Pro is proline, Arg is arginine, Asp is aspartic acid, γ-Glu represents γ-glutamic acid).