JP2001151796A - New tachykinin-related peptide and its precursor polypeptide and gene encoding the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a tachykinin-related peptide isolated from the brain of Octopus vulgaris and to provide a new approach to medicines and agrochemicals through research on structural activity correlation on the molecular level. SOLUTION: This new peptide is a tachykinin-related peptide represented by the amino acid sequence formula, R1-Phe-Xaa-Yaa-Zaa-Arg-NH2 (R1 is a peptide having 5 or 6 amino acid residues; Xaa is Leu, Ile, Met, Val or Gln; Yaa is Gly or Pro; Zaa is Ser or Thr) and especially the following amino acid sequence formulae (1) to (3): H-Thr-Val-Ser-Ala-Asn-Ala-Phe-Leu-Gly-Ser-Arg- NH2 (1), H-Leu-Asn-Ala-Asn-Ser-Phe-Met-Gly-Ser-Arg-NH2 (2) and H-Ala-Ser- Leu-His-Asn-Thr-Phe-Ile-Pro-Ser-Arg-NH2 (3).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to novel neuropeptides, and more particularly to octopus ( Octopus v).
ulgaris ), a novel tachykinin-related peptide having activity to enhance the heartbeat of molluscs, and its precursor polypeptides and the genes encoding them.

[0002]

2. Description of the Related Art As invertebrates develop,
The neuroendocrine system is developing and various endogenous peptides are known to act as neurotransmitters, neuromodulators or neurohormones.

The nervous system of mollusks is generally simpler than higher animals, and can be used for elucidating the mechanism of information processing. In particular, if the knowledge about the information processing mechanism obtained from mollusks is related to the mechanism at the subcellular level, the knowledge can be generalized to elucidate the information processing mechanism in the nervous system of higher animals. The search for neurotransmitters in mollusks has been vigorous.

[0004] For example, Japanese Patent Application Laid-Open No. 1-221392 discloses an endogenous neuropeptide obtained from mussels. JP-A-2-286696 discloses a novel peptide containing a D-amino acid as an endogenous neurotransmitter obtained from the ganglion of the African snail, and JP-A-6-56890 discloses Hungary. Disclosed are neuropeptides obtained from the ganglia of the worm.

[0005] In addition, from mollusks, H-Ala-P
ro-Gly-Trp-NH ₂ , myomodulin-CA
Numerous neuropeptides have been isolated and identified, such as RP, small myocardial peptides (SCP), and baccarines (M.
Kobayashi and Y. Muneoka, Zo
ol. Sci. , 7, 801, 1990; Mune
oka and M.S. Kobayashi, Exper
ientia 48,448,1992; Yojiro Muneoka,
Journal of the Japanese Society of Pesticides 18, S191, 1993; Mu
neoka, T .; Takahashi, M .; Kobay
ashi, "Perspective in Comp"
arative Endocrinology ", Na
tional Research Council
f Canada 1994, p109).

[0006] In addition, the open sea musk octopus ( Eledo)
ne moschata and E. et al . aldrovand
i ) from the posterior salivary glands of eredoisin
V. & Anastasi, A .; : Expertient
ia, 18, 58, 1962), and the term "tachykinin" was defined because this peptide has the effect of rapidly contracting the guinea pig ileum. Later, the structure of substance P was revealed based on the structure of eledoisin, and today tachykinin is
As a common amino acid residue at its C-terminus: -Phe-Aa
a-Gly-Leu-Met-NH ₂ , wherein Aaaa is an aromatic amino acid (Phe, Tyr) or a branched amino acid (Val, Ile),
It is a general term for bioactive peptides that exhibit blood pressure lowering, salivation promoting action, and the like. Tachykinins include substance P (substance P), eledoisin, fizalamine,
Neurokinin A, Neurokinin B, Casinin, etc. (Biological Dictionary (4th edition), Iwanami Shoten, 199
7).

On the other hand, a peptide related to tachykinin has been isolated from insects and reported as locust tachykinin (Schoofs, L., Holman,
G. FIG. M. Hayes, T .; K. , Nachman,
R. J. & De Loof, A. : FEBS Let
ters, 261, 397, 1990). JP-A-6-32798 discloses a tachykinin-related neuropeptide derived from a worm. These tachykinin-related peptides are known to have a contractile action on the hindgut of insects (Nassel.DR: Pept).
ides, 20, 141, 1999).

Through research on such tachykinins and tachykinin-related peptides, it has been revealed that there are tachykinins acting on vertebrates and tachykinin-related peptides acting on invertebrates, and studies on their structure-activity relationships have been conducted. I have. The results of the study revealed that the difference in amino acid residues in the peptide is an important factor. Depending on whether the C-terminal amino acid is a Met residue or an Arg residue, vertebrate It was found to be classified into those that act and those that act on invertebrates. That is, it is considered that tachykinins can be classified into Met-type tachykinins and Arg-type tachykinins.

[0009] However, in order to clarify the structure-activity relationship and species specificity of these tachykinin-related peptides and obtain information that can be generalized to the nervous system of higher animals, tachykinin-related peptides can be obtained from a greater number of animal species. There is a need to find peptides and their precursor peptides.

[0010]

Accordingly, the present invention provides
We discovered a new invertebrate tachykinin-related peptide from invertebrates, clarified its structure, and elucidated the effects of the tachykinin-related peptide in animals themselves and the effects on other invertebrates. It is an object of the present invention to obtain a reagent for elucidating an information processing mechanism in a nervous system of a higher animal, and a novel tachykinin-related peptide that can be used as a basic compound in agrochemical or drug development. Furthermore, an object of the present invention is to clarify such a tachykinin-related peptide precursor polypeptide and a gene encoding the same, and to provide a method for producing such a tachykinin-related peptide.

[0011]

Among the mollusks that represent invertebrates, the octopus, which is classified as cephalopod, has a highly developed brain and has a motor function and a higher level than other molluscs. It has evolved dramatically in perceptual function. In particular, the circulatory system is the only closed blood vessel in molluscs, and it is thought that substances made in the brain are transported by blood vessels to peripheral tissues.

The present inventors have reported that octopus ( Oct)
Opus vulgaris ) has found vertebrate tachykinins from the posterior salivary glands and clarified their physiological effects. However, it was speculated that octopus species may also contain invertebrate tachykinin-related peptides. This time, the present inventors studied to obtain a new neuropeptide from octopus, and isolated and purified an invertebrate tachykinin-related peptide of octopus from octopus brain using its activity on the heart as an index. Thus, the present invention has been completed.

Further, the present inventors have clarified the precursor polypeptide of the tachykinin-related peptide thus obtained, and the gene encoding the same, and have clarified the precursor polypeptide and the tachykinin-related peptide using the gene. The manufacturing method has been completed.

Accordingly, the present invention provides an amino acid sequence represented by the following formula (I): R ¹ -Phe-Xaa-Yaa-Zaa-Arg-NH ₂ (I) wherein R ¹ is 5 or 6 amino acid residues. Xaa represents Leu, Ile, Met, Val or Gln, Yaa represents Gly or Pro, and Zaa represents Ser or Thr.

In a more specific embodiment, the present invention provides the following amino acid sequence formula (II): R ² -Aaa-Baa-Phe-Xaa-Yaa-Zaa-Arg-NH ₂ (II) ² represents a peptide having 3 or 4 amino acid residues, Aaa represents Asn, Tyr or Leu, Baa represents Ala, Ser, Thr or Asp, Xaa represents Leu, Ile, Met, Val or Gln; Yaa represents Gly or Pro, and Zaa represents Ser or Thr).

That is, to obtain an invertebrate tachykinin-related peptide, the present inventors have proposed octopus ( Octopus).
us vulgaris ), a search was conducted using the activity of the octopus on the heart of octopus as an index. As a result, among the amino acid sequence formulas (I) and (II), the following amino acid sequence formulas (1), (2) and (3):

H-Thr-Val-Ser-Ala-Asn-Ala-Phe-Leu-Gly-Ser-Arg-NH ₂ (1) (hereinafter, this peptide is referred to as compound (1))

H-Leu-Asn-Ala-Asn-Ser-Phe-Met-Gly-Ser-Arg-NH ₂ (2) (hereinafter, this peptide is referred to as compound (2))

H-Ala-Ser-Leu-His-Asn-Thr-Phe-Ile-Pro-Ser-Arg-NH ₂ (3) (hereinafter, this peptide is referred to as compound (3))

The peptide represented by the formula was isolated and purified, its chemical structure was determined, and its structure and its physiological activity against invertebrates were confirmed by total synthesis.

Furthermore, the present inventors prepared primers based on the above amino acid sequence, and used a reverse transcription polymerase chain reaction (RT-PCR) method or the like on total RNA prepared from octopus brain. By combining the means for analyzing the gene sequence, it was revealed that the entire primary amino acid sequence of the polypeptide which is the precursor of the tachykinin-related peptide is represented by SEQ ID NO: 4. Also,
The gene encoding the precursor polypeptide was found to have the nucleotide sequence shown in SEQ ID NO: 5.

A partial structure presumed to be a tachykinin-related peptide was searched based on the entire primary amino acid sequence of the precursor polypeptide of the tachykinin-related peptide thus determined. In addition to the amino acid sequence formulas (1) to (3), the C-terminal is Ar
Amino acid sequence formulas (4), (5) and (6) corresponding to the tachykinin-related peptide that is g residue:

H-Val-Asn-Pro-Tyr-Ser-Phe-Gln-Gly-Thr-Arg-NH ₂ (4) (hereinafter, this peptide is referred to as compound (4))

H-Ser-Asp-Ala-Leu-Ala-Phe-Val-Pro-Thr-Arg-NH ₂ (5) (hereinafter, this peptide is referred to as compound (5))

H-Tyr-Ser-Pro-Leu-Asp-Phe-Ile-Gly-Ser-Arg-NH ₂ (6) (hereinafter, this peptide is referred to as compound (6))

It was revealed that the peptide represented by Therefore, the precursors of these peptides were excised and amidated at the C-terminus, and the physiological activities of these peptides were examined.
It was confirmed that it had the same physiological activity as the tachykinin-related peptide of (3).

The present inventors further examined the entire primary amino acid sequence of the precursor polypeptide, and obtained the following amino acid sequence formula (7) in which the C-terminal was a Lys residue other than an Arg residue:

H-Arg-Met-Asn-Ser-Leu-Ser-Phe-Gly-Pro-Pro-Lys-NH ₂ (7) (hereinafter, this peptide is referred to as compound (7))

It was confirmed that the peptide represented by the formula (1) was a peptide having the same physiological activity as the tachykinin-related peptide, although the activity was slightly reduced.

Accordingly, in another aspect, the present invention provides an amino acid sequence represented by SEQ ID NO: 4 or an amino acid sequence in which a part thereof is deleted or substituted, or an amino acid in which the amino acid sequence or a part thereof is deleted or substituted. Disclosed is a tachykinin-related peptide precursor polypeptide having an amino acid sequence in which one or more amino acids are added to the sequence.

In still another aspect, the present invention provides a precursor polypeptide of such a tachykinin-related peptide and a method for producing the tachykinin-related peptide. In another aspect, the present invention provides a precursor polypeptide of such a tachykinin-related peptide Provided are a gene encoding a peptide, and a method for producing a precursor polypeptide and a tachykinin-related peptide using the gene.

[0032]

BEST MODE FOR CARRYING OUT THE INVENTION The novel peptides provided by the present invention are tachykinin-related peptides that enhance the pulsation of the octopus heart, and they include octopus ( Octopus vulg).
aris ) can be isolated and purified, for example, by the following method.

That is, the octopus brain is extracted with hot water, acetic acid is added to the extract to a concentration of 3%, and the mixture is cooled and centrifuged to obtain a crude extract. This crude extract is applied to a C18 cartridge (for example, Sep-Pak (registered trademark) Vac).
After adsorbing on C18 Cartridges: Waters, 0.1% trifluoroacetic acid (hereinafter TF)
A) and eluted with 60% methanol containing the same to separate the peptide fraction and subject it to ion exchange chromatography, reverse phase chromatography, etc. to separate and purify the desired peptide. Can be.

Further, since the peptide of the present invention is a peptide having 10 or 11 amino acid residues, a solid phase using a conventional peptide synthesizer (eg, a peptide synthesizer 433A manufactured by PE Biosystems Japan) is used. Synthesis methods,
It can be easily synthesized by ordinary organic synthetic chemistry techniques. The crude peptide obtained by these methods can be purified, if necessary, by ordinary purification techniques such as reversed-phase high-performance liquid chromatography and crystallization.

On the other hand, the amino acid sequence of the precursor polypeptide of the tachykinin-related peptide and the nucleotide sequence of the gene encoding the polypeptide can be determined as follows. That is, a primer was prepared based on the amino acid sequence of the tachykinin-related peptide obtained as described above, and a reverse transcription polymerase chain reaction (RT-PCR) was performed on total RNA prepared from octopus brain to obtain about 600 base pairs. , And subsequently, the 5'-end and 3'-end gene sequences can be elucidated using the 5'-RACE method and the 3'-RACE method. In the present invention, according to this method, the precursor polypeptide of the octopus tachykinin-related peptide is represented by SEQ ID NO: 4.
And that the gene encoding the precursor polypeptide has the base sequence shown in SEQ ID NO: 5.

Accordingly, the tachykinin-related peptide precursor polypeptide and tachykinin-related peptide of the present invention can also be produced by a gene recombination method.
That is, when the production is performed by a gene recombination method, for example, a vector incorporating the gene represented by SEQ ID NO: 5 is prepared, and after transforming a host with the vector, the host is cultured or grown. The target precursor polypeptide may be isolated and purified from the host. Then, in order to obtain the target tachykinin-related peptide from the precursor polypeptide, after excision (processing) from the precursor polypeptide using an enzyme or the like, the C-terminal is modified (amidation) using an amidating enzyme or the like. Then, if necessary, isolation and purification may be performed.

[0037]

The peptide provided by the present invention is a tachykinin-related neuropeptide, which is not only useful as a biochemical reagent for elucidating the neurotransmission system, but also a new approach to the development of medicines and agricultural chemicals. Can be used as a compound that gives

[0038] For example, as a medicament containing the peptide of the present invention as an active ingredient, an orally or parenterally in a suitable dosage form such as a capsule, a tablet, an injection or the like together with an excipient commonly used in pharmaceutical preparations. Can be administered in a controlled manner. In particular,
Capsules can be prepared by mixing the peptide of the present invention with excipients such as lactose, starch or derivatives thereof, and cellulose derivatives, and filling the resulting mixture with gelatin capsules.

Tablets are kneaded with a binder such as sodium carboxymethylcellulose, alginic acid, gum arabic, etc. in addition to the above-mentioned excipients, kneaded, and if necessary granulated as granules. Tablets can be prepared by adding a lubricant such as magnesium acid and using a conventional compression tableting machine.

Further, for parenteral administration, the peptide of the present invention can be dissolved in sterile distilled water or sterile physiological saline together with a solubilizing agent and enclosed in an ampoule to prepare an injectable preparation. In this case, if necessary, a stabilizer, a buffer substance, and the like may be contained. Alternatively, the powder may be filled in a vial, and the preparation may be dissolved in sterile distilled water before use. These parenteral preparations can be administered intravenously or intravenously.

The dose when the peptide of the present invention is used as an active ingredient is determined in consideration of various factors, for example, the condition to be treated, the condition and severity of the patient, the age of the patient, and the administration route. May be set as appropriate. In general, in the case of oral administration, the active ingredient is usually in the range of 0.1 to 1000 mg / day / human, preferably 1 to 500 mg / day / human. The dose can be appropriately selected and administered within a range of about 1/100 to 1/2 of the dose in that case.

[0042]

The present invention will be further described with reference to the following examples, but the scope of the present invention is not limited to these examples.

Example 1: Separation of peptides that enhance the pulsation of octopus heart from octopus (a): Crude extraction The brain (including the optic lobe) was excised from 100 octopus ( Octopus vulgaris ), and the mixture was extracted with liquid nitrogen. And stored frozen. The frozen excised tissue (180 g) was placed in 900 ml of boiling distilled water and boiled for 10 minutes. After cooling, acetic acid was added to a concentration of 3% and homogenized.
The supernatant was obtained by centrifugation at 12,000 xg for 30 minutes at ° C. Meanwhile, 200 ml of 3% acetic acid was added to the precipitate,
After homogenizing again, centrifugation was performed under the same operating conditions to obtain a supernatant. The collected supernatant was concentrated under reduced pressure to about 200 ml to obtain a crude extract.

(B): Adsorption on C18 cartridge To the crude extract obtained in (a), 5 ml of 6N-HCl was added, and the mixture was centrifuged at 12,000 xg at 4 ° C for 30 minutes. The obtained supernatant was used for Sep-Pak (registered trademark) Va.
c Passed through a C18 cartridge (10 g, 35 cc, Waters). 0.1% TFA2 cartridge
After washing with 00 ml, the retentate was reduced to 60% methanol /
The eluate was eluted with 100 ml of 0.1% TFA, and the eluate was concentrated under reduced pressure and freeze-dried to obtain 0.150 g of a dried product.

(C) Reversed-phase high-performance liquid column chromatography (1) The dried product obtained in (b) was subjected to Capcell pak.
The mixture was subjected to reversed-phase high-performance liquid column chromatography (reverse-phase HPLC) using C18 UG80 (5 μm, Φ10 × 250 mm, manufactured by Shiseido) at a flow rate of 1.5 ml / min.
Elution was performed with a linear gradient of 0% to 60% acetonitrile in 0.1% TFA (pH 2.2) for 60 minutes. 2
The mixture was fractionated into 3 ml portions by monitoring the ultraviolet absorption at 15 nm.

Each fraction was subjected to a bioassay in accordance with the method described in Example 7 described later. As a result, the fraction eluted with acetonitrile at a concentration of 26 to 30% showed heartbeat-enhancing activity with respect to the heart of octopus.

(D) Cation exchange column chromatography The active fraction obtained in (c) was subjected to TSKgel SP-5.
It was subjected to cation exchange column chromatography (cation exchange HPLC) using PW (10 μm, Φ7.5 × 75 mm, manufactured by Tosoh Corporation), and at a flow rate of 1.0 ml / min, 1
0M in 60 mM phosphate buffer (pH 7.0) for 60 minutes
To a 0.6M NaCl linear gradient.

When a bioassay was performed on the fractions of 2 ml each,
Fraction eluted at a 0.08 M NaCl concentration (hereinafter referred to as fraction A), a fraction eluted at a 0.12 M NaCl concentration (hereinafter referred to as fraction B), and 0.16 M NaC
Activity was observed in the fraction eluted at 1 concentration (hereinafter referred to as fraction C).

Example 2 Purification of Peptides from Active Fraction (Part 1: Purification from Active Fraction A) With respect to the active fraction A obtained by the separation operation of Example 1, the following operation was further performed. The peptides were purified.

(A) Reversed phase HPLC (1) The active fraction A obtained in (d) of Example 1 was
11 pak C18UG80 (5 μm, Φ4.6 × 1
Reversed phase HPLC using 50 mm, manufactured by Shiseido Co., Ltd. was performed at a flow rate of 1.0 ml / min and 0.1% TFA (pH 2.0).
In 2), elution was performed with a linear gradient of 10 to 30% acetonitrile in 40 minutes. Fractionation was performed in 1 ml portions, and activity was observed in the fraction eluted at an acetonitrile concentration of about 19%.

(B) Reversed phase HPLC (2) The active fraction obtained in (a) was subjected to Capcell pak
C18 UG80 (5 μm, Φ4.6 × 150 mm,
Reverse phase HPLC using Shiseido), and the flow rate was 0.5 m.
At 1 / min, developed with acetonitrile 18% in 0.1% TFA (pH 2.2). A single compound was obtained at a retention time of 14 minutes, and was designated as Compound (1). This reversed phase HPL
The development of C is shown in FIG.

Example 3 Purification of Peptides from Active Fraction (Part 2: Purification from Active Fraction B) With respect to the active fraction B obtained by the separation operation of Example 1, the following operation was further performed. The peptides were purified.

(A) Reverse phase HPLC (1) The active fraction B obtained in (d) of Example 1 was
11 pak C18UG80 (5 μm, Φ4.6 × 1
Reversed phase HPLC using 50 mm, manufactured by Shiseido Co., Ltd. was performed at a flow rate of 1.0 ml / min and 0.1% TFA (pH 2.0).
In 2), elution was performed with a linear gradient of 10 to 30% acetonitrile in 40 minutes. The fractions were fractionated by 1 ml, and the activity was observed in the fraction eluted at an acetonitrile concentration of about 18%.

(B) Reversed phase HPLC (2) The active fraction obtained in (a) was subjected to Capcell pak
C18 UG80 (5 μm, Φ4.6 × 150 mm,
Reverse phase HPLC using Shiseido), and the flow rate was 0.5 m.
Developed with 16% acetonitrile in 0.1% TFA (pH 2.2) at 1 / min. A single compound was obtained at a retention time of 16 minutes, and was designated as Compound (2). This reversed phase HPL
The development of C is shown in FIG.

Example 4 Purification of Peptides from Active Fraction (Part 3: Purification from Active Fraction C) The following procedure was further performed on the active fraction C obtained by the separation operation of Example 1, The peptides were purified.

(A) Reversed phase HPLC (1) The active fraction C obtained in (d) of Example 1 was
11 pak C18UG80 (5 μm, Φ4.6 × 1
Reversed phase HPLC using 50 mm, manufactured by Shiseido Co., Ltd. was performed at a flow rate of 1.0 ml / min and 0.1% TFA (pH 2.0).
In 2), elution was performed with a linear gradient of 10 to 30% acetonitrile in 40 minutes. The fractions were fractionated by 1 ml, and the activity was observed in the fraction eluted at an acetonitrile concentration of about 18%.

(B) Reversed phase HPLC (2) The active fraction obtained in (a) was subjected to Capcell pak
C18 UG80 (5 μm, Φ4.6 × 150 mm,
Reverse phase HPLC using Shiseido), and the flow rate was 0.5 m.
Developed with 16% acetonitrile in 0.1% TFA (pH 2.2) at 1 / min. A peak was observed at a retention time of 16.7 minutes, but it was possible that another substance was contaminated. Therefore, reverse phase HPLC was performed again under the same conditions, and a single compound was observed at a retention time of 16.7 minutes. This was designated compound (3). FIG. 3 is a development view of this reverse phase HPLC.

Example 5: Identification of neuropeptides The structures of the compound (1) purified in Example 2 above, the compound (2) purified in Example 3 and the compound (3) purified in Example 4 are shown below. The analysis was carried out using a Shimadzu PSQ-1 gas phase sequencer (manufactured by Shimadzu Corporation). The obtained amino acid sequences are shown in Table 1 below.

[0059]

[Table 1] Amino acid sequence of peptide (pmol)

The molecular weight of compound (1), compound (2) and compound (3) was determined by MALDI TOF-MS (Voy
ager Elite, manufactured by PE Biosystems Japan). The measured values are shown in Table 2 below.

[0061]

[Table 2] MS data of peptides

Based on the above instrumental analysis data, the active fraction A
Compound (1), which is a more isolated and purified octopus neuropeptide, has the following amino acid sequence formula (1): H-Thr-Val-Ser-Ala-Asn-Ala-Phe-Leu-Gly-. It was revealed that the compound was represented by Ser-Arg-NH ₂ (1).

The compound (2), which is an octopus neuropeptide isolated and purified from the active fraction B, has the following amino acid sequence formula (2): H-Leu-Asn-Ala-Asn-Ser- It was revealed that the compound was represented by Phe-Met-Gly-Ser-Arg-NH ₂ (2).

Further, compound (3), which is an octopus neuropeptide isolated and purified from the active fraction C, has the following amino acid sequence formula (3): H-Ala-Ser-Leu-His-Asn- It was found to be represented by Thr-Phe-Ile-Pro-Ser-Arg-NH ₂ (3).

Experimental Example 6: Synthesis of Madako Neuropeptides by Solid Phase Method Each of the octopus neuropeptides was synthesized by the FastMoc (registered trademark) method using a fully automatic peptide synthesizer 433A manufactured by PE Biosystems Japan. Synthesized.

The synthesis of compound (1) was performed using Fmoc
Fmoc-Thr (tBu), Fmoc-Va, using -NH-SAL-A resin (manufactured by Watanabe Chemical Industry Co., Ltd.) as a carrier.
1, Fmoc-Ser (tBu), Fmoc-Ala,
Fmoc-Asn (Trt), Fmoc-Phe, Fm
oc-Leu, Fmoc-Gly and Fmoc-A
rg (Pmc) was used.

For the synthesis of compound (2), Fmoc
-NH-SAL-A resin (manufactured by Watanabe Chemical Industry Co., Ltd.) as a carrier, Fmoc-Leu, Fmoc-Asn (Tr
t), Fmoc-Ala, Fmoc-Ser (tB
u), Fmoc-Phe, Fmoc-Met, Fmoc
-Gly and Fmoc-Arg (Pmc) were used.

Further, for the synthesis of compound (3), Fmo
Using c-NH-SAL-A resin (manufactured by Watanabe Chemical Industry Co., Ltd.) as a carrier, Fmoc-Ala, Fmoc-Ser (tB
u), Fmoc-Leu, Fmoc-His (Tr
t), Fmoc-Asn (Trt), Fmoc-Thr
(TBu), Fmoc-Phe, Fmoc-Ile, F
moc-Pro and Fmoc-Arg (Pmc) were used.

However, Fmoc is 9-Fluoreny.
lmethycarbonyl, tBu is tB
util, Trt is trityl, Pmc is 2,
2,5,7,8-pentamethyl chroma
2 shows an-6-sulfonyl.

For the separation and deprotection of the crude peptide from the peptide resin after the reaction, 4.3% of phenol / 1,2
-Ethanedithiol 2.1% / thioanisole 4.3%
/ Water 4.3% / TFA 85% was used. The reaction mixture was filtered, ether was added to the filtrate to precipitate the peptide, and the precipitate was washed three times with ether to obtain about 100 mg of the crude peptide. Approximately 10 mg of the crude peptide was replaced with reverse phase HPL.
Purification by C yielded about 6 mg of each purified peptide.

Each of the obtained purified peptides was Capcel
In reverse phase HPLC using 1 pak C18, the retention times were completely consistent with each of compounds (1), (2) and (3). Also, in the heartbeat activity of the octopus heart, the synthetic compounds were similar to their natural products.

Example 7: Measurement of the heartbeat activity of octopus heart The heartbeat activity of octopus heart was determined by Morishita et al. (Fumihiro)
Morishita, Biochem. Biophy
s. Res. Commun. , 240, 354-35
8, 1997). That is,
The heart of the octopus was excised, both atriums were cut, and a cannula was inserted from each atrioventricular into the ventricle, and the chamber (capacity: 80 ml) was tied with a cotton thread and fixed. Also, sandwich the aorta so as not to prevent the solution in the ventricle from flowing out,
The test specimen was connected to a tension transducer.
The artificial seawater (containing 1% glucose) was set to flow at 1 to 2 ml / min from the cannula. The sample to be assayed was dissolved in 1 ml of the same artificial seawater, added to reach the inside of the heart from the cannula, and the change in heart beat was recorded.

The results are shown in FIGS. As is clear from the results shown in each figure, compound (1) [FIG. 4], compound (2) [FIG. 5] and compound (3) [FIG. 6] each enhance the pulsation of octopus heart. .

Example 8: Measurement of the contractile activity of the cockroach made of cockroaches The contractile activity of the cockroach made of cockroaches was measured by Cook et al.
B. J. et al. , Comp. Biochem.
Physiol. , 61C, 291-295, 199
This was performed according to the method of 8). That is, the intestinal tract of the cockroach was removed, the both ends of the hindgut were tied with a cotton thread, one end was fixed to a chamber (capacity: 2 ml), and the other end was connected to a tension transducer to prepare a test sample.
The sample to be assayed was dissolved in physiological saline, added to the chamber, and the change in length due to contraction was recorded.

The results are shown in FIGS. As is clear from the results shown in each figure, the compound (1) [FIG. 7], the compound (2) [FIG. 8] and the compound (3) [FIG. 9] each contract the hindgut of the cockroach.
From this, it was revealed that the peptides of the present invention, that is, the compounds (1), (2) and (3) are tachykinin-related peptides acting on invertebrates.

Example 9: Determination of the entire amino acid structure of the precursor polypeptide and the nucleotide sequence of the gene coding for it (1) Preparation of octopus brain total RNA About 1 g of octopus brain is ground in liquid nitrogen and 10 ml of T
After dissolving in RIzol (registered trademark) reagent (GIBCO BRL), it was homogenized. After standing at room temperature for 5 minutes, 1 ml was dispensed into Eppendorf tubes,
After adding 200 μl of chloroform and stirring, the mixture was centrifuged (15,000 rpm) using a cooling centrifuge (manufactured by Sakuma Seisakusho).
m, 15 minutes, 4 ° C). The upper aqueous layer was collected in an Eppendorf tube, 0.5 ml each of isopropanol was added, and the mixture was allowed to stand at room temperature for 10 minutes. After centrifugation (15,000 rpm, 10 minutes, 4 ° C.) with a cooling centrifuge, the supernatant was removed, 1 ml of 75% ethanol was added, and the mixture was centrifuged again (10,000 rpm, 5 minutes, 4 ° C.). The supernatant was removed, air-dried for about 10 minutes, 10 μl of DEPC-treated water was added, and the mixture was incubated at 60 ° C. for 10 minutes to obtain RNA.
Was dissolved. Approximately 250 μg of total
RNA was obtained.

(2) degenerate 3'-RA
CE The following degenerate primers were designed based on the sequence of the peptide isolated from the octopus brain and synthesized by a conventional method. Oct-B-Tac-1: 5'-TIAA (C / T) GCIAA (C / T) (T / A) (C / G) IT
T (C / T) ATGGG-3 'Oct-B-Tac-2: 5'-TT (C / T) ATGGGI (T / A) (C / G) I (C / A) GI
GGIAA-3 '(English characters in each formula are based on "Nucleotide abbreviation table" (Cell Engineering separate volume "Bio-Experimental Illustrated": Shujunsha); the same applies to the following formulas.)

Next, the 5 '/ 3'-RACE Kit (B
oehringer Mannheim), and degenerate 3'-RAC according to the following procedure.
E was performed. That is, 2 μg of total RNA,
4 μl of cDNA synthesis buffer
r, 2 μl dNTP mix, 1 μl oligo
dT-anchor primer (12.5 pmol
/ Μl), 1 μl of AMV reverse trans
cryptase (20 units / μl), DEPC
-Mix treated water to make a total volume of 20 μl, incubate at 55 ° C for 60 minutes, then treat at 65 ° C for 10 minutes, 1s
t-strand cDNA was synthesized.

Next, under the following conditions, 1st-3'-RAC
E was performed. That is, 3 μl of the first-strand
cDNA, 5 μl of 10 × PCR buffer, 8
μl dNTP mix, 3 μl Oct-B-Tac
-1 (100 pmol / μl), 1 μl of PCR an
chor primer (12.5 pmol / μl),
0.5 μl of TaKaRa Ex Taq®
(Takara Shuzo Co., Ltd.) and water to make a total volume of 50 μl,
5 minutes at 94 ° C. for 30 seconds, 48 ° C. for 30 seconds, 72 ° C.
The reaction was performed at 30 ° C. for 2 minutes and 30 seconds and then at 72 ° C. for 5 minutes. The polymerase chain reaction (PCR) involves G
eneAmp PCR System2400 the
rmal cycler (Perkin-Elmer) was used.

Subsequently, the first-PCR product was applied to a spin column (MicroSpin (registered trademark) S-400, Am
and 2nd-3′-RACE under the following conditions. That is, 3 μl of the 1st-PCR product, 5 μl of 10 × PC
R buffer, 8 μl dNTP mix, 3 μl
Oct-B-Tac-2 (100 pmol / μl),
1 μl of PCR anchor primer (12.
5 pmol / μl), 0.5 μl of TaKaRaEx
Taq (registered trademark) (manufactured by Takara Shuzo Co., Ltd.) and water are mixed to make a total volume of 50 μl.
The reaction was performed at 5 ° C for 30 seconds, at 72 ° C for 1 minute and 30 seconds for 30 cycles, and then at 72 ° C for 5 minutes. Add 5 μl of the reaction solution to 1.5
As a result of electrophoresis on a% agarose gel, amplification of a PCR product of about 600 bp was observed.

(3) Ligation reaction of PCR product (ligat
ion) The PCR product was purified by spin column, and 8 μl and 2 μl of the TA cloning vector pCR2.1 (In
Vitrogene), 10 μl Ligatio
n high (manufactured by Toyobo Co., Ltd.) was mixed and ligation was performed at 16 ° C. for 1 hour.

(4) Transformation of Escherichia coli 20 μl of the ligation reaction solution obtained in the above (3) was applied to competent cells Competent high E. coli . coli
The mixture was mixed with DH5α (manufactured by Toyobo Co., Ltd.), allowed to stand on ice for 30 minutes, and then subjected to heat shock at 42 ° C. for 50 seconds.
After cooling in ice for 2 minutes, 1 ml of SOC media
m, and incubated at 37 ° C. for 30 minutes. Subsequently, LB / Amp. (LB containing 50 μg / ml ampicillin) 35 μl of X-gal was spread on an agar medium, and then 10 μl of a transformant was sown. The remaining transformants were also spun down at 10,000 rpm for 1 minute to a volume of 100 μl.
1 and reduce the total amount to LB / Amp. Plated on agar medium. The medium was cultured at 37 ° C. overnight.

(5) Colony PCR Colony PCR was performed using the obtained colony as a template under the following conditions. That is, E. coli cells and 5 μl of 10 × Reaction Buffer
r, 5 μl of 2 mM dNTP mix, 3 μl of 25
mM MgCl ₂ , 0.5 μl of M13FW prim
er (100 pmol / μl), 0.5 μl of M13R
V primer (100 pmol / μl), 0.5μ
1 rTaq DNA polymerase (manufactured by Toyobo Co., Ltd.) and water to a total volume of 50 μl. Allowed to react for minutes.
5 μl of the reaction solution was electrophoresed on a 1.5% agarose gel. The M13FW primer used in this reaction was used.
r and M13RV primer were synthesized by a conventional method. The sequence is shown below.

M13FW: 5'-GTAAAACGACGGCCAGTG-3 'M13RV: 5'-GGAAACAGCTATGACCATG-3'

(6) DNA Sequence A colony (colon) of a desired size (about 800 bp)
y) The PCR product is purified by a spin column to give DNA S
EQing Kit (PE Biosystem)
(manufactured by MS Co., Ltd.). The sequence includes the ABIPRISM 310 Genetic
Analyzer (manufactured by PE Biosystems)
Was used. The obtained sequence is used for gene analysis software GENE.
As a result of analysis using TYX-MAC (manufactured by Software Development Corporation), a partial cDNA sequence of about 600 bp was clarified.

(7) 5'-RACE The following primers were synthesized from the base sequence of the partial cDNA. TKRP-5'-1R: 5'-GTAAGATCGTTTAGTTGGGG-3 'TKRP-5'-2R: 5'-TTTAGTTGGGGCTTTTCACC-3'TKRP-5'-3R:5'-TTTCACCGAGTAAGGTAACC-3'

Next, the 5 '/ 3'-RACE Kit (B
5′-RACE was performed using Oehringer Mannheim) according to the following procedure. First, 2 μg of total RNA and 4 μl of cDNA synthe
sis buffer, 2 μl dNTP mix, 1
μl of TKRP-5′-1R (12.5 pmol / μl
l) 1 μl of AMV reverse transsc
riptase (20 units / μl), DEPC-
Mix the treated water to a total volume of 20 μl, incubate at 55 ° C. for 60 minutes, treat at 65 ° C. for 10 minutes, and
t-strand cDNA was synthesized. Then 1s
After purifying the t-strand cDNA with a spin column, 2.5 μl of reaction buffer,
2.5 μl of 2 mM dATP was added and the mixture was treated at 94 ° C. for 3 minutes, and 1 μl of terminal trans was added thereto.
Add ferase (10 units / μl) and add 37 ° C
And then treated at 70 ° C. for 10 minutes to synthesize dA-tailed cDNA.

Next, 1st-PCR and 2nd-P
CR was performed under the following conditions. 1st-PCR: 5 μl of dA-tailed c
DNA, 5 μl of 10 × PCR buffer, 8 μl
DNTP mix, 1 μl TKRP-5′-2R
(12.5 pmol / μl), 1 μl oligo d
T-anchor primer (12.5 pmol /
μl), 0.5 μl of TaKaRa Ex Taq (registered trademark) (manufactured by Takara Shuzo Co., Ltd.) and water are mixed to a total volume of 50 μl.
5 minutes at 94 ° C, 30 seconds at 94 ° C, 3 ° C at 55 ° C.
The reaction was carried out at 0 ° C. for 30 cycles at 72 ° C. for 2 minutes and then at 72 ° C. for 7 minutes.

2nd-PCR: 3 μl spin column purified 1st-PCR product, 5 μl 10 × PCR
buffer, 8 μl dNTP mix, 1 μl T
KRP-5'-3R (12.5 pmol / μl), 1μ
l PCR anchor primer (12.5p
mol / μl), 0.5 μl of TaKaRa Ex T
aq (registered trademark) (manufactured by Takara Shuzo Co., Ltd.) and water were mixed to make a total volume of 50 μl, and the same cycle as 1st-PCR was performed.

When the 2nd-PCR product obtained by the above method was electrophoresed on a 1.5% agarose gel, a band of about 400 bp was confirmed. This 2nd-PCR
The product was sequenced according to the methods described in methods (3), (4), (5) and (6) above.

As a result of the sequence analysis, the 5 ′ side was incomplete. Therefore, the following primers were synthesized by a conventional method, and 5′-RACE was performed again.

TKRP-5'-4R: 5'-TTCAGCAGATTCTGAGTCGG-3 'TKRP-5'-5R: 5'-TCTACTTTCGATAGTGTTGGC-3' TKRP-5'-6R: 5'-GACGTTATTACTGTCCAACTC-3 '

That is, 2 μg of total RNA,
4 μl of cDNA synthesis buffer
r, 2 μl dNTP mix, 1 μl TKRP-
5′-4R (12.5 pmol / μl), 1 μl of AM
V reverse transscriptase (2
0 units / μl), mixed with DEPC-treated water to make a total volume of 20 μl, incubated at 55 ° C. for 60 minutes, and then treated at 65 ° C. for 10 minutes to give 1st-strand
cDNA was synthesized.

Next, 1st-strand cDNA
Was purified by a spin column, and 2.5 μl of react
ion buffer, 2.5 μl of 2 mM dATP
And treated at 94 ° C. for 3 minutes, into which 1 μl of ter
minal transfer (10 units
/ Μl) and incubate at 37 ° C for 20 minutes.
DA-tailed cDNA by treating at 0 ° C for 10 minutes
Was synthesized.

Next, 1st-PCR and 2nd-P
CR was performed under the following conditions. 1st-PCR: 5 μl of dA-tailed c
DNA, 5 μl of 10 × PCR buffer, 8 μl
DNTP mix, 1 μl TKRP-5′-5R
(12.5 pmol / μl), 1 μl oligo d
T-anchor primer (12.5 pmol /
μl), 0.5 μl of TaKaRa Ex Taq (registered trademark) (manufactured by Takara Shuzo Co., Ltd.) and water are mixed to a total volume of 50 μl.
5 minutes at 94 ° C, 30 seconds at 94 ° C, 3 ° C at 55 ° C.
The reaction was carried out at 0 ° C. for 30 cycles at 72 ° C. for 2 minutes and then at 72 ° C. for 7 minutes.

2nd-PCR: 3 μl spin column purified 1st-PCR product, 5 μl 10 × PCR
buffer, 8 μl dNTP mix, 1 μl T
KRP-5'-6R (12.5 pmol / μl), 1μ
l PCR anchor primer (12.5p
mol / μl), 0.5 μl of TaKaRa Ex T
aq (registered trademark) (manufactured by Takara Shuzo Co., Ltd.) and water were mixed to make a total volume of 50 μl, and the same cycle as 1st-PCR was performed.

When the 2nd-PCR product obtained by the above method was electrophoresed on a 1.5% agarose gel, a band of about 500 bp was confirmed. This 2nd-PCR
The product is prepared according to methods (3), (4), (5) and (6) above.
Were sequenced according to the method described in (1).

As a result, the size of the cDNA encoding the precursor polypeptide of the tachykinin-related peptide (about 1.
7 kb), and the number (266 amino acid residues) and sequence of the deduced amino acids could be determined. The amino acid sequence is shown in SEQ ID NO: 8, and the sequence of cDNA is shown in SEQ ID NO: 9.

The precursor polypeptide of the tachykinin-related peptide represented by SEQ ID NO: 8 determined as described above was examined to cut out a portion having an amino acid sequence corresponding to the tachykinin-related peptide provided by the present invention. As a result, it was found that in addition to the compounds (1) to (3) isolated in Examples 1 to 5, the compounds (4) to (7) presumed to be tachykinin-related peptides were included. These are shown in FIG. 10 at the portion corresponding to the underline.

Compounds (4) to (4) obtained by excision from the precursor polypeptide based on this sequence formula and modification of the C-terminus
For (7), the heartbeat activity of the heart of the octopus described in Example 7 and the contraction activity of the cockroach of the cockroach described in Example 8 were measured.
Activity similar to (3) was observed. In addition, compound (7)
Has an activity intensity of 1 / 1,0 compared to compounds (1) to (3).
It was about 00, which seems to be due to the C-terminal being Lys.

Example 10: Formulation Example Tablet Composition: Compound (1), (2) or (3) 10 g Lactose 125 g Microcrystalline cellulose 25 g Corn starch 25 g 5% hydroxypropyl methylcellulose 100 ml Magnesium stearate 5 g After kneading, granulating, drying and tableting,
A tablet weighing 190 mg containing 10 mg of compound (1), compound (2) or compound (3) as an active ingredient in the tablet was obtained.

[0102]

Industrial Applicability The peptides of the present invention are neuropeptides having the effect of enhancing the heartbeat of octopus hearts and contracting the hindgut of the cockroach, and are useful as biochemical reagents for elucidating the neurotransmission system. is there.

Furthermore, the peptides of the present invention are tachykinin-related neuropeptides obtained from the mollusk octopus, and provide a new approach to medicines, agricultural chemicals and the like.

The tachykinin-related peptide precursor polypeptide obtained by the present invention and the gene encoding the same are important tools for producing the tachykinin-related peptide of the present invention.

[0105]

[Sequence List] SEQUENCE LISTING <110> SUNTORY LIMITED <120> TACHYKININ RELATED PEPTIDE <130> SN186 <141> 2000-05-11 <150> JP11-260982 <151> 1999-09-14 <160> 9 <170> PatentIn Ver. 2.1 <210> 1 <211> 11 <212> PRT <213> OCTOPUS VULGARIS <400> 1 Thr Val Ser Ala Asn Ala Phe Leu Gly Ser Arg 1 5 10 <210> 2 <211> 10 <212> PRT <213> OCTOPUS VULGARIS <400> 2 Leu Asn Ala Asn Ser Phe Met Gly Ser Arg 1 5 10 <210> 3 <211> 11 <212> PRT <213> OCTOPUS VULGARIS <400> 3 Ala Ser Leu His Asn Thr Phe Ile Pro Ser Arg 1 5 10 <210> 4 <211> 10 <212> PRT <213> OCTOPUS VULGARIS <400> 4 Val Asn Pro Tyr Ser Phe Gln Gly Thr Arg 1 5 10 <210> 5 <211> 10 <212> PRT <213> OCTOPUS VULGARIS <400> 5 Ser Asp Ala Leu Ala Phe Val Pro Thr Arg 1 5 10 <210> 6 <211> 10 <212> PRT <213> OCTOPUS VULGARIS <400> 6 Tyr Ser Pro Leu Asp Phe Ile Gly Ser Arg 1 5 10 <210> 7 <211> 11 <212> PRT <213> OCTOPUS VULGARIS <400> 7 Arg Met Asn Ser Leu Ser Phe Gly Pro Pro Lys 1 5 10 <210> 8 < 211> 265 <212> PRT <213> OCTOPUS VULGARIS <400> 8 Met Tyr A la Ile Arg Val Ser Ser Ser Arg Arg Ile Ser Cys Phe Leu 1 5 10 15 Gln Val Ser Leu Leu Phe Ser Gly Leu Leu Asn Thr Leu Gln Trp Gly 20 25 30 Ala Phe Ala Ser Tyr Leu Pro Lys Gln Leu Glu Pro Gln Gln Lys Asn 35 40 45 Tyr Glu Leu Asp Ser Asn Asn Val Asn Ser Ser Arg Tyr Pro Gly His 50 55 60 Glu Glu Ile Met Glu Phe Leu Arg Lys Leu Gly Phe Leu Ala Asn Thr 65 70 75 80 Ile Glu Ser Arg Ala Ser Ile Asp Asp Thr Asp Ser Glu Ser Ala Glu 85 90 95 Arg Met Lys Lys Val Asn Pro Tyr Ser Phe Gln Gly Thr Arg Gly Lys 100 105 110 Arg Leu Asn Ala Asn Ser Phe Met Gly Ser Arg Gly Lys Arg Val Leu 115 120 125 Val Asn Asp Lys Arg Thr Val Ser Ala Asn Ala Phe Leu Gly Ser Arg 130 135 140 Gly Lys Arg Leu Val Thr Leu Leu Gly Glu Lys Pro Gln Leu Asn Asp 145 150 155 160 Leu Thr Gly Val Ile Asp Lys Lys Ser Asp Ala Leu Ala Phe Val Pro 165 170 175 Thr Arg Gly Arg Ser Gln Ser Thr Asn Asp Glu Leu Ala Tyr Thr Gly 180 185 190 Pro Met Leu Leu Arg Glu Gly Arg Arg Met Asn Ser Leu Ser Phe Gly 195 200 205 Pro Pro Lys Gly Lys Lys Tyr Se r Pro Leu Asp Phe Ile Gly Ser Arg 210 215 220 Gly Lys Lys Ser Thr Phe Glu Asp Tyr Val Leu Asn Thr Gln Asn Asp 225 230 235 240 Ser Pro Lys Glu Asn Tyr Glu Arg Arg Ala Ser Leu His Asn Thr Phe 245 250 255 Ile Pro Ser Arg Gly Lys Arg Ser Thr 260 265 <210> 9 <211> 1660 <212> DNA <213> OCTOPUS VULGARIS <220> <221> CDS <222> (384) .. (1178) <400> 9 caactctaat ttctgcagac gatctttttt tccgaacata caagcagtta catttattga 60 aaaaaaaaag atatatatat atagagagag aacaaagaaa aacttaaaca cttttttaaa 120 gaactgcata ttttcatatt tatttaataa ctttcttcac tttcatccct gtctccactt 180 agctctgctt ttcctttgtt ttattctaca tcatctacct atctagctat ctctctacct 240 actcaatgta tctttctgtt tatctgtctg tctgtctgtc tattcaatca acacacacat 300 atatttacgc gcacacacac cccagttttc tttctttcta gcattccttc tttctttctt 360 ttcgtctttc ttcttcttcc ctc atg tat gca att aga gta tca agc tca agg 413 Met Tyr Ala Ile Arg Val Ser Ser Ser Arg 1 5 10 cga ata agc tgt ttt tta caa gta tcc ttg cta ttt tcc gga ctt ttg 461 Arg Ile Ser Cys Phe Leu Gln Val Ser Leu Leu Phe Ser Gl y Leu Leu 15 20 25 aac act ttg caa tgg ggt gcg ttt gcc agt tat cta ccc aag caa ttg 509 Asn Thr Leu Gln Trp Gly Ala Phe Ala Ser Tyr Leu Pro Lys Gln Leu 30 35 40 gaa cca cag cag aag aac tat gag ttg gac agt aat aac gtc aac agt 557 Glu Pro Gln Gln Lys Asn Tyr Glu Leu Asp Ser Asn Asn Val Asn Ser 45 50 55 agt agg tac cca ggc cat gaa gaa att atg gaa ttt tta cgc aaa ctg 605 Ser Arg Tyr Pro Gly His Glu Glu Ile Met Glu Phe Leu Arg Lys Leu 60 65 70 ggt ttc ctg gcc aac act atc gaa agt aga gct tca att gac gac acc 653 Gly Phe Leu Ala Asn Thr Ile Glu Ser Arg Ala Ser Ile Asp Asp Thr 75 80 85 90 gac tca gaa tct gct gaa aga atg aaa aaa gtt aac ccc tac agt ttc 701 Asp Ser Glu Ser Ala Glu Arg Met Lys Lys Val Asn Pro Tyr Ser Phe 95 100 105 caa ggt acc aga ggc aaa aga tta aac gca aac agt ttc atg ggt tcc 749 Gln Gly Thr Arg Gly Lys Arg Leu Asn Ala Asn Ser Phe Met Gly Ser 110 115 120 cga ggt aaa aga gtc ctc gtt aat gac aaa cgg act gtc agt gct aac 797 Arg Gly Lys Arg Val Leu Val Asn Asp Lys Arg Thr Val Ser Ala Asn 125 130 135 gct ttc ctc gga tcg agg ggt aaa cgt ctg gtt acc tta ctc ggt gaa 845 Ala Phe Leu Gly Ser Arg Gly Lys Arg Leu Val Thr Leu Leu Gly Glu 140 145 150 aag ccc caa cta aac gat ctt acg ggagt att gac aaa aaa tca gat 893 Lys Pro Gln Leu Asn Asp Leu Thr Gly Val Ile Asp Lys Lys Ser Asp 155 160 165 170 gct tta gca ttc gtc cca aca aga ggc aga tca caa tca acc aat gac 941 Ala Leu Ala Phe Val Pro Thr Arg Gly Arg Ser Gln Ser Thr Asn Asp 175 180 185 gaa ctt gct tac acg ggt ccg atg tta ttg aga gaa ggc cga aga atg 989 Glu Leu Ala Tyr Thr Gly Pro Met Leu Leu Arg Glu Gly Arg Arg Met 190 195 200 aat tcc tta tcg ttt ggt ccc cca aaa ggc aaa aaa tac agt cca ctt 1037 Asn Ser Leu Ser Phe Gly Pro Pro Lys Gly Lys Lys Tyr Ser Pro Leu 205 210 215 gac ttc att gga tcg cgt ggc aag aaa agt act ttt gaa gattat gtg 1085 Asp Phe Ile Gly Ser Arg Gly Lys Lys Ser Thr Phe Glu Asp Tyr Val 220 225 230 tta aat acc caa aac gat tca cca aag gag aac tac gaa agg cgg gcg 1133 Leu Asn Thr Gln Asn Asp Ser Pro Lys Glu Asn Tyr Glu Arg Arg Ala 235 240 245 250 tct tta cac aac aca ttc ata cct tcg cga ggt aag cga tcc aca 1178 Ser Leu His Asn Thr Phe Ile Pro Ser Arg Gly Lys Arg Ser Thr 255 260 265 taaagagacc cgagcaacca ttgactgatca ctgact accattacat tcctcccaac taccactacc actaccactc acatcactat taccaccact 1298 gattaaccaa ttaaccaaac aaactaatca accgtgattg ctgccatctt gcaaattgtc 1358 cagtggttac gaccagatac aatgagattc gaagagattc gacaacatat agtctgcttt 1418 aaaaacgctt cgcgtattaa ctgtttgacg taataatctc atgttacaca aaaacttgtg 1478 cgtgtaattt acagaatgtt tttgtcgttt ttttttaatt ataattttcc agtgttttcc 1538 ataatggatc ctgcgattac tttaggttgt atgacaataa ataaaaaaaa ctgttttctt 1598 ttggaataaa tcattcgtta aggaatcatg aactaaaaaa aaaaaaaaaa aaaaaaaaaa 1658 aa 1660

[Brief description of the drawings]

FIG. 1 is a developed view of reverse phase HPLC showing the final elution pattern of compound (1) which is a peptide of the present invention in Example 2.

FIG. 2 is a developed view of reverse phase HPLC showing the final elution pattern of the peptide (2) of the present invention in Example 3.

FIG. 3 is a development view of reverse phase HPLC showing the final elution pattern of compound (3), which is a peptide of the present invention, in Example 4.

FIG. 4 is a graph showing the results of enhancing the pulsation of the octopus heart of the octopus of the compound (1) of the present invention in Example 7, wherein the amount of the compound (1) is equivalent to one octopus; Shows the results when was added to the chamber.

FIG. 5 is a graph showing the results of enhancing the pulsation of the heart of octopus of compound (2), which is the peptide of the present invention, in Example 7; Shows the results when was added to the chamber.

FIG. 6 is a graph showing the results of enhancing the pulsation of the octopus heart of the octopus of the compound (3) of the present invention in Example 7, wherein the amount of the compound (3) corresponds to one octopus; Shows the results when was added to the chamber.

FIG. 7 is a graph showing the contraction activity of the cockroach of the cockroach of the cockroach for the compound (1) of the present invention in Example 8.
The result when 1 × 10 ⁻⁶ M compound (1) was added to the chamber is shown.

FIG. 8 is a graph showing the contraction activity of the cockroach of the cockroach made of cockroaches of compound (2) of the present invention in Example 8.
The result when 1 × 10 ⁻⁶ M compound (2) was added to the chamber is shown.

FIG. 9 is a graph showing the contraction activity of the cockroach of the cockroach of the cockroach for the compound (3) of the present invention in Example 8.
The result when 1 × 10 ⁻⁶ M compound (3) was added to the chamber is shown.

FIG. 10 is a diagram showing one-letter amino acids for explaining a part of the amino acid sequence corresponding to the tachykinin-related peptide in the amino acid sequence formula of the precursor polypeptide of the tachykinin-related peptide of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/435 ZNA C12N 1/15 4H045 C12N 1/15 1/19 1/19 1/21 1/21 C12P 21/02 C 5/10 (C12P 21/02 C 15/09 ZNA C12R 1:19) C12P 21/02 A61K 37/02 // (C12P 21/02 C12N 5/00 A C12R 1:19) 15/00 ZNAA (72) Inventor Kyoko Kiwawa 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka Prefecture F-term (reference) 4B024 AA01 AA07 AA11 BA80 CA04 CA11 DA06 EA04 GA11 GA19 4B064 AG01 BA10 BH09 CA02 CA19 CC24 DA12 DA13 4B065 AA26X AA90Y AB01 BA02 CA24 CA46 CA48 4C084 AA01 AA03 BA17 BA18 BA23 CA51 ZA011 ZA012 4H011 AC01 AC02 AF01 BB06 BC18 BC19 DA03 DC11 DH10 4H045 AA10 AA20A 0 BA10 BA15 BA16 CA52 EA06 EA21 EA50 FA34 FA74 GA01 GA15 GA23 GA25 HA03

Claims (15)

[Claims] 1. The following amino acid sequence formula (I): R ¹ -Phe-Xaa-Yaa-Zaa-Arg-NH ₂ (I) (wherein R ¹ represents a peptide having 5 or 6 amino acid residues) , Xaa represents Leu, Ile, Met, Val or Gln, Yaa represents Gly or Pro, and Zaa represents Ser or Thr). 2. The following amino acid sequence formula (II): R ² -Aaa-Baa-Phe-Xaa-Yaa-Zaa-Arg-NH ₂ (II) wherein R ² is 3 or 4 amino acid residues. Aaa represents Asn, Tyr or Leu, Baa represents Ala, Ser, Thr or Asp, Xaa represents Leu, Ile, Met, Val or Gln, Yaa represents Gly or Pro, Zaaa Represents Ta or Ser). The tachykinin-related peptide according to claim 1, wherein 3. Octopus vulgar
The peptide according to claim 1 or 2, which is obtained from the brain of ( is ). 4. The following amino acid sequence formulas (1) to (6): H-Thr-Val-Ser-Ala-Asn-Ala-Phe-Leu-Gly-Ser-Arg-NH ₂ (1) H- Leu-Asn-Ala-Asn- Ser-Phe-Met-Gly-Se r-Arg-NH 2 (2) H-Ala-Ser-Leu-His-Asn-Thr-Phe-Ile-Pr o-Ser-Arg _{-NH 2 (3) H-Val} -Asn-Pro-Tyr-Ser-Phe-Gln-Gly-Th r-Arg-NH 2 (4) H-Ser-Asp-Ala-Leu-Ala-Phe-Val- Pro-Th r-Arg-NH 2 (5) H-Tyr-Ser-Pro-Leu-Asp-Phe-Ile-Gly-Se r-Arg-NH claim represented by ₂ (6) To peptide according to any of the three. 5. The following amino acid sequence formula (I): R ¹ -Phe-Xaa-Yaa-Zaa-Arg-NH ₂ (I) wherein R ¹ represents a peptide having 5 or 6 amino acid residues. , Xaa represents Leu, Ile, Met, Val or Gln, Yaa represents Gly or Pro, and Zaa represents Ser or Thr). 6. The following amino acid sequence formula (II): R ² -Aaa-Baa-Phe-Xaa-Yaa-Zaa-Arg-NH ₂ (II) wherein R ² is 3 or 4 amino acid residues. Aaa represents Asn, Tyr or Leu, Baa represents Ala, Ser, Thr or Asp, Xaa represents Leu, Ile, Met, Val or Gln, Yaa represents Gly or Pro, Zaaa Represents Ser or Thr), and a tachykinin-related peptide represented by the formula (I): 7. Tachykinin having an amino acid sequence represented by SEQ ID NO: 8, or an amino acid sequence modified by adding, deleting, and / or substituting one or more amino acids to the amino acid sequence with another amino acid Precursor polypeptide of related peptide. 8. An amino acid sequence identical to the polypeptide of SEQ ID NO: 8, or 1
Tachykinin-related peptide obtained from a culture of a transformed cell constructed by genetic recombination, having an amino acid sequence modified by the addition, deletion, and / or substitution of another amino acid or amino acids Precursor polypeptide. 9. A method comprising the steps of: using a polynucleotide that hybridizes with a polynucleotide encoding at least one of the amino acid sequences shown in SEQ ID NOs: 1 to 3, from a culture of a transformed cell created by a genetic recombination technique. A precursor polypeptide of the tachykinin-related peptide according to any one of claims 1 to 4, which is obtained. 10. A gene encoding the amino acid sequence of the precursor polypeptide of the tachykinin-related peptide according to claim 7. 11. A vector comprising the gene according to claim 10. A host transformed with the vector according to claim 11. 13. A host according to claim 12, wherein the host is cultured or grown, and a tachykinin-related peptide precursor polypeptide is collected from the host, and a tachykinin-related peptide precursor is excised from the precursor polypeptide. ,
A method for producing a tachykinin-related peptide, comprising subsequently amidating the C-terminus. 14. A tachykinin-related peptide obtained by the method of claim 13. 15. The following amino acid sequence formulas (1) to (7): H-Thr-Val-Ser-Ala-Asn-Ala-Phe-Leu-Gly-Ser-Arg-NH ₂ (1) H- Leu-Asn-Ala-Asn- Ser-Phe-Met-Gly-Se r-Arg-NH 2 (2) H-Ala-Ser-Leu-His-Asn-Thr-Phe-Ile-Pr o-Ser-Arg —NH ₂ (3) H-Val-Asn-Pro-Tyr-Ser-Phe-Gln-Gly-Thr-Arg-NH ₂ (4) H-Ser-Asp-Ala-Leu-Ala-Phe-Val- Pro-Th r-Arg-NH 2 (5) H-Tyr-Ser-Pro-Leu-Asp-Phe-Ile-Gly-Se r-Arg-NH 2 (6) H-Arg-Me -Asn-Ser-Leu-Ser- Phe-Gly-Pr o-Pro-Lys-NH tachykinin peptide according to claim 14 represented by ₂ (7).