JPH03120298A - Mollusk-muscular contraction-modifying tetradecapeptide - Google Patents

Abstract

NEW MATERIAL:Peptides of formula I (X is H, H-Val, H-Arg-Val, etc.; Y is Phe or Tyr). EXAMPLE:A peptide of formula II. USE:An experimental reagent useful for an experiment in which the nerve system of a mollusk is used as a model system for studying human-brain mechanism on a neuron level. And useful for isolation of an analogous peptide from an animal. PREPARATION:For example, a massive ganglion separated from a mollusk prosobranch Fusinus perplexus is placed in acetone cooled by ice and homogenized. The obtained homogenate is then subjected to centrifugal separation and acetone in the resultant supernatant is evaporated to obtain an acetone- extract of the massive ganglion of Fusinus perplexus. The obtained extract is then subjected to gel permeation chromatography and purified by HPLC.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a peptide, particularly a tetradecapeptide, which exhibits physiological activity on mollusc muscle. More specifically, the present invention provides a neuropeptide derived from the ganglion of the mollusc protobranchia, Chrysanthemum sinensis, a neuropeptide derived from the ganglion of the African snail, a mollusc pneumocan, and one or more of these neuropeptides. This invention relates to bioactive vebboudo synthesized by changing the amino acid residues. Furthermore, the present invention also relates to methods for producing and using such bioactive peptides.

The tetra-f-force peptide of the present invention is a peptide with a completely new structure that does not resemble any physiologically active peptide known so far. However, these peptides show strong activity in various muscles of molluscs, and this family seems to be widely distributed, at least in molluscs. Furthermore, in general, peptides that are widely distributed in a certain animal phylum are also often distributed in other animal phyla.
Considering that the butraburic peptide of the present invention exhibits activity in the body wall muscles of leaf beetles, these members may also exist in other animal phyla. For these reasons, the peptides of the present invention can be used not only as useful experimental reagents in the fields of biology and basic medicine, but also to develop useful drugs in the biological production and pharmaceutical fields based on these peptides. It is possible to do so.

[Prior Art] The progenitor cypress is an easily available animal, and its ganglia have relatively large amounts of various physiologically active peptides. Therefore, it is a convenient animal as a research material for the discovery of unknown molluscan neuropeptides. The present inventors have also conducted research to isolate physiologically active peptides and determine their structures using the ganglia of this animal as material, and were able to discover several peptides7. In this process, the present inventors discovered a type of tetradecapeptide that enhances single contraction at low m degrees and induces contraction itself at slightly higher concentrations in the radula retractor muscle. This is one type of peptide according to the present invention.

By the way, in general, molluscans belonging to the pulmonate family have many giant neurons, and these can be identified. Therefore, pneumophiles are often used to study information processing mechanisms in the nervous system. The African snail is also a type of pneumophila, and its central nervous system has recently been well investigated1-4). However, neurotransmitters, which are substances that mediate information between neurons or between neurons and muscle cells, have not been investigated in detail. In particular, the role of peptide transmitters, which have recently attracted attention, is largely unknown.

The present inventors have also conducted experiments to clarify the information processing mechanism in the central nervous system of the African snail, and therefore it was necessary to identify neuropeptides present in the nervous system. Normally, in vertebrates, neurotransmitters that act centrally also exhibit activity in peripheral muscles. Therefore, the present inventors investigated the effect of the above-mentioned =1 Naganisitetrade force bebutide on the penile retractor muscle of the African snail, and found that it enhances the contraction of this segment. Furthermore, when the activity of the crude fraction of African snail ganglion extract was investigated on penile traction pain, both fractions showed activity very similar to that of Chrysanthemum peptide. Therefore, we thought that a homolog of the above-mentioned tetrade force bebuboud exists in the ganglion of African snail, and attempted to isolate the homologue and determine its structure using the retractor penis muscle as a bioactivity assay system.

As a result, 2
I was able to discover the species Tetrade Force Bebujido. These are also peptides according to the invention.

[Problems to be Solved by the Invention] As will be described later, the three types of tetradecapeptides have effects not only on the retractor muscle of the tongue of the tongue and the retractor of the penis of the African snail, but also on the heart of the clam, which is a bivalve. Shows strong activity. In other words, it shows activity even against taxonomically distant muscles of the soft body.

From this fact, it seems that these peptide families are widely distributed, at least in molluscs. Furthermore, these peptides also show activity on the body wall muscles of the leafhopper, and considering this, these peptides are
It may also exist in animals other than molluscs. Therefore, these three types of tetradecapeptides and their synthetic homologues are considered to be useful as experimental reagents in fields such as neurobiology, biological production, and basic medicine.

For the above-mentioned reasons, the main object of the present invention is to provide tetradecapeptides that are present in D. thaliana and African snails and exhibit physiological activity on mollusk muscle, and their homologs that exhibit biological activity.

Another object of the present invention is to provide a method for producing such a peptide.

Furthermore, the present invention is also directed to various uses of such peptides.

[Means for Solving Problem E] The present inventors isolated 11 types of tetradecapeptides that cause contraction of the radula retractor muscle of this animal from the oil exudate of the ganglion of this animal, and determined their structure. did. At low concentrations (10-10 to 10'M), this peptide potentiates the single contraction of the retractor lingulae muscle and exhibits strong inhibitory activity on cardiac activity in clams. Therefore, this peptide was converted into molluscan muscle contraction-modifying tetradecapeptide Δ (Holluscan
MlloilOdLIlatOryTetradec
It was named apeptide A (hereinafter abbreviated as MMTPA). MMTPA has the following amino acid sequence formula.

MMTPA: H-Gly-Phe-Arg-Net
-^5n-3er-Ser-Asn-Aro-Val
-A 1a-111s-G Iy-Phe-H1t2
On the other hand, the present inventors isolated two types of tetradecapeptides that enhance the tension of the penis retractor muscle of this animal from extracts of ganglia of African snail, and determined their structures. These have a structure very similar to the above-mentioned MMTPA,
Furthermore, like MMTPA, it exhibits strong inhibitory activity on cardiac activity in clams. Therefore, we added these to molluscan muscle contraction-modifying tetradecapeptides B and C (HolluS
CanHyomodulatory Tetradec
apeptide B and C, hereafter M M T P
B and MMTPC). MMTPB and M
Each MTPCG has the following 7 amino acid sequence formula.

H) 4TP8: It-Gly-Phe-Arg
-Gln-8sp-^1a-A Ia-Ser-^r(
1-Val-8Ia-11is-Gly-Tyr-NH
12HHTPC: H-Gly-Phe-^rg-Gl
y-Ast1-^1a-Ala-3er-^rg-Va
l -A I a-1t is-G I y-Ph
e-NH12 The above three types of tetradecapeptides are clearly homologous peptides, as can be seen from their structures,
Both exhibit almost the same activity. No peptide with a structure similar to this structure has been discovered so far, and since these peptides show strong activity against the heart of bivalves, new peptides containing the three peptides listed in E are available for molluscs. It is thought that a family exists.

By the way, the C-terminus of both MMTPA and MMTPC is -P.
It has a structure of he-NH2, but that of MMTPB has a structure of -Tyr-NH. Phe and T
Considering the structural similarity of yr, MMTPA and MMT
It is thought that a peptide in which Phe at the C-terminus of PC is replaced with Tyr and a peptide in which 'ryr at the C-terminus of MMTPB is replaced with Phe also exhibits the same activity as MMT PA-C. Therefore, as a result of synthesizing the peptides shown below and examining their activities, all of these peptides were found to be MMTP.
It showed the same activity as A-C.

”Tyr-HHTPA: H-Gly-Phe-^r
Q-1'1et-Asn-Ser-3er-Asn-^
rg-Val-Ala-old 5-Gly-Tyr112 ”Phe-HHTPB: 1(-Gly-Phe-^
rlJ-Gln-^5p-Ala-Ala-ser-^
rg-Val-^1a-His-Gly-Phe112 14Tyr-HHTPC: If-Gly-Phe-
^r! J-Gly-ASI)-Ala-81a-ser
-^rg-Va I-^1a-Old 5-Gly-TyrH
2 Therefore, the present invention also relates to the above three types of peptides.

Furthermore, the C-terminal 6 amino acid residues of MMTPA%MMTPB%MMTPC are C-terminal Phe or Ty.
All are composed of the same amino acids except r. Therefore, when fragment peptides based on MMTPA shown in the following formula were synthesized and their activities were examined, they all showed attenuated activity. Therefore, the present invention also relates to the following three types of fragment peptides.

9-14 HHTPA: H-to rO-Val
-Ala-formerly 5-Gly-Phe-N) 1210-14
n: )l-Val-Ala-His-Gl
y-Phe-811211-14〃:H-^1a-11
is-Gly-Phe-882 Specific examples of the mollusc muscle contraction-modifying peptide of the present invention are as follows. , () is an abbreviation.

H-Gly-Phe-^rg-net-Asn-ser
-ser-^5n-Ar13-Val-Ala-His
-Gly-Phe-NHl (H
HTPA) H-Gly-Phe-^rg-Hot-^5
n-ser-ser-^sn-^rg-va+-Ala
-His-Gly-Tyr-NHl (14Tyr-HH
TPA) Gly-Phe-Ar(1-Gln
-Asp-A I a-A 1a-Ser-Ar(J-
Va l -^1a-His-Gly-Tyr-NH2
(HHTPB)H-Gly-Phe-^rg-Gln-
^5p-Ala-Ala-Ser-^rg-Val-^
1a-His-Gly-Phe-N12 (“Phe-H
HTPB) Gly-Phe-^ro-GIy-Asp
-Ala-^1a-3er-^r(1-Val-^1a
-His-Gly-Phe-NH2(HHTPC)
ly-Phe-Ara-Gly-Asp-^la-^1
a-3er-Arg-Val-^Ia-11is-Gl
y-Tyr-NH12 ("Tyr-HHTPC) and Ar
g-Val-^1a-His-Gly-Phe-NHl
(9-148HTPA)If-Val-Ala-11i
s-Gly-Phe-NHl, (10-1
4HHTPA)H-Ala-11is-Gly-Phe
-882 < 11-148 HTPA) These peptides all potentiated single contractions of the retractor dentoglosus muscle at low concentrations (10-10 to 1G-7M) and at high concentrations (10'
M or higher) causes contraction in this muscle. They also enhance the tensile strength of the penile retractor muscle in African snails 10 (e4 explants 10 to 1).
G'M). In addition, it suppresses the heart activity of Amaguri11.
(Threshold II value: 10-10 to 1 G-'M) and enhances single contractions of the body wall muscles (threshold: 10-9 to 10'7M).

Among the peptides of the present invention, MMTPΔ-G can be produced as vJ by purifying extracts of Chrysalis ganglion or African snail ganglion by conventional methods such as HPLC, as shown in the Examples below. can. Organic solvents such as acetone are used for extraction. In addition, MMTP
Synthesized amount of A-C 41414 rry-HHTPA, Phe-11T
P8. Although rry-HHTPC has not yet been discovered as a natural product, it is possible that they will be discovered in molluscs and other sources.

Furthermore, the peptide of the present invention can also be synthesized, for example, by a conventional solid-phase peptide synthesis method, as shown in Examples below.

[Efficacy of the Invention FA] Like all neuropeptides, the peptide of the present invention not only exhibits effects in the muscular system of mollusks;
It is certain that it also has effects on the central nervous system. The nervous systems of molluscs, especially those of pneumophora and catfishes, which have huge neurons, are also used in experiments as good model systems for investigating our brain mechanisms at the neuron level5. -6), the peptides of the invention are used as useful reagents in these experiments.

The peptides of the present invention and their related peptides appear to be widely distributed in molluscs, and may also be distributed in other animal phyla. Therefore, if an antibody to the peptide of the present invention is made, it can be used to isolate related peptides from other animals, and immunohistochemical techniques can be used to isolate the peptide of the present invention and its analogs. The localization of peptides in the nervous system can be investigated.

The tetradecapeptides of the present invention have 3@ amino acid residues in common on the N-terminal side. In addition, there is only an exchange of phe and TVr in the six amino acid residues on the C-terminal side, and they are almost completely common. That is, it is considered that active expression sites exist in these parts. Therefore, it will be possible to develop antagonists of the peptides of the invention based on the structures of these parts.

If an antagonist is obtained, this could be used as a pesticide (eg for eradication of African snail). It could also be used to propagate aquatic products.
・-1,.

It is also known that the peptide, a relative of the peptide discovered in non-promoting animals, also exists in non-promoting animals, as seen in the example of a peptide called FM RF side7). It is possible that relatives of the peptide of the present invention also exist in animals, and therefore, the peptide of the present invention may also be used for research in the medical field.

[Example] Hereinafter, the present invention will be specifically explained with reference to Examples.

Example 1: Isolation and purification of D. chinensis MMTPA D. D. nigra, a promolluscan catfish, was collected from Hiroshima ditch. In Ko, Naganishi, other ganglia except for the visceral ganglia exist as a single mass. Ganglion masses were separated from approximately 1,100 individuals of P. aeruginosa, immediately frozen on dry ice, and stored at -20°C.

The frozen ganglion mass was placed in ice-cold acetone and immediately homogenized, the homogenate was centrifuged, and the acetone in the supernatant was evaporated using a vacuum evaporator. Add hydrochloric acid to the remaining aqueous solution to a final concentration of 0.1N.
The mixture was added to the solution, stirred, and centrifuged again to remove insoluble matter. This was converted into a C-18 cartridge (Waters, 5
After washing the cartridge with 4% acetic acid, the retained material was eluted with methanol and the eluate was lyophilized. Dissolve this sample in distilled water and
Place on a X G-15 column (2,6 x 40 cIl) and
．． It was fractionated by flowing 1NH acid.

Each fraction was lyophilized and then used for bioactivity assay.

Bioassays were performed using the retractor muscle of both tongues. Approximately 2
The muscle specimen was attached to an experimental container of m, and electrical stimulation (20V,
2 m5ec, 0.2 Hz, 5 pulses) were applied to produce a set of 5 single contractions every 10 minutes, and the effect of each fraction on this contraction was investigated. Bioassays in each step of peptide purification described below were also performed in the same manner.

As a result of bioassay of each fraction obtained by gel filtration with 5cphadex G-15, three excitatory peaks were observed, with the maximum activity for single contraction enhancement occurring at the 22nd, 45th, and 50th fractions, respectively. Obtained. Also, 28 for both
A peak of inhibitory activity was obtained with maximum activity at ~30 hrs. Therefore, we divided both groups into two groups, numbers 22-40 and numbers 41-54, and purified the active substances for each using l-IPL G. As a result, MMTPA was discovered in the former group. I was able to do that.

Below, the procedure for purifying MMTPA by HPLC will be described.

Gel filtration fractions 22 to 40 were collected and concentrated to obtain C-1.
8 reversed phase column (TSK-Gel ODS-80TM), 0.1% TFA (1)H2,2), 0-6
Gradient elution was performed using HPLC (JASCO TR1-Roter IV) under the condition of 0% acetonitrile (60 minutes). As a result, a peak showing contraction-inducing activity and a peak showing single contraction-inhibiting activity were obtained.

Next, the peak showing the contraction-inducing activity was extracted using a C8 reverse phase column (F
ine-Pak SIL C8-5) and 0.1
When gradient elution was performed under the conditions of %TEA (902,2) and 0-30% acetonitrile (60 minutes), two single peaks of contraction-enhancing activity appeared. The effect of the first peak of activity eluted was small and was only visible at high concentrations, but the effect of the second peak was strong and itself caused contraction at slightly higher concentrations.

Therefore, the sample obtained from the second peak was then injected into a cation exchange column (TSK-Gel 5P-5PW) and treated with 20 mH phosphate buffer (pH 6,0), 0-0
．． Gradient elution was performed under the conditions of 7HNaCl (70 minutes),
Finally, the activity peak obtained at that time was again
18 reverse phase column, 0.1% TFA (+) + 42
．． 2'), 10-20% acetonitrile (20 minutes)
, followed by gradient elution under conditions of 20-60% acetonitrile (20 min). As a result, a single absorption peak at 220 nm (the peak indicated by the arrow in Figure 1)
An activity peak (bottom of Figure 1) corresponding to this was obtained.

The activity at this time was investigated using the retractor muscle of the genus dentoglosus, as described above. First, electrical stimulation (20V,
2isec%0.2 fiz, 5 pulses),
After producing a single contraction in each individual, purified MMTPA (3 4 min/d artificial seawater) was applied (upward arrow) to observe the induction of contractions 8 minutes before the next electrical stimulation. Next, 8
Minutes later, electrical stimulation was applied and the muscles were washed immediately after observing the enhancement of single contractions in the presence of MMTPΔ (downward arrow).

J3, the generated MMTP△ was determined by repeated electrical stimulation (IOV, 11 sec, 10
11z.

It enhanced the tension wire for 1 sec) and the single contraction of the body wall muscle of the clam body in response to a single electrical stimulation (20 V, 5 m5 ec), and suppressed the automatic activity of the clam's heart. The threshold values for various physiological activities of MMPTA are summarized below.

] Naganishi radula Contraction-inducing action retractor muscle Threshold 10'M Clam heart Contraction inhibition,, 10-10 M
Murasa Y mussel Suppresses transient contraction of anterior byssus retractor muscle by electrical stimulation 10-9M African porpoise penis retractor muscle 10'M African porphyry penis retractor muscle 5L Muscle body wall muscle 10'M 10'M Molluscum present The African snail, a lung species, was caught in Okinawa.
They used those that were airlifted to Hiroshima. African snail 1
From 600 individuals, the red mass containing the cranial ganglion and subesophageal ganglion was separated, frozen and stored at -20° C. in the same manner as in Example 1 for D. chinensis.

The frozen-preserved African snail ganglion was placed in water-cooled acetone, extracted in the same manner as in the case of the African snail ganglion in Example 1, and gel filtration was performed in the same manner. Both aliquots obtained by gel filtration were frozen and then used for bioactivity assay.

The bioassay was performed using the retractor penis muscle of the African snail. A Wb marker was attached to the experimental B device of approximately 2 d, and repeated electrical stimulation (IOV, 1 m5ec, 1011z, 1
sec) to cause transient tetanic contraction, and the effects of each component on this contraction were investigated. Bioassays in each step of peptide purification described below were also performed in the same manner.

As a result of bioassay of each fraction obtained by gel filtration,
Contraction enhancing activity with peaks at both the 27th and 41st positions was observed. Therefore, I divided myself into two groups, 24th-30th and 37th-52nd, and purified the active substances for each using HPLC.
As a result of investigating the structure of the obtained substance, it was found that the MM of the present invention
TPB and M M 1-PC were found from the previous group. Therefore, the purification procedure will be described next.

First, fractions 24-30 were collected and concentrated, and then converted into C-
18 reverse phase column (TSK-gel, oos-go-rM)
0.1%-[Δ(1]112.2>,0
Gradient elution was performed using a HPtC system similar to that used for Rhinoceros chinensis under conditions of −60% acetonitrile (60 min). As a result, we obtained a peak that enhanced the contraction of the penile retractor muscle.As a second step, this sample was applied to a cation exchange column (TSK-gel, 5P-5PW).
10 a+M phosphate buffer (all 6.7>,
Gradient elution was performed under the conditions of 0-0.7 H Macl (70 minutes), and two contraction-enhancing activity peaks were obtained.

Next, in the third step [1], the sample of the peak eluted later in the second step was loaded onto the above-mentioned reverse phase column,
As a result of gradient elution under the conditions of 1% TFΔ (pH 2,2) and 15-30% acetonitrile (30 minutes), two contraction-enhancing activity peaks were obtained that corresponded to two almost perfect single absorption peaks. .

Therefore, as a final step, the one eluted first in the third step was treated with 0.1% TFA (roll 2.2), 15
% ace1-dyl-lyl condition C1 and the one eluted later was 0.1% TFΔ(pII 2.2), 16% 7
Perform isocratic elution under setonitrile conditions,
Peaks of enhanced activity were obtained, each corresponding to a complete absorption peak. The former is MMTPL3. The latter is MMTPC.

FIG. 2 shows the HPLC chroma l~g of the final flj of MMTPB and the potentiating activity (inset) for tetanic contraction of the African porpoise penis retractor muscle. Tetanic contraction is a repetitive electric charge *
(IOV, i m5ec, 1oz, 1se
c) was given every 10 minutes, and MMTPB was administered 8 minutes before contractions and washed out immediately after contractions were recorded.

FIG. 3 shows a 1'' PLC chromatogram of the final stage of MMTPC and no contractile enhancement activity. How to check the enhancing activity:
This is similar to the case of MMTPB.

In each case, the concentrations used correspond to those obtained from 10-quarter African snails. The composition of the physiological saline solution used in these bioassays is as follows. N
aC161m)l, KCN 3.3mM, CaC1
10.7mH, MOCj! 213 mH1 glucose 5
mM, Hebesu Na Ot-110mH, pH 7.5
゜Purified MMT P 13 and MMTPC's single contraction of the retractor muscle of the radula, autoactivation of the clam heart.
The activity of the body wall muscle for instep contraction was almost the same as in the case of MMTP△.

The threshold values of various physiological activities that MMTPB and MMTPC do not show are summarized below.

Afrikaans contraction-enhancing milo muscle Afrikaans penis retractor muscle threshold 1O-10 n, o-10 Murasaki mussel Byssus-like retractor muscle suppresses transient contractions by electrical stimulation II 10'M
Clam heart Contraction inhibition n io'M Konaganishi radula Contraction inducing retractor muscle 10'M Yumushi resting wall muscle Contraction enhancing action 10-9M pressure in sealed tube 100IIf! Hydrolyzed with constant boiling point hydrochloric acid (containing 0.1% lol) at 110°C for 24 hours, and dried under reduced pressure to give 0.0
It was dissolved in 100 pi of 2N salt and subjected to Hitachi automatic amino acid analysis model 1IL-8500. The results are shown in Table 1. In Table 1, MMTPA and MMTPC are phe-2,
MMTPB was calculated using phc-=1.

The number 1tl in parentheses does not indicate the nearest integer ratio.

Example 3: Structure Determination of Neuropeptides The structures of the active substances obtained in Examples 1 and 2 were determined as follows.

Amino acid composition Amino acid analysis uses approximately 1 nanomole of a sample, and the same amount of sample as used for the amino acid sequence amino acid analysis shown in Table (11) is run on an automatic protein sequencer (Applied Biosystems 47).
Type 7A). The obtained phenylthiohydantoin-amino acid (PTH-amino acid) is
Identification was performed using a PTH-amino acid analyzer (Model 120A manufactured by Applied Biosystems) connected online to the PTH-amino acid analyzer. The results are shown in Table 2 for MMTPA and MMTPB.
The details are shown in Table 3, and the information about MMTPC is shown in Table 4.

Amino acid sequence of MMTPA Amino acid sequence of MMTPC Table Amino acid sequence of MMTPB (iii) Dan AB
[OL JIS HX-100 type] was used.

The (M+1) ion of MMTPA is 1578.5 (theoretical molecular weight 1577, 7>, and the (M+1) of MMTPB
The ion is 1377.4 (theoretical molecule ff11376.6
), the (M+1)” ion of MMTPC is 1446.5
(Theoretical molecular weight: 1445.7).

From the above results, MMTPA% MMTPB, MMTPC
The structural formulas of each were estimated as follows.

HHTPA: H-Gly-Phe-Arg-Net-Asn-3er
-3er-^5n-Ara-Val-Ala-His-
Gly-Phe-NH4I-Gly-Phe-＾rg-
Gln-Asl)-Ala-Ala-3er-Arg-
Val-^1a-11+5-GIy-Tyr-NH4I
-Gly-Ph(3-Arri-Gly-Asp-Ala
-Ala-8Or-^rg-Va, I-81a-Old 5-
G to y-Pt+e-NH2HHTP8: HHTPC: Example 4: Synthesis of all peptides All peptides were synthesized by in-phase peptide synthesis using a 7Pride Biosystems peptide synthesizer 430A.
This was done using a protected amino acid manufactured by the same company. The HF-anisole method was used to remove the 84 fat and the protective group, and the lyophilized product after resin removal was subjected to HPLC using a reversed phase column.
It was purified by

As in the case of natural products, the structure of the synthetic product was confirmed by amino acid analysis, amino acid sequence analysis, and FAB analysis, and the behavior of the synthetic product was compared with that of the natural product on l-I PLC. It was subjected to the same activity test. As a result, the synthetic MMTPA%MMTPB, MMTPC was found to be superior to the natural MMTPAlMMTPB, MMTPC in HPLC behavior using a reversed phase system and a cation exchange column, respectively.
Matched with PC. Furthermore, the synthetic product and the natural product are the same in terms of biological activity, and the above-mentioned M M T l)△, MM
The chemical structure of TP[3 and MMTPC was confirmed.

Example 5: Neuropeptide Congeners and Fragment Bevbuds Using a method similar to Example 4, the following amino 11if
Synthesis of 3 types of f-transformed peptides and 3 types of fragment peptides,
When purified and examined for biological activity, 14■yrHHTP8,
"Phe-HHTPB" and "Tyr-HHTPO" were found to have almost the same activity as MMTPA, MMTPB and MMTPC, respectively, and 9-14.10-14.11-14M
MTPA showed a reduced activity (1/10 to 1/100 or less).

14Tyr-HHTPA: H-Gly-Phe
-8rg-Hct-Asn-3er-3cr-Asn-
^ro-Va 1-Ala-11is-Gly-Tyr
-NH1214Phe-H14TPB: H-Gly
-Phe-Arg-Gln-^sp-^1a-^1a-
3er-Arg-Val-^1a-11is-Gly-
Phe-NH2”Tyr-HHTPC: H-Gly-
Phe-^ro-Gly-Asp-Ala-^la-6
er-Arg-Val-Ala-old 5-Gly-Tyr
-NH29-14HHTPA: H-Arg-Va
l-^+a-His-Gly-Phe-Ni1210-
14: H-Val-AIaJlis-Gl
y-Phe-88211-14#:H-^1a-H
is-Gly-Phe-NH2 References The documents cited in the specification are listed below.

(1) J, Exp, Riot, 137.31
9 (1988).

■ Experience, 43.918 (f9
871゜■ J, [xp, Riot, 129.2
95 (1987).

(4) J, Fxp, Biol, 129.27
9 (1987).

■Science, 51.10 (1981) ■Science, 51.109 (1981).

(7) Peptides, 9.915 (198
8)

[Brief explanation of drawings]

Figure 1 is a diagram showing the activity on the HPL retractor tongue muscle (bottom) in the final stage of MMTPA purification, and Figure 2 is a diagram showing the activity of the HPL tongue retractor muscle in the final stage of MMTPA purification.
FIG. 3 is a diagram showing the HPLC chromatogram of the final stage of PB purification and the activity of the purified product against the African porpoise retractor muscle (inset); FIG. It is a figure showing the activity (inset) for the penile retractor muscle.

Claims (6)

[Claims] (1) Formula: (X)-Ala-His-Gly-(Y)
-NH_2 [wherein, X is H-, H-Val, H-Arg
-Val, H-Gly-Phe-Arg-Met-As
n-Ser-Ser-Asn-Arg-Val, or H
-Gly-Phe-Arg-(Z)-Asp-Ala-
Ala-Ser-Arg-Val, Y is Phe or T
yr, Z is Gln or Gly] A physiologically active peptide. (2) The peptide according to claim 1, which exhibits contraction-inducing, contraction-enhancing, and/or contraction-inhibiting physiological activity in mollusc muscle. (3) The peptide according to claim 2, wherein the mollusk is the probranchial Chrysalis spp. and the pneumonic Snail African snail. (4) Gel filtration of an organic solvent extract of the ganglion of Chrysanthemum or African snail, followed by high performance liquid chromatography (HPL) using a reverse phase column and an ion exchange column.
C) The method for producing a tetradecapeptide according to any one of claims 1 to 3, which comprises performing a treatment. (5) The method for producing a peptide according to any one of claims 1 to 3, which comprises synthesis by a conventional solid-phase peptide synthesis method. (6) An experimental reagent comprising one or more peptides according to any one of claims 1 to 3.