JPH0674280B2 - Novel bioactive peptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The novel peptide represented by the above formula (I) of the present invention has the effect of distancing harmful fish such as sharks due to its toxicity. By using peptides, it is possible to safely perform diving work and fish and shellfish collection in areas where harmful fish inhabit. The peptide of the present invention also has a strong lethal toxicity to fish.

[Conventional technology]

Certain species of the marine fish Ushi-noshita release a mucous secretion from a special organ present in the fins, which acts as a fish poison for other fish and shark repellent. It is known to act as a substance. (E. Clark, Envi. Biol. Fish. 1979, Vol. 4, 103
page). Southern Mussel (Pardachirus) collected in Okinawa
Tachibana et al. isolated a steroid glycoside as the above-mentioned substance from the secretory fluid of Pavoninus) and reported its structure (Tachirahedron, Tetrahedron, 1985, 41).
Volume, 1027, Tachibana et al., Science, 1984, 226
Vol., P. 703). In addition, Primor of Israel and others are closely related to the Red Sea, Pardachirus marmoratus (Pardachi).
A protein with a molecular weight of 17.000 daltons was isolated from rusmaraoratus and named Pardaxin, but its structure has not yet been confirmed (N.Primor et al., Toxicon.) 1975 , Volume 13,
227).

[Problems to be solved by the invention]

The inventors of the present invention have used the Okinawan minnow (Pardachirus pa)
As a result of intensive studies on the secretory fluid of voninus), it was confirmed that the secretory fluid has a strong fish venom activity and shark repellent action in addition to the saponin fraction, and the clarification of its main body was planned.

Marine organisms differ greatly from terrestrial organisms in their living environment, leading to the discovery of new types of compounds.
It is expected to expand into the development of agricultural chemicals and foods. For example,
Research on marine product poisons, etc. helps prevent disasters such as poisoning and is important as part of the food problem.

[Means for solving problems]

INDUSTRIAL APPLICABILITY The novel peptide of the present invention has fish venom activity and shark repellent action, and is useful for safely performing diving work in the area where sharks inhabit and collecting fish and shellfish.

The present invention provides the following formula (I) H-Gly-Phe-Phe-Ala-X-Ile-Pro-Lys-Ile-Il.
e-Ser-Ser-Pro-Y-Phe-Lys-Thr-Leu-Leu-Ser
-Ala-Val-Gly-Ser-Ala-Leu-Ser-Ser-Ser-Gly
-Z-Gln-Glu-OH (I) (wherein X is Leu or Phe, Y is Leu or Ile, and Z is Gly.
Or Gln), or a salt thereof.

In the above formula (I), Gly is L-glycine and Phe is L-
Phenylalanine, Ala is L-alanine, Ile is L-isoleucine, Pro is L-proline, Lys is L-lysine, Ser is L-
Serine, Thr is L-threonine, Leu is L-leucine, Val is L-valine, Glu is L-glutamic acid, Gln is L-glutamine. Represents

Production method Southern blue porridge (Pardachirns pavoninus) is bred in an aquarium and mucus is collected once a day, and immediately frozen for storage. The frozen mucus is collected, freeze-dried and then fractionated with acetone, and the produced precipitate is separated by gel filtration chromatography. Fractions having fish venom activity are collected, freeze-dried and then subjected to ion exchange chromatography. As the ion exchange resin used here, various anion exchange resins are possible, but DEAE / Toyopearl (manufactured by Toyo Soda Co., Ltd.)
Is preferable, or the ion exchange chromatography can be replaced by isoelectric focusing chromatography (chromatofocusing). As the column for isoelectric focusing, Falmatia PBE94 (manufactured by Falmatia) is most suitable. The fish venom active fraction is separated into two fractions (fraction-1 and fraction-2) by the above-mentioned ion exchange chromatography or isoelectric focusing chromatography. Both fractions are subjected to hydrophobic chromatography, and finally separated and purified by high pressure liquid chromatography (HPLC) to obtain the compound of the present invention. Phenyl-Sepharose column (CL-4B (Pharmacia)) for hydrophobic chromatography, and Hypersil (Hxpers) for HPLC.
il) WP300-butyl-5 (Shando
n)) is most preferred.

From the above-mentioned Fraction-1 by the above method, the formula (Ib) H-Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-I
le-Ser-Ser-Pro-Ile-Phe-Lys-Thr-Leu-Leu-S
er-Ala-Val-Gly-Ser-Ala-Leu-Ser-Ser-Ser-G
The peptide represented by ly-Gly-Gln-Glu-OH (Ib) is represented by the formula (Ia) H-Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-I from Fraction-2.
le-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-S
er-Ala-Val-Gly-Ser-Ala-Leu-Ser-Ser-Ser-G
Ply represented by ly-Glu-Gln-Glu-OH (Ia) and formula (Ic) H-Gly-Phe-Phe-Ala-Phe-Ile-Pro-Lys-Ile-I
le-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-S
er-Ala-Val-Gly-Ser-Ala-Leu-Ser-Ser-Ser-G
A peptide represented by ly-Glu-Gln-Glu-OH (Ic) can be obtained. In the present specification, the above-mentioned novel peptide Ia is referred to as pardaxin P in the following.
-1 (PardaxinP-1), Ib to PardaxinP-2 (Par
daxinP-2), Ic to pardaxin P-3 (PardaxinP-
3).

Paldaxin P-1, P-2, and P-3 according to the above method
However, it goes without saying that it is more advantageous to obtain it by a synthetic method. As a synthetic method, a commonly used liquid phase method or solid phase method can be used.

For example, when synthesizing Paldaxin P-1 and P-2 by the solid phase method, the solid phase peptide synthesis method of Merrifield (Merifield, J, Am. Chem. Soc., 196
3 years, 85, 2149; Biochmistry, 1964, 3, 1385). That is, a phenylacetate methyl resin PAM resin derived by copolymerization of styrene and divinylbenzene; Starting with a resin called. This PAM resin is a commercial product, and it is also a protected glutamate PAM resin Is also available. Using this protected glutamic acid PAM resin as a starting material, a peptide synthesizer is used to sequentially condense protected amino acids according to the amino acid sequences of pardaxin P-1 and P-2 by a conventional method. As protected amino acids, all α-amino groups are protected by tert-butyloxycarbonyl groups (Boc groups), hydroxyl groups of serine and threonine are benzyl groups (Bgl groups), and gamma of glutamic acid is γ.
The -carboxylic acid group is preferably used as a benzyl ester (O-Bgl), and the? -Amino group of lysine is preferably protected by a 2-chlorobenzyloxycarbonyl group (Cl-Z group).

The protected pardaxin P-1 · PAM resin and the protected pardaxin P-2 · PAM resin thus obtained were treated with hydrofluoric acid to remove the protecting groups and cleaved from the PAM resin to obtain crude pardaxin P-1 and Obtain P-2. The crude pardaxin P-1 and P-2 can be purified by HPLC to obtain the compounds of the present invention pardaxin P-1 (Ia) and pardaxin P-2 (Ib).

Operation 1. Fish venom activity Two medaka (Oryzias latipes) were placed in aquariums containing test substances with different concentrations, and the time until death was measured. The test substance was dissolved in 5 mM phosphate buffer (pH 7.2) and added. The results are shown in Table-2.

2. Shark repellent activity The compound of the present invention, pardaxin P-1, pardaxin P-2, is placed in the oral cavity of a shark (Triaendor obesus) having a body length of about 60 cm.
The shark showed a strong repellant reaction to the seawater and the parudaxin P-3 in seawater.

Examples Example 1. Paldaxin P-1, P from Minamiushinoshita
-2, Separation of P-3 Five minamiushinoshitas with a body length of about 20 cm collected in Kabira Bay, Ishigaki Island, Okinawa Prefecture were bred in an aquarium, and mucus was collected once a day. To collect mucus, raw fish was placed in a shallow dish, the roots of the dorsal fin and hip fin were lightly pressed to collect the mucus from the mucous glands, and immediately frozen and stored. Lyophilization of the collected mucus yielded 27 g of powder. 1.0 g of this powder was suspended in 10 ml of 1 M acetic acid, 90 ml of cold acetone was added, the mixture was left standing at 4 ° C. overnight, the precipitate was collected by filtration, and this operation was repeated again to obtain 420 mg of precipitate. . This precipitate was dissolved in 5 ml of 0.1 M ammonium bicarbonate solution and subjected to Separation with the same solution to Sephadex G-150 (Pharmacia 2.5 × 95 cm) gel perchromatography to fractionate 100 drops. did.
Fractions having fish venom activity (fraction No. 62-77) were collected and freeze-dried to obtain 47.4 mg of powder. The powder thus obtained was collected, 190 m was dissolved in 5 ml of 0.025 M imidazole hydrochloride solution (pH 7.4), and the solution was leveled to obtain isoelectric focusing chromatograph (Farmacia PBE94,1c).
m × 30 cm). It was washed with 60 ml of the solution and diluted 9-fold with Polymafer PB74 (pH 4.
Elution at 0); Fraction 100 divided into fractions-1 (fraction N
o.3-13) and Fraction-2 (Fraction No. 36-62) were obtained. Fraction-1 was added to ammonium sulfate to make it 80% saturated, and then applied to a phenyl-sepharose column (CL-4B made by Pharmacia, 1 cm x 30 cm) equilibrated with an 80% saturated ammonium sulfate solution, and washed with 6 ml of the same solution. After that, it was eluted with a 0.1 M ammonium bicarbonate solution and fractionated by 125 drops. Fish poison active fraction (Fluxion N
51-65) were collected and freeze-dried to obtain 22 mg of powder.
This powder was subjected to high pressure liquid chromatography (HPLC) (column: Hypersil-WP300-butyl-5) for purification to obtain 4.5 mg of pardaxin P-2. As a solvent for HPLC, 0.1% trifluoroacetic acid aqueous solution (A) and 1
Acetonitrile (B) containing 10% trifluoroacetic acid (B) was used, and a solvent according to a linear gradient method of A: B mixing ratio of 8: 2 to 3: 7 was used to elute at a flow rate of 3 ml / min. Also, HPLC is 220n
It was monitored by ultraviolet absorption of m, and the main peak eluted at a retention time of 33.1 minutes was collected to obtain pardaxin P-2.

Then, for fraction-2, exactly the same as for fraction-1, phenyl sepharose column chromatography and HPLC
By attaching to PALDAXIN P- with a retention time of 32 minutes.
3 (2.0 mg) with a retention time of 33 minutes and pardaxin P-1
(13.1 mg) was obtained.

Circular dichroism of Paldaxin P Amino acid analysis In a 13 × 100 mm Pyrex glass test tube, 10 to 50 μg of pardaxin P-1, P-2 and P-3 were taken and freeze-dried. 100μC constant boiling hydrochloric acid (Pierce)
(Manufactured by the company, containing 0.1% phenol) was added, the pressure was reduced to 10 μmHg or less, and hydrolysis was performed at 110 ° C. for 24 hours or 72 hours. About 5
After drying under reduced pressure in a water bath at 0 ° C., the product was dissolved in 300 μm of 0.02N hydrochloric acid and analyzed by Hitachi Amino Acid Analyzer 835-50. The column is Hitachi Custom Ion Exchange Resin # 2619 (2.6 mm x 150 m
m), and the eluent is Wako Pure Chemical's 835 buffer pH-1,2,3,4
And a PH-RQ was used for the stepwise gradient method. The detection was by the ninhydrin method. The analysis results are shown in Table-1.

The presence or absence of cysteine was analyzed as cysteic acid after oxidation of the test substance with the test substance, and the presence or absence of tryptophan was analyzed by the UV-method, but they were not present.

Amino acid sequence About 10 μg of the freeze-dried sample was dissolved in 30 to 50 μ of 70% trifluoroacetic acid (manufactured by Abride Biosystems) and immediately subjected to Abride Biosystems (Applied Bi
osystems) gas phase sequencer 470 type. Phenylthiobitantoin (PT obtained from a sequencer
H) -amino acids are analyzed by HPLC (Spectra Fusion (S
Pectra-Physics)) analysis, and as a result, the amino acid sequences of pardaxin P-1, P-2 and P-3 were determined as follows.

Pardaxin P-1 (Ia) H-Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-I
le-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-S
er-Ala-Val-Gly-Ser-Ala-Leu-Ser-Ser-Ser-G
ly-Glu-Gln-Glu-OH Pardaxin P-2 (Ib) H-Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-I
le-Ser-Ser-Pro-Ile-Phe-Lys-Thr-Leu-Leu-S
er-Ala-Val-Gly-Ser-Ala-Leu-Ser-Ser-Ser-G
ly-Gly-Gln-Glu-OH Pardaxin P-3 (Ic) H-Gly-Phe-Phe-Ala-Phe-Ile-Pro-Lys-Ile-I
le-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-S
er-Ala-Val-Gly-Ser-Ala-Leu-Ser-Ser-Ser-G
ly-Glu-Gln-Glu-OH Example 2 Synthesis of Pardaxin P-1 and P-2 1) Synthesis of Pardaxin P-2 Commercially available protected glutamate PAM resin [Boc Glu (OBgl) PAM]
Resin, manufactured by Afluoride Biosystems] 0.6 g (0.
5 mM) and a peptide synthesizer (Model 430A, manufactured by Abride Biosystems, Inc.) were used for the synthesis.

Condensation reaction is a conventional method: Boc-amino acid PAM resin is treated with 50% trifluoroacetic acid in methylene chloride to release the terminal amino group, and the free amino group and protected amino acid are condensed in the presence of dicyclohexylcarbodiimide (DCC). Repeated. As a protected amino acid, Boc-Thr (Bg
l), Boc-Sev (Bgl), Boc-Glu (OBgl), Boc-Gly, Boc
-Ala, Boc-Val, Boc-Ile, Boc-Leu, Boc-Phe, Boc-Ly
s (Cl-z), and Boc-Pro, using pardaxin P
According to the amino acid sequence of -2, they were sequentially condensed from the C terminus.

The protected Paldaxin P-2 resin (2.
06g) 1.01g was swollen with 0.5ml anisole,
After treatment with 3 ml of hydrofluoric acid at 60 ° C. for 60 minutes, excess hydrofluoric acid was distilled off under reduced pressure. To the residue was added 130 ml of 10% acetic acid aqueous solution for extraction, the extract was washed with methylene chloride and then freeze-dried to obtain 410 mg of crude pardaxin P-2.
This crude pardaxin P-2 was used under the conditions used in Example 1.
HPLC gave 121.6 mg of Paldaxin P-2.

2) Synthesis of Paldaxin P-1 Under completely the same conditions as in 1), it was condensed with Boc-amino acid according to the amino acid sequence of Paldaxin-P-1, and after the release of crude Paldaxin P-1 by hydrofluoric acid, HPLC. Was purified by to obtain Paldaxin P-1.

Paldaxin P-1 and P-2 produced by the above synthesis are
HPLC, circular dichroism and amino acid analysis were in perfect agreement with those of natural origin.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Hiroyuki Minamikata 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka Prefecture Suntory Institute for Biological and Organic Sciences (72) Ichiro Kubota Shimamoto-cho, Mishima-gun, Osaka Prefecture 1-1-1 Wakayamadai Suntory Ltd. Biomedical Research Institute

Claims (4)

[Claims] 1. Formula (I) H-Gly-Phe-Phe-Ala-X-Ile-Pro-Lys-Ile-Il
e-Ser-Ser-Pro-Y-Phe-Lys-Thr-Leu-Leu-Ser
-Ala-Val-Gly-Ser-Ala-Leu-Ser-Ser-Ser-Gly
-Z-Gln-Glu-OH (I) (wherein X represents Leu or Phe, Y represents Leu or Ile, and Z represents Gly or Glu) or a salt thereof. 2. The peptide according to claim 1, wherein X is Leu, Y is Leu, and Z is Glu, or a salt thereof. 3. The peptide according to claim 1, wherein X is Leu, Y is Ilu, and Z is Gly, or a salt thereof. 4. The peptide according to claim 1, wherein X is Phe, Y is Leu, and Z is Glu, or a salt thereof.