JPH0225499A - Avian-derived physiologically active peptide - Google Patents

Abstract

NEW MATERIAL:A peptide expressed by formula I (X is absent or amino acid sequence expressed by formula II). USE:A diuretic and hypotensive agent. PREPARATION:For example, a chicken heart is finely cut and treated with boiling water for 5min to inactivate proteases. One M acetic acid and 20mM hydrochloric acid are then added to homogenize the finely cut heart in a mixer, etc. The resultant homogenate is subsequently centrifuged and acetone is added to the supernatant to form precipitates, which are then removed to concentrate the supernatant. The resultant concentrate is then subjected to silica gel column to provide an eluate, which is treated by ion exchange chromatography, eluted with 2M pyridine-acetic acid, etc., to afford an active fraction containing a basic peptide using relaxant activity of a chick rectal specimen as an index. The resultant fraction is successively subjected to gel filtration and ion exchange reversed phase high-speed liquid chromatography and purified to afford the chicken atrial natriuretic active peptide (gamma-chANP) expressed by formula I.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of the Invention] The present invention relates to a novel physiologically active peptide derived from birds having diuretic, natriuretic and antihypertensive effects.

[Conventional technology]

Atrial natriuretic active peptide (ANP) is a hormone synthesized, stored, and secreted in mammalian atrial tissue.
) is a substance that regulates the homeostasis of body fluids and blood pressure. In recent years, research on mammalian (human, rat, etc.) ANP has shown that ANP is first biosynthesized in the atrium as γ-ANP, which consists of 126 residues, and then processed by processing enzymes, which consists of the C-terminal 28 residues of T-8NP. It became clear that α-ANP was generated and that this was circulating in the blood (de B
old, A, J,.

5science, 230, 767-770, 1985;
Cantin, M., & Gerrest, J.
, , [1ndocrine” Reviews +
6 + 107-12L 1985; Matsuo
, M., and Nakazato, ft., , Endocri.
nology and Metabolism C11
nics of north america,
16+8trialNatriuretic Fact
er (eds Rosenblatt, M. and J.
acobs, J, W, )+p, 43-61), A
The amino acid sequence of NP is broadly similar in mammals from rodents to humans. In particular, α-ANP
was found as a substance with the same sequence (identical form) in higher animals including humans, dogs, and pigs. Also, only one amino acid differs in the rodent neck (Kangawa, K.
and Matsuo, It.

Biochem, Biophys, Res, Co
mmun, , 118.131-139.1984
; Flynn, T. G., de Bold, M., and de Bold A. J.;

Biochem, Biophys, Res, Co
mmun, , 147.859-865.1983
; Oikawa, S., et al. + Biochem, Biop
hys, Res, Common, +132+862
-898, 1985; Nemer, M et al. Nature
, 312, 654-656゜1984; Ong, H
,,Life Sci,, 3B, 13094315.
1986; Seidman, C.E., et al., 5ci
ence, 226.1206-1209.1984)
.

On the other hand, histological and pharmacological studies have suggested the presence of ANP-like substances in the atria and ventricles of non-mammalian animals (de Bold et al.
et al., Can, J., Physiol, Pharmacol.
,,61+127-130+1983: Benscom
E, S. A., and Berger, J. M., Meth.
Oddsand Achievements in Ex
Perimental Pathology.

vol, 5.173-213.1971). In particular, some immunohistochemical and biological activity data have been shown for chicken among birds, suggesting that chicken-derived ANP (chANP) is different from that of mammals. (Benscome, S.
, 8, and Berger, J.

M.,Methods and Achievemen
ts in Experimental Patholo
gy, vol, 5.173-213, 1971; de
Bold, A.J.

and 5alerno, T. A., , Can, J., Phy.
siol, Pharmacol.

61.127-130, 1983; Gregg, C.
M, and -tdeman, Jr -+11, F,, 8m, J, Physio1. , 251, 543-551.
1985; Chapeau.

C., Gutkrvska, J., and Cantin,
M., J., Histochem.

Cytochem, 33.541-550.19
86>. Based on these facts, it is also possible for birds and chickens to
Although it is thought that a P-like substance exists, its structure is not yet clear, only that it has been pointed out that it may be different from human A and NP.

[Problem to be solved by the invention]

Therefore, the present inventors conducted an intensive search for ANP derived from chicken heart using the same isolation method as mammalian (human) ANP, using the chick rectal relaxant activity as an indicator. We have developed novel physiologically active peptides that have a blood pressure lowering effect, namely, a peptide consisting of 29 amino acid residues (α-chANP) and a peptide containing it at the C-terminus and consisting of 116 amino acid residues, chicken ANP' (r-chANP). They discovered, isolated, purified, and succeeded in determining its structure. Furthermore,
We also succeeded in cloning cDNAs corresponding to these peptides, and by analyzing this cDNA, we determined that chAN
The structure of the P precursor was determined and the present invention was completed. Therefore, an object of the present invention is to provide a novel physiologically active peptide that is similar in structure to mammalian ANP, but is clearly distinct from them.

[Means for Solving the Problems] 1. Production of chANP The physiologically active substance in the present invention is obtained by homogenizing chicken heart tissue in an appropriate acidic solvent, such as acetic acid or acetic acid containing hydrochloric acid, and using the method of Currie et al. (Curri
et al., Nature, 2'21.1-13.1983)
Accordingly, using the relaxant activity of a chick rectal specimen as an indicator, various treatments commonly used for peptide purification, such as centrifugation, acetone precipitation, solvent extraction, ultrafiltration, gel filtration, and adsorption chromatography, and α-chANP antibody were used. It can be obtained by separating peptides using appropriate combinations of radioimmunoassay (RIA) and the like. In the present invention, as shown in FIG. 2, the relaxant activity of the chick rectal specimen was observed in the portions corresponding to molecular weights 3K and 12.

A, α-chANP with a molecular weight of 3 is purified using ion exchange and reversed phase high performance liquid chromatography (IIPI, C). As a result of amino acid analysis, the physiologically active substance in both cases was found to have 29 amino acids and was named α-chANP. Furthermore, it goes without saying that if the structure (amino acid sequence) of the physiologically active peptide is clear, it is more advantageous to use a chemical synthesis method for industrially obtaining it. α-chANP of the present invention can be produced by an in-phase method or a liquid-phase method commonly used for peptide synthesis. For example, cJ+ANP can be synthesized by solid phase synthesis method such as Merrifield et al.
can be synthesized. When this method is applied to the present invention, the α-amino group of any amino acid residue is protected with a tert-butyloxycarbonyl group (Boc group), the guanidino group of arginine is a tosyl group (Tos group), and the serine The hydroxyl group of is a benzyl group (Bzl group), and the β-carboxylic acid group of aspartic acid is an O-benzyl group (0
-Bzl group).

First, a protected derivative of asparagine, which is a C-terminal amino acid; Boc-Asn is introduced into chloromethyl polystyrene resin, and then amino acids are sequentially extended to obtain protected chANP.
- synthesize a resin and treat it with hydrogen fluoride to cleave the protected chANP from the resin and simultaneously deprotect it;
By reducing this, Cys723-(S-Ac
m)-chANP (8 cm is an acetamidomethyl group protecting the thiol group of cysteine) can be obtained. Next, by oxidizing this with iodine, the thiol protecting group S-Acm-Boc is removed, and at the same time, 7
By forming an intramolecular disulfide bond with the thiol group of cysteine at position and 23,
NP is obtained, and the desired α-chANP can be obtained by purifying it using reverse phase, ion exchange HPLC, etc.

B, r-chANP After purification by ion exchange and reversed phase high performance liquid chromatography (IIPLc), both components in 12 above are further purified using immunoaffinity chromatography using α-chANP antibody. During purification, detection of the physiologically active fraction is performed by RIA using α-chANP antibody. As a result of amino acid analysis, the physiologically active substance in these two components was found to have 116 amino acids and was named γ-chANP.

1) Preparation of chANP antiserum α-chANP' (5-29) (4
g Iobu I 1n) (8■) and used as an antigen. This antigen solution was added to an equal volume of Freund's a
New sealant is mixed with djuvant, and the mixture is subtly injected into white rabbits to immunize them to obtain antiserum.

2) Immunoaffinity chromatography From 2 ml of the antiserum obtained by the above method, protein A
- Immunoglobulin GOgG) fraction is prepared using a Sepharose CL-4B (manufactured by Pharmacia) column.

This was further added to 0.1M sodium phosphate buffer y-(p)
17.4) in AFFI-GRLIO (Bio-Rad
) (2,5ml) and used for chromatography as chANP antibody binding resin. Next, the sample is dissolved in 0.1 M sodium phosphate buffer (pH 7.4), applied to the column (total volume 2.2 ml), and washed with the same buffer.
Elute with M acetic acid.

3) Radioimmunoassay (RIA) α-chAN
P (10.17 g) was dissolved in 0.4 M sodium acetate buffer and subjected to lactoperoxidase method (Y, M, Yachi, J, ll, Vaituka) in the presence of Na I'25.
Itis, E.

N1eschla, M, B, I, 1psett, J
, CI in, Endocrinal, + 3423
-28.1972), 1125-labeled α-chA
After synthesis and purification by HPLC, it is used as a tracer.

RIA using the above antiserum and tracer according to the conventional method.
system was established. 0.5 fmol/tu due to nature
chANP of be can be measured.

As described in the examples below, total c in acidic solvent extraction
Most of the parts with hANP immunoreactivity are γ-chANP
Therefore, like mammalian ANP, it is stored in the T form, and when α-chANP is needed as a hormone, the T form is first processed from the precursor, and then converted to the α form and secreted. (See Figure 14).

2. Structure A1 of chANP The novel physiologically active substance chANP in the present invention is the first
It has the amino acid sequence shown in the figure; γ-chANP (1-
116), α-chANP (position 88-116).

B0 molecular weight: 3000 Daltons (α-chANP)
12,000 daltons (r-chANP) C9 Distinction between acidic, neutral and basic: basicity, amino acid composition (according to amino acid analysis; γ^NP, see Table 1) E, Comparison of amino acids 1) α-chANP especially Among the 17 amino acid residues between the cysteine residues that are thought to be necessary for ANP activity, there are only four positions, namely, glycine at position 10 of rat ANP becomes arginine, alanine at position 17 becomes serine, and glutamic acid at position 18 becomes leucine. , only the leucine at position 21 has been changed to methionine.

In addition, human ANP has five locations, i.e., in addition to the above, h
Methionine at position 12 of ANP has been changed to isoleucine. This strongly indicates that the sequence between cysteine residues, which is essential for expression of activity, is maintained in birds and mammals, and that this part is important for having diuretic effect.

On the other hand, the N-terminal and C-terminal parts of chANP have features that differ from mammalian sequences. That is, the asparagine at position 3 is not found in mammals, and the C-terminal sequence is considered to be essential for mammalian ANP to exhibit its activity; Asn-3Asn
-3er-Phe-Ar A<Asn-Gly-3er
Changed to Arg-Lys-Asn (see Figure 1)
.

α-chANP is homologous to mammalian ANP, but there are considerable differences in the overall structure of the molecule.

2) r-chANP The C-terminal part (88-116) of γ-chANP is α-ch
It shows a molecular structure similar to the mammalian γ-ANP/α-ANP relative relationship in that it corresponds to the ANP structure and indicates the possibility that circulating α-chANP is biosynthesized from heart-stored γ-chANP. However, as shown in Figure 15, r
The structural similarity between -ANP and γ-chANP is that the C-terminal region (
α-chANP site) and the N-terminal Asn-Pro.

Other than the fact that Pro(86)-Arg(87) is common, the overall homology between the two is not very high. Asn-
LeuSer (5-7), 85n-Gly-5er (1
11-113), an amino acid sequence capable of glycosylation is observed, but the presence of sugar chains is not observed in r-chANP.

To summarize the above, the amino acid sequences of α-chANP and γ-chANP are expressed as follows.

X-Met-Met-8rg-Asp-3er-Gly
^rg-Lys-8sn In the above amino acid sequence, X is absent in α-chANP, and in γ-chANP, X is the following amino acid sequence: Asn-Pro-11e-Tyr- to 5n-Leu-S
er-Pro-8la-LysGlu-Leu-Ala
-3er-Met-Glu-Ala-Leu-Leu-
G], u to rg-Leu-Glu-Asp-Lys-P
he-Ala-Leu-11e-Glu8I-Leu
-Glu-3er-Asn-Pro-Asp-Leu-
Gin-GluPro-Gln-Thr-Gln-Gl
u-Glu-I Ie-Pro-Pro-GluLeu
-Thr-Asp-Asp-5er-Asp-Glu-
Gln-Lys-Ala-Glu-Pro-Lys-L
eu-Ala-3er-Asn-Thr-Pro-Le
uSer-Tyr-8rg-Asn-Pro-Phe-
Leu-Lys-8rg-LeaArg-Gly-Va
1-Gln-Met-Pro-Arg.

3. Isolation of chANP cDNA Total RNA was prepared from chicken heart according to a conventional method, and then po
Prepare ly (A) + RNA. Next, using the poly (A) + RNA obtained here,
et al.'s method (Gubler, U., & Hoffman, B.
, J., Gene+25.2630983), c
Create DNA. Next, after binding the EcoRT adapter, a portion of it (70 ng of poly (A) + RN
A) was previously treated with EcoRI and dephosphorylated.
gt 10 DNA (Promega Biotec)
and perform ligation. After ligation, half of it was injected into Gigapace Plus (Stratagen
Create a library by 1n vitro packaging using e). Next, in order to isolate the desired chANP cDNA from the cDNA library obtained here, the phosphoramidite method (Beau
cage, S. L., and Caruthers.

M,H,,Tetrahedron L'ett,,
22.1859-1862.1981) using three oligodeoxynucleotide probes (Probe Ia
, probe Ib, probe ■) are prepared (see FIG. 11). Next, the 3′ end of each probe (α-32P)
A single cD corresponding to chANP purified from chicken heart was radiolabeled with dCTP (2-5XX107cp/pmol) and screened for cDNA by Brake hybridization.
NA clones can be obtained.

4. cDNA analysis of chANP A plasmid is isolated from the single clone obtained according to a conventional method and cut with various restriction enzymes to create a restriction enzyme map of the cDNA. Next, in order to determine the DNA base sequence of this cDNA, we first cut the cDNA with various restriction enzymes.
The NA fragments were subcloned into M13 phage, and the DNA base sequence of the cDNA portion of each clone was determined by 7-zoaza sequence scanning (Takara Shuzo) using 2'-deoxy-7-diaza-GTP. In addition, the transcription initiation site at the 5' end is determined by the primer extension method (prim
er extension; Maniatis, T., et al., Molecular Cloning: A labor
Atory manual+cold Sprig Ha
rbor Laboratory+New York.

1987, Nakayama, et al., Nature.
310.699-701.1984).

5. Pharmacological properties of chANP The peptide chANP of the present invention has diuretic, natriuretic, and antihypertensive effects. Note that in the present invention, natriuresis refers to diuresis that selectively excretes sodium ions relative to potassium ions.

〔Test method〕

Male SD rats (body weight 300-400 g) are anesthetized with 60.7 kg of bentoharbital by intraperitoneal administration. To secure the airway, use a tracheal cannula (PH-240C).
An arterial cannula (PH-50) for blood pressure measurement is inserted into the femoral artery, and a venous cannula (PH-50) for administering Ringer's solution is inserted into the femoral vein.

A constant infusion is performed through this venous cannula at a rate of 1.2 ml/hr. Silaste ink tube (0602 inch inner diameter,
Urine was collected into a test tube through a bladder cannula with an outer diameter of 0.03 finch (manufactured by Dow Corning), and this urine collection was performed for 15 minutes before administering the test substance and for 5 minutes after administration, or over time thereafter, and the urine sample was analyzed. The effect of the test substance is determined by comparing the values.

The predetermined amount of the test substance fANP was 0. After dissolving in IN acetic acid, neutralize with 1/10 volume of 1.3 M) lis-acetic acid solution. This is diluted with 50 μl of sterile physiological saline and administered through the jugular vein. As a control, human I-ANP (α-hANP), which is a known natriuretic, is used.

Hereinafter, the present invention will be explained in detail with reference to Examples.

The hearts (720 g) of 90 chickens made of ANP with a grain size of 0 x 1 - 1 cm were removed, chopped, and treated with 7 volumes of boiling water for 5 minutes to inactivate the protease. . After cooling, acetic acid and hydrochloric acid were added to make the final concentration of acetic acid IM and hydrochloric acid 20 mM. The treated tissues were homogenized at 4°C using a Polytron mixer, centrifuged at 2400 × g for 30 min, and then incubated at 6°C.
Precipitated with 6% acetone. After removing the precipitate, the supernatant was concentrated. The obtained concentrate was dissolved in 0.5M acetic acid and applied to a C18 silica gel column (32x 500m, C-
5ORB SPW-C-ODSChemco). After washing with 3 volumes of 0.58M acetic acid, the concentrate was reduced to 0.1%.
Elution was with 60% acetonitrile (C)I, Ct+) containing trifluoroacetic acid (TFA). After concentrating the eluate, it was dissolved again in 1M acetic acid and equilibrated with 1M acetic acid.
P-Sephadex C-25 column (H+-form,
1. 6 x 60an, 10ml). Elution was carried out sequentially with 1M acetic acid, 2M pyridine, and 2M pyridine-acetic acid (pH 5,0), and the eluents sp-r, sp-
n, and sp-m were obtained. After freeze-drying, the chick rectal specimen SP-2 (dry weight 269.9), which has a relaxing effect and contains a basic peptide, was placed on a Sephadex G-50 column (fine, 1.8 x 135 cm, Pharmacia).
Gel filtration using (solvent: 1M acetic acid, flow rate: 7.5
ml/hr, fraction size; 5 ml/tub
e). Next, each fraction was assayed for its relaxing effect on a chick rectal specimen, and this effect was observed in the portions corresponding to daltons with molecular weights of 3K and 12 (
(See Figure 2 A, B).

1) Fraction ff155-62, which has a small molecular weight among both components having the above action (total amount 21.5
After freeze-drying, the fraction was purified on a TSKCM-25W column (7,6X 30
Ion exchange reverse phase high performance liquid chromatography using (in, Tosoh) E (HPI, C) C solvent; A;
10mM HCOONII4 (pH 6,6): C
H3CN =90: 10 (v/v), BRIMHC
OON)l, (pH6,6): CH3CN =9
0: 10 (v/v), linear concentration gradient (time) A-B
;140 minutes, flow rate;2. Oml/hr, fraction size;
5 ml/1 tube)). Elution time 70-7
The active fraction at 6 minutes (arrowed part in Figure 3) was collected and further added to C
hmcosorb 30DS-H column (8,0x75
It was purified by reverse phase HPLC using fin, chemco) (solvent: 1lzO: CH3CN: 10
%TFA = (A) 90:10:1, (B) 40
: 60 : 1 (v/v), flow rate i2. oml/
hr, linear concentration distribution (time) A-B; 120 minutes). Finally, for final purification, the active fraction with an elution time of 34 minutes (the asterisked area in Figure 4A) was purified using a 219Tp54 phenyl column (4
, 6X 250mm, manufactured by Vydac)
It was purified using PLC (solvent; H2O:CH3CN
:t.

%TFA-(A) 90:10:1, (B)
40:60: 1 (v/v), flow rate; 1. Oml
/hr, linear concentration gradient (time) A → B; 120 minutes)
(See Figure 4B).

As shown in the examples below, amino acid analysis and amino acid sequencing revealed that the purified peptide was a peptide with 29 amino acids, and this was named α-chANP (see Figure 5). ).

2) Next, 12 of the active fractions corresponding to the Dalton region (fraction numbers 34-37) were subjected to PTA to elute the immunologically active peptide (r-chANP). Both fractions were collected and lyophilized (dry weight: 50 μm), and then subjected to immunoaffinity chromatography using the anti-α-chANP IgG-AFFI-GEI column (2.2 ml). ) 1M containing 13CN
After elution with acetic acid, it was applied to reverse-phase HPLC using a former-Pore RP-318 column (4,6X 25On+, Bio-Rad) [solvent; HzO: ClI
3CN: 10% TFA-(A>90:10:
1, (B) 40:60:1 (v/v), flow rate: 1.5ml/hr, linear concentration gradient (time) A:
B=100: O-80:20 (5 minutes), A:
B=80:20→O:100 (80 minutes)]. Both parts of the peak appearing at a retention time of about 52 minutes had immunoreactivity (see Figure 6). As a final purification, μmBo
ndasphere C-18 column (3,8X 15
Reverse phase liver LC using 0mm, 300A, Water)
[Solvent; 1120': CH3C
N: 10% TFA-(A)100: O:
1, (B) 40:60: 1(v/v
), flow rate; 1. Oml/hr, linear concentration gradient (time) AB (60 minutes), see Figure 7]. Total acid rate is 24nm
It was OL.

As shown in the examples below, the number of amino acids in both purified peptides was determined to be 1 by amino acid analysis.
16 peptides, and this was identified as γ-chA.
It was named NP. The results of amino acid composition are shown in Table 1.

To analyze the amino acid composition of the peptide, first hydrolyze the peptide at 110'C for 20 hours in 6N hydrochloric acid containing 0.1% phenol. After that, the analysis was performed using a Hitachi 835 amino acid sequence analyzer.As a result, the amino acid composition was 8 sp; 4.0
5(4), Ser: 3.69(4), Gly; 6.01
(6), Cys; + (2), Met; 2.82 (3)
, Ile; 2.12 (2), Leu; 1.07 (1).

Phe: 10.7 (1), Lys: 0.92 (1), 8rg: 5.25 (5).

This revealed that α-chANP is a peptide consisting of 29 amino acids. Next, the amino acid sequence of this peptide was analyzed using a gas phase automatic amino acid sequence analyzer (Applied Bi) using the Edman degradation method.

System, Model 4708/120Δ).

The results are shown in FIG. When the peptide chemically synthesized by the method described above based on the amino acid sequence clarified as described above was subjected to reverse-phase HPLC, it eluted at exactly the same position as the natural one. In addition, chemically synthesized α-
When the relaxant effect of chANP in chick rectal specimens was measured, it was found to have an activity comparable to that of natural chANP.

2) r-chANP First, purified γ-chANP was analyzed using a gas phase automatic analyzer (manufactured by Δppied Biosystem, model 47).
When the amino acid sequence was measured using 0A), the N
We were able to determine the amino acid sequence from the end to position 41. Next, we analyzed the amino acid composition of γ-chANP.
First, the peptide was incubated at 110°C in 6N hydrochloric acid containing 0.1% phenol and 0.02% mercaptoethanol.
After 4 hours of hydrolysis, analysis was performed using a Hitachi 835 amino acid sequence analyzer. The results are shown in Table 1.

Margin below Furthermore, the entire sequence was determined as follows.

First, the amino acid sequence of γ-chANP was determined from two enzymes, lysyl endopeptidase (Wako Pure Chemical Industries) and Staphylococcus aureus (Staphyl).

coccus aureus) V 8 protease (
Peptide fragments (Ll~■, 6.■1~V5, V6°, V7') obtained by treatment with
was analyzed.

That is, purified r-chANP (1,8 nmol
l) with 200 ng glue endopeptidase and 100 ng
The reaction was carried out in 50mM N-2-hydroethylpiperazine-N-2ethane-sulfuric acid (HEPES) (pH 7,5) at 37°C for 3 hours. Next, μmBondas
here C-18 column (3,8X 150in,
300A, Water); H2O: CH3CN: 10% TFA - (
A) 100: O,: 1, (B) 40: 60
: 1 (v/v), flow rate; 1. 0ml/hr, linear concentration gradient (time) AB (60 minutes)] to obtain L1 to L6 (see Figure 7). on the other hand,
Purified 7-chANP (1,8 nm) was added to 0.5μ
V1 to V5 were obtained by reacting with g of ■8 protease in 100 μl of 0.1 mM ammonium acetate (pH 4,0) at 37°C for 2 hours, and then adding 1 μg of enzyme and leaving it for 4 hours. . In addition, peptides V6° and v
7” was left to stand for 4 hours, then added with 50 μl of 0.1 M ammonium bicarbonate (p
This is the peptide fragment obtained by reacting at 37° C. for 24 hours while adding 1 μg of enzyme every 6 hours in 1+ 7.7°C. By amino acid analysis of each peptide fragment obtained as described above (see Table 1) and determination of their amino acid sequences, the entire amino acid sequence of r-chANP was determined (Figure 9).

Implementation ■Main 薊ANP■ Kume embedding method By the above method, the pharmacological activity of α-chANP was
Compared with ANP.

First, when the chick rectal relaxing activity was used as an index, the effect of chANP was about 1/30 that of α-hANP.

Next, the diuretic effect and excretion effect of each ion (Na'', K゛, Cβ → by intravenous injection of chANP into anesthetized rats are as follows.
The effect was strongest 5 minutes after injection, and after that, the effect was similar to that of α~hANP until 30 minutes after injection (Table 2, Table 10).
(see figure).

Table 1 - Diuretic and natriuretic effects of α-chANP (α
-hANP) α-hANP 3.0 (9)
501±82 592±79 276±25 65
0±144◎The numerical value is the value 15 minutes after injection compared to the value 15 minutes before injection after intravenously injecting the test substance to anesthetized rats.

These results indicate that it has diuretic and natriuretic effects. However, as shown in Table 2, the effect of both was weaker than that of hANP, and the ratio was also about 1/30.

Furthermore, when chANP is injected into anesthetized rats, it also exhibits a hypotensive effect. That is, one injection of chANP (60 μg
/kg) lowers arterial blood pressure (15-20 mmHg)
lasts for 15-20 minutes.

a DNA partial probe (probe I) corresponding to the amino acid sequence of positions 1-5 and 20-25 of cDNA cloning α-chANP
a, Ib, and ■) were synthesized (Fig. 11), and the cDNA library obtained by the method described above was screened. First, the library (approximately 1×10 5 clones) was screened using probe ①, and 29 positive clones were obtained. Next, for nine of them, a mixture of probes Ia and Ib (mixing ratio;
When screened with Ja:1b-1:2), eight positive clones were obtained. Of these, about 8
A clone having 00bp cDNA was transformed into λchANP-
It was named E. Regarding this λchANP-E, the 12th
The DNA sequence was determined according to the strategy shown in the figure, and the DNA sequence was determined to be 75, which contains polyA and EcoRI adapters at both ends.
It was a 5 bp DNA sequence. The DNA sequence of chANP and the amino acid sequence deduced from the DNA sequence are shown in FIG. In Figure 13, DNA numbers 334-420
corresponds to α-chANP consisting of 29 amino acids,
DNA numbers 1-72 correspond to the signal peptide.

〔Effect of the invention〕

As described above, the present inventors isolated a novel peptide using the chick rectal relaxant activity as an indicator from the heart of birds (Japanese chicken).
We succeeded in purifying this peptide, determined its structure, and found that this peptide has diuretic, natriuretic, and antihypertensive effects. That is, in the present invention, the existence of a novel physiologically active peptide that is similar in structure to mammalian ANP but is clearly distinct from them has been revealed, which indicates that it is essential for the activity of the peptide that has the above-mentioned action. It greatly contributes to the elucidation of the structure of amino acid sequences and their activity relationships, and is useful for the production of useful peptides that exhibit this effect.

[Brief explanation of the drawing]

Figure 1 shows the amino acid sequences of α- and γ-chANP.
) shows the elution progress of gel filtration and the chick rectal relaxing effect of each of the two parts. FIG. 3 shows the elution progress of ion exchange HPLC. FIGS. 4A and 4B show the elution progress of chANP reverse phase HPLC. FIG. 5 shows the yield of each PTH-amino acid of α-chANP by a gas phase analyzer using the Edman decomposition method. Each amino acid is represented by a single letter code. The amino acid sequence is shown. FIG. 6 shows the elution course of α-chANP purified by immunoaffinity chromatography in reversed-phase liver LC. Figure 7 shows α-ch after lysyl endopeptidase treatment.
Figure 8 shows a comparison of the amino acid sequences of chANP, rANP, and hANP. Figure 9 shows the amino acid sequence of r-chANP and the peptides treated with two types of enzymes. Fig. 10 shows the diuretic effect of ty-chANP and α-hANP, and the time course of Na'', K゛, and Cρ- excretion.
DNA synthesis probes corresponding to the locations are shown. Figure 12 shows the sequencing strategy for clone λchANP4. FIG. 13 shows the eight cDN sequences of chANP. FIG. 14 shows processing of chANP. Figure 15 shows the precursor of chANP (amino acids are 1
character notation). A, αBet ANP B, YBetN Italy

Claims (1)

[Claims] 1. The following amino acid sequence: [There is an amino acid sequence] (wherein X is absent or the following amino acid sequence: [There is an amino acid sequence] ) Peptide represented by.