JPH0225500A - Novel physiologically active peptide - Google Patents

Abstract

NEW MATERIAL:A peptide expressed by the formula [(1) and (2) or (3) and (4) are directly bonded; X is absent or represents amino acid sequence Ser- Ser-Asp]. USE:A diuretic and hypotensive agents. PREPARATION:For example, a frog 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 homogenizer. The resultant homogenate is then centrifuged to subject the supernatant to ion exchange chromatography. Adsorbed substances are subsequently eluted with 2M pyridine-acetic acid, etc., to provide an active fraction containing a basic peptide using relaxant activity of a chick rectal specimen as an index. Furthermore, the resultant fraction is successively subjected to gel filtration and affinity chromatography using an antibody column and purified to afford the frog atrial natriuretic active peptide (fANP) expressed by the formula.

Description

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

[Conventional technology]

Atrial natriuretic active peptide (AN) is a hormone synthesized, stored, and secreted in the atrial Mi tissue of mammals.
P) is a substance that regulates the homeostasis of body fluids and blood pressure. In recent years, research on mammalian (human) ANP has revealed that it exists in the atrium as γANP, which consists of 126 residues, and that α-ANP, which consists of 28 residues at the C-terminus, circulates in the blood (de Bold, A.J., 5c
ience, 230,767770.1985; Ca
ntin, M., and Genest, J., Endo
crineReviews, 6, 107-127, 19
85; Matsuo, M. and Nakazat.

H, Endocrinology and Meta
bolism C11nics of North A
merica, 16. Trial Natriur
etic Factor (eds Rosenbla
tt, M, and Jacobs, J, W,)+p,
43-61].

The amino acid sequence of ANP is broadly similar in mammals from rodents to humans. In particular, α-ANP
was found as a substance with the same sequence (identcal form) in higher mammals including humans, dogs, and pigs. Also, only one amino acid sequence differs in the rodent neck (Kangawa et al. and Matsuo et al.)1. ,Biochem,
Biophys, Res.

Commun, 118131-139, 1984;
Flynn, T.G., deBold, M.I.
and de Bold A, J., Bioc.
hem, Biophys.

Res,Commun,, 147.859-865,1
983; Oikawa, S. etal, Bioc.
hem, Biophys, Res, Commun,,
132, 862-8981985; Nemer, M.
et al Nature, 312, 654-65.
6, 1984; Ong, H., Life Sci, 3
8.1309-1315, 1986; Seidma
n.

C, E, et al, 5science, 226.
1206-1209 1984).

On the other hand, histological and pharmacological studies suggest that ANP-like substances exist even in the hearts of non-mammalian animals (de Bold, Aj, et al.
Can, J., Physiol, Pharmacol.
61, 127-130.1983).

In addition, Netcbitailo et al.
AN in the heart of Ranaridibunda)
It has been reported that there is P-like immune activity (Netch
itailo, P, etat, American 5
society of l1ypertension S
ynposiumSeries, 1. Biologic
al Active Trial Pepti
des (eds Brenner, B. M., et.
al), p, 166-169) a Based on these facts, it is thought that amphibian ANP is structurally related to mammalian ANP, but its structure is still unclear.

cProblems to be Solved by the Present Invention] As a result of intensive research by the present inventors in order to find a substance that has diuretic effects from amphibians, the present inventors discovered that the frog (Rana cat
esbiana) We have successfully isolated and purified a new peptide with diuretic effects from the heart, determined the structure of this peptide, and discovered that this peptide has diuretic, natriuretic, and antihypertensive effects, and have developed the present invention. completed. Therefore, an object of the present invention is to provide a novel bioactive peptide that is similar in structure and pharmacological activity to mammalian ANP, but is clearly distinct from them. In this specification, this novel physiologically active peptide is referred to as fANP (frog atrial
It is called Natriuretic Peptide;).

[Failure to solve the problem]

A. The bioactive peptide of the present invention is a frog (Rana).
catesbiana) heart tissue in a suitable acidic solvent,
For example, homosinate in acetic acid or acetic acid containing hydrochloric acid, the method of Currie et al.
Various methods commonly used for purification of peptides, such as centrifugation, acetone precipitation, solvent extraction, ultrafiltration, and gel filtration adsorption chromatography, were carried out using the relaxant activity of chick rectal specimens as an indicator, according to ``Nature, 221.1-1.3.1983''. It can be obtained by separating peptides by appropriately combining treatments. In the present invention, as shown in FIG.
Chick rectal sample relaxant activity was observed in the parts corresponding to Daltons K and 2 to 5, and the region with the smallest molecular weight of both parts was analyzed by reversed-phase high performance liquid chromatography (HPLC).
Two peaks were obtained (see Figure 2). As a result of amino acid analysis, the number of amino acids was 21 and 24, and they were named fANP-21 and fANP-24, respectively. Furthermore, it goes without saying that if the structure and amino acid sequence of the physiologically active peptide are known, it is more advantageous to use chemical synthesis to obtain it industrially.

The fANP of the present invention can be produced by a solid phase method or a liquid phase method commonly used for peptide synthesis.

For example, fANP can be synthesized by the solid phase synthesis method of Merrifield et al.
can be synthesized. When this method is applicable to the present invention, the α-amino group is protected with tert-butyloxycarbonyl 1 (Boc group) for any amino acid residue, and the guanidino group of arginine is protected with a tosyl group (Tosyl group).
It is preferable to protect the hydrogen group of serine with a benzyl group (1'tz I group) and the β-carboxylic acid group of aspartic acid with an O-benzyl group (0-Bzl group). First, a protected derivative of phenylalanine, a C-terminal amino acid;
By synthesizing NP-resin and treating it with hydrogen fluoride, 1fANP is cleaved from the resin, deprotected at the same time, and reduced, resulting in Cys" 20-(S-
Acm)-fANP (Acm 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, disulfide bonds are formed by the thiol groups of cysteine at the 4- and 20-positions, thereby producing a crude synthetic fAIJP.
is obtained, and the desired fANP can be obtained by purification using reverse phase, ion exchange HPLC, etc.

B, HA Raccoon■Episode■) The novel physiologically active peptide fANP in the present invention has the following amino acid sequence; Table 1 (4)-Arg-8rg-Phe (wherein (1) and (2), and (3) and (4) are directly linked and X is absent or does not represent the amino acid sequence 5er-3er-Asp). Among these, fANP in which X does not exist is called fANP -21, and if X is 5
fANP which is er-5er-8 sp is fANP-3
It is called 4.

2) Molecular weight: fANP-24:2564; fANP
-21:22753) Distinction between oxidized, neutral, and basic: Basic 4) Amino acid composition (by amino acid analysis) fANP
-21 and fANP-24 are as follows:
Shown in the table.

Numbers in parentheses indicate the closest integer value to the measured value.

(Cys)z: Cystine 5) Comparison of amino acids fANP has high homology with rat ANP (rANP), and for the 17 amino acid residues between the cysteine residues, glycine at position 10 of rANP Leucine at position 12 has been changed to serine and methionine. Furthermore, human ANP differs from human ANP in that methionine at position 12 of hANP is changed to isoleucine in three places, in addition to the above. This indicates that fANP exhibits cross-activity with anti-α-hANP antiserum that recognizes the core of hANP. Furthermore, the sequence between cysteine residues is highly conserved across mammals and amphibians (
This strongly indicates that this part is fundamentally important for having a diuretic effect. However, the N-terminal and C-terminal portions of fANP have features that differ from mammalian sequences.

That is, the asparagine at position 3 is not found in mammals, and the sequence As at the C-terminus, which is considered to be essential for mammalian ANP to exhibit its activity.
n-3er-Phe-^rg-Tyr is Gly-Arg
-Arg-Phe (see Figure 5).

It has diuretic and antihypertensive effects.

(Test method) Male SD rats (body weight 300 to 400 g) are anesthetized with 60 mg/kg of bendoparvicool by intraperitoneal administration. To secure the airway, use a tracheal cannula (PE-240C1).
An arterial cannula (PE-50) for blood pressure measurement is inserted into the femoral artery, and a venous cannula (PE-50) for administering Rinkel's solution is inserted into the femoral vein. l through this venous cannula, 2 ml/h
Constant injection at a rate of r
on). Silastic tube (inner diameter 0.0
Urine was collected into a test tube through a bladder cannula of 2 inches, outer diameter 0.03 inches, manufactured by Dow Corning, Inc., and this urine collection was performed for 15 minutes before administering the test substance and for 5 minutes after administration, or over time thereafter. The effect of the test substance is determined by comparing the analysis of urine samples.

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) lithium hydrochloric acid solution. Dilute this with 50 μl of sterile physiological saline,
Administer through the jugular vein. As a control, Hi-1-ANP (α-ANP), which is a known soot/slum diuretic, was used.

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

After decapitating 154 ANP frogs (Rana catesbiana), the hearts (142 g) were immediately removed. After shredding, it was treated with 10 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 tissue was homogenized with a Polytron homogenizer for 10 minutes, centrifuged at 14,500×g for 40 minutes, and then filtered with a GF/B filter. The supernatant was transferred to a 018 column (90 ml, Ch
emco LC-3OLB, SPW-C-00sCh
emco) and 0.1% trifluoroacetic acid (T
7SeI-nitrile (CHiCN) containing 60% of FA)
) "ill:' was eluted. After concentrating the eluate, it was applied to an sp-Sephadex C-25 column equilibrated with 1M acetic acid (
It was subjected to ion exchange chromatography using It'-formlontt. 1M acetic acid, 2M pyridine,
Sequentially eluted with 2M pyridine-acetic acid (pl+5.0), ? in each solution. Eluate sp-r, 5p-n,
sp-■ was obtained.

After freeze-drying, the sp-m fraction, which has a relaxing effect and contains basic peptides, is extracted from the chick rectal specimen. jE25mg) on a Sephadex G-50 column (fine, 1.8X
134 cm, manufactured by Pharmacia) (solvent: 1M acetic acid, flow rate: 1 Q m7/hr, fraction size: 5 ml/1 tube). Next, each fraction was assayed for its relaxing effect on a chick rectal specimen, and the effect was observed in the portions corresponding to the molecular weights 12K and 2 to 5 (see Figure 1) 6. In order to purify both fractions N0.51-57, which is the region with the smallest molecular weight among the two fractions,
After lyophilizing both parts, anti-α-hANPIgGAFFI
- Immunoaffinity chromatography using a GEL 10 column. [The IgG fraction used in the chromatography can recognize the center of α-hANP, and anti-α-hANP antiserum (#125-8
) using a Protein A Sepharose CL-4B (manufactured by Pharmacia) column.

The IgG was also added to 2 ml of AFF4-GEL 1 in 0.1 M sodium phosphate buffer (I2) (pl+7.4).
0 (manufactured by Bio-Rad), and this was packed into the column for use. ] In other words, the sample is 0.1
It was dissolved in M striium phosphate buffer (pH 7,4), adsorbed onto a column, and washed with the same buffer. Most of the active fractions were retained in the column, and the peptides in both fractions were eluted with 1M acetic acid containing 10% CH3CN. Next, a C-18 column (Chemco
sorb 30DS-118, OX751m, Che
(manufactured by mco) using reversed phase ((circle, C<flow rate; 2 m
l/min, solvent conditions, HzO:Ctl:+CN:
10% TFA = (A) 90:10:1, (B
) 40:60:1 (V/V) linear concentration gradient; 80 minutes) (Fig. 2a).

As shown in FIG. 2a, two peaks (I, ■) having the above-mentioned effects were obtained. In order to ensure the degree of purification of both components, we used a C-18 column (μBondasphere) again.
; 3.9 X 150 n+, 300A, Water
Reversed phase HPLC (flow rate: 1ml/11
i1, solvent conditions; HzO: C11aCN: 10%
TFA - (8)90:10:1, (B)40
: 60 : 1 (V/V) linear concentration gradient;
90 minutes) (No. 2b, No. 20.). As shown in the Examples below, the peptides in the two purified fractions were analyzed by amino acid analysis to determine the number of amino acids or the peak
There were 24 and 21 peptides at peak ■ and 21, respectively (Table 1), and they were named fANP-24 and fANP-21, respectively. In addition, fANP from frog heart tissue (142 g')
The yield of fANP-24 is 2.30 nmol, fA
NP-21 was 0.9 nmol.

The analysis of the amino acid sequence was carried out by first hydrolyzing the peptide in 6N hydrochloric acid containing 0.1% phenol at 110°C for 20 hours, followed by the Hitachi 835 amino acid sequence. This was done using an analyzer. Furthermore, fANP reduced S-carboxymethylation (S-carboxymethylation; R
CM) and 10 mM dithiothreyl (DTT)
(0,5 M) lithium-hydrochloric acid buffer (p) containing
18.5) for 4 hours at 37'c, then 20 m
The RCM-fANP obtained by treatment with sodium M iodoacetate for 5 minutes was subjected to reverse phase HPLC (conditions:
Same as in Example 1) and purified using a gas phase automatic analyzer (A
Manufactured by ppied Biosystem, model 470A
The amino acid sequence was analyzed by the Edman degradation method using 12OA). Figure 3 shows the results of successive identification of PTI (- amino acids toward the C-terminus) using the above method, that is, the yield and amino acid sequence of PT old amino acids at each stage of Edman degradation. The peptide synthesized by the above method based on the sequence was subjected to reverse phase 11
When subjected to PLC, L was eluted at the same position as the natural product (Figure 2, c), and it had the same relaxing effect on chicken rectal specimens. Also, fANP -21 is fANP
The amino acid sequence was the same as the amino acid sequence in which the N-terminal three residues of -24 were deleted.

By the method described above, fANP-21 and fANP-24 were isolated.
The pharmacological activity of α-hANP was compared with that of α-hANP.

First, when we used chick rectal relaxant activity as an index, we found that fANP-21 was 2/3 compared to α-hANP;
fANP-24 was approximately F/13.

Next, the diuretic effect and excretion effect of each ion (Na*, K゛, Cd) due to intravenous injection of fANP into anesthetized rats are strongest during 5 minutes after injection, and then α
- It exhibited the same effect as hANP (Fig. 4). These results indicate that both fANPs have diuretic and natriuretic effects. However, the second
As shown in the table, the effect of both is weaker than that of hANP, and the ratio is 1/10 for fANP21 and 1/10 for fANP -2.
4 was 1/100.

*The upper row of numerical values shows the results 15 minutes before administration of the test substance, and the lower row shows the results 15 minutes after administration. Values represent the mean Shinji standard error obtained from 3 animals.

Furthermore, when fANP is injected into anesthetized rats, it also exhibits a hypotensive effect. That is, one injection of fANP-21 (30μ
g/kg), the decrease in arterial blood pressure (1520 mmHg) lasts for 15-20 minutes. On the other hand, fANP-24 has an even lower antihypertensive effect, with a single injection (300 μg/kg
), the decrease in systolic blood pressure (approximately 10 mm) (Ig) only lasted for 10-15 minutes.

〔Effect of the invention〕

As described above, the present inventors have discovered that amphibians (frogs; Ran
We succeeded in isolating and purifying a new peptide from the atrium of A. a. I found it. 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. This will greatly contribute to the elucidation of the structure of amino acid sequences and their activity relationships, and will be useful in the production of useful peptides that exhibit this effect.

[Brief explanation of the drawing]

Figure 1 shows the acidic solvent extract (SP-1) from frog heart.
11) shows the elution progress of the gel filtration of 11) and the elution progress of the reversed phase 11PLc of the fractions purified by H1 of each fraction. 4+(b) is fANP-24 and synthetic f8NP-2
4 shows the elution progress of No. 4 by reverse phase HPLC.噸十(C) is fANP-21 and synthetic fANP-21
The elution progress of reverse phase HPI, C is shown. FIG. 3 shows the yield and amino acid sequence of each PTH-amino acid by Edman degradation of fANP-21 and fANP-24. Each amino acid is represented by a single letter,
C9 represents an S-carboxymethyl cysteine residue. Figure 4 shows fANP-21, fANP-24 and α
- diuretic effect of hANP and Na'', K'' and cr
- shows the time course of emissions; Figure 5 shows fANP, rA
NP, indicates amino acid sequence comparison. and hANP

Claims (1)

[Claims] The following amino acid sequence: [There is an amino acid sequence] [In the formula, (1) and (2), and (3) and (4) are directly bonded, and X is absent or or the amino acid sequence Ser-Ser-Asp].