JPH07289283A - Angiotensin i transferase-inhibiting peptide and method for production the same - Google Patents

Abstract

PURPOSE:To obtain an angiotensin I transferase-inhibiting peptide low in side effects, and to provide a method for suitably producing the same. CONSTITUTION:An angiotensin I transferase-inhibiting peptide having a structure of Glu-Lys-Lys-Val-lie-Asn. This peptide is produced by separating a peptide from rat erythrocyte and subsequently purifying the peptide, or can also be produced by a chemical synthesis method. The Glu-Lys-Lys-Val-lie-Asn lowers blood pressure because of exhibiting the angiotensin I transferase-inhibiting action.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an angiotensin I converting enzyme inhibitory peptide and a method for producing the peptide.

[0002]

BACKGROUND ART Hypertension has a maximum blood pressure of 160 mmHg.
The above is the minimum blood pressure of 95 mmHg or more or both of them. It is said that the number of patients in Japan is about 20 million, which is a disease with a high morbidity. Hypertension is known to cause various complications across a wide range of organs such as cerebral hemorrhage, cerebral infarction, subarachnoid hemorrhage, angina, myocardial infarction, nephrosclerosis, renal failure, and retinal sillosis. Therefore, effective therapeutic agents are desired.

One of the mechanisms for controlling blood pressure in the living body is the renin-angiotensin system, which is a pressor system, and the kallikrein-quinine system, which is a hypotensive system. In the renin-angiotensin system, the enzyme renin is produced in renal juxtaglomerular cells (JG cells) and acts on angiotensinogen, which is a renin substrate in blood vessels, to produce angiotensin I. The enzyme that converts angiotensin I into angiotensin II is an angiotensin I converting enzyme, and the resulting angiotensin II acts on arterioles to cause contraction.

[0004] Angiotensin II also acts on the adrenal cortex, promotes the synthesis and secretion of aldosterone, promotes sodium reabsorption in the kidney, and maintains the body fluid volume. Thus, angiotensin II raises blood pressure. On the other hand, in the kallikrein-quinine system,
The proteolytic enzyme kallikrein acts on the substrate, kininogen, to produce quinine. Kinins have the functions of dilating blood vessels and lowering blood pressure, but they are degraded by kininase II. Kininase II is known to be the same substance as angiotensin I converting enzyme.

From the above, it is considered possible to treat hypertension by inhibiting angiotensin I converting enzyme. Based on this idea, synthetic drugs such as captopril, enapril, and alacepril are currently being developed. In addition, it is known that peptides derived from natural products such as casein, gelatin, fish meat, and blood of domestic animals also have an inhibitory effect on angiotensin I converting enzyme (JP-A-59-44324 and JP-A-64324). -5497
No. 01-313498, No. 5-3062
No. 95).

[0006]

[Problems to be Solved by the Invention] Captopril exhibits a strong hypotensive action when taken orally. However, it is expensive because it is made by a synthetic method, and it is difficult to obtain it cheaply. Moreover, improper use of the drug leads to renal dysfunction and hypotension.
Further, it is said that the SH group existing in the molecule causes rash and taste abnormality.

[0007] Peptides derived from casein, gelatin, fish meat and the like, which are angiotensin I-converting enzyme inhibitors derived from inexpensive natural products, are difficult to separate and purify, and the yields are poor. On the other hand, animal blood is not effectively used and is mostly treated as waste. Angiotensin I from pig blood cells
Although a peptide having a converting enzyme inhibitory activity has been found, it is considered that the blood of other animals also contains the same peptide, and the development of a new blood pressure-lowering peptide with few side effects has been awaited (Japanese Patent Laid-Open No. 3-30083). 66626, JP-A-6-49096 and the like).

[0008]

Means for Solving the Problems In the present invention, a peptide that inhibits angiotensin I-converting enzyme was found in rat blood as an animal other than pig, the structure was clarified, and a method for producing the peptide was established to complete the present invention. did. That is, the present invention is a method for producing an angiotensin I converting enzyme inhibitory peptide having the following structure, and a method for enzymatically decomposing red blood cells, separating and purifying the angiotensin I converting enzyme inhibiting peptide.

Gly-Lys-Lys-Val-Ile-Asn The present invention relates to an angiotensin I-converting enzyme inhibitory peptide derived from rat blood, which is obtained by hemolyzing, denaturing and hydrolyzing erythrocytes of animal blood, It is obtained by separating the obtained peptide-containing composition.
As a hydrolysis method, there is a method using a proteolytic enzyme. As the proteolytic enzyme, any proteolytic enzyme derived from animals, plants, microorganisms or fungi, which can produce the peptide corresponding to the present invention from hemoglobin which is a red blood cell protein, can be used alone or in a mixture.

As a method for separating the peptide-containing composition from the above hydrolyzate, there are the following methods. Filter and separate with an ultrafiltration of 1000 to 50,000 daltons.
The pH is adjusted to near the isoelectric point of the protein, the heme portion is precipitated, and the supernatant is separated by centrifugation. The fractions containing various peptides derived from the heme protein obtained in or were first adsorbed on a packing material C ₁₈ having a octadecyl group for reverse phase chromatography (silica gel having an octadecyl group chemically bonded). After washing the filler with distilled water, the acetonitrile concentration was gradually increased to 15, 30, 6
The adsorbate is eluted stepwise by increasing it to 0,100%. The 15 to 60% fractions were further subjected to C ₁₈ reverse phase HPLC to elute the adsorbate by continuous change of acetonitrile concentration, and further gel filtration HPLC.
The desired peptide can be obtained by purification with.

As blood, rat blood can be used, but blood from all animal species having a corresponding amino acid sequence in hemoglobin can also be used. It can also be obtained from hemoglobin obtained by purifying these bloods. Further, the peptide of the present invention is Fmoc (9
-Fluorenylmethyloxycarbonyl) -amino acid, t-Boc (t-butoxycarbonyl) -amino acid, and other solid-phase synthesis methods, known chemical synthesis methods such as peptide synthesis methods using protease [Biochemistry Laboratory 1, Protein Chemistry IV Part II Peptide Synthesis (P. 20
5), Japanese Society for Biochemistry, Tokyo Kagaku Doujin, Genus Biochemistry Laboratory 2 Under Protein Chemistry, Chapter 20, Peptide Synthesis (P.
641), edited by The Japanese Biochemical Society, Tokyo Kagaku Dojin].

[0012]

EXAMPLES The present invention will be specifically described with reference to the following examples.

[0013]

Example 1 10 g of water was added to 10 g of rat dark blood cells containing 35% of protein to hemolyze erythrocytes. To this, 30 g of water was added and the pH was adjusted to 11 with a 5N aqueous sodium hydroxide solution. After standing at room temperature for 1 hour, the pH was adjusted to 9.0 with a 1 M aqueous citric acid solution, and Noval Alcalase 0.6L
0.124 g was added and enzymatic decomposition was performed at 50 ° C. During enzymatic decomposition, the pH is adjusted to 8.7-with a 5N sodium hydroxide aqueous solution.
I kept it at 9.3. After the completion of the enzymatic reaction, the temperature was lowered to 20 ° C., 10 ml of water was added, and ultrafiltration module of a hollow fiber type having a separation limit of 10,000 daltons was used for ultrafiltration to obtain about 40 ml of a filtrate.

The obtained filtrate is dried by a spray dryer,
About 2 g of a peptide-containing composition powder was obtained. This peptide-containing composition was adjusted to 2 g / 100 ml,
C _{1 8} filler; it was added (particle size 55~105μm Wotazu Ltd.) 4g, and stirred for 1 hour. The solution is filtered using a glass filter, washed with distilled water, and then 0, 15, 30,
Elution was performed at a concentration of 60% acetonitrile, and the eluate was concentrated to dryness. This 15% acetonitrile elution fraction was dissolved in water and separated by ion exchange HPLC. HPL
The C condition is shown in HPLC condition 1. HPLC condition 1 column TSK-gel SP-5PW (21.5 mm x 7.5 cm, manufactured by Tosoh Corporation) (strong cation exchange column) flow rate 4.0 ml / min column temperature room temperature detection UV 210 nm eluent A: 20 mM acetate buffer (PH 5.5) B: A + 500 mM Na ₂ SO _{4 A} 100% linear gradient from 40% to B100% after 40 min Under this condition, the peptide having angiotensin I converting enzyme inhibitory ability was eluted at a retention time of about 25 minutes.
The peptide was further purified by reverse phase HPLC to purify. The conditions at this time are shown in HPLC condition 2. HPLC condition 2 column LichroCART 250-10 HPLC-Cartridge Lichrospher 100 RP 18 (e) (10 μm) Merck flow rate 4.0 ml / min column temperature room temperature detection UV 210 nm eluent A: 0.05% trifluoroacetic acid B : 0.05% trifluoroacetic acid 50% acetonitrile A linear gradient from 100% A to 120% B after 120 min Under condition 2, a peptide having a high angiotensin I converting enzyme inhibitory ability was eluted at a retention time of 28 minutes.

This peak is referred to as Applied Biosy.
gas phase protein sequencer (mode)
1-470A) and online high performance liquid chromatography were used to carry out Edman degradation reaction to identify the PTH amino acid obtained in each cycle. As a result, it was found to have a structure of Gly-Lys-Lys-Val-Ile-Asn. [Measurement of Angiotensin I-Converting Enzyme Inhibitory Activity] Angiotensin I-converting enzyme inhibition is measured by the method of Kashman et al. (Biochemical Pharmacology, 20 P. 1).
637-1648 (1971)) improved method of Maruyama et al. (Agricultural Biological Chemistry, 46 [5] P. 1393-1394 (1982))
Obeyed.

30 μl of the peptide aqueous solution of the present invention in a test tube
Then, 250 μl of boric acid buffer containing 8.3 L-hypril histidyl leucine (manufactured by Sigma) and NaCl as an enzyme substrate and having a pH of 8.3 was added and preincubated at 37 ° C. for 10 minutes. Then Angiotensin I
100 μl of the converting enzyme-containing solution was added to start the enzymatic reaction. At this time, the concentration of borate buffer was 0.1M, L-
Hipryl histidyl leucine concentration is 5 mM, NaCl3
The enzyme activity when it is 00 mM and no inhibition is
8 mU.

After the reaction was carried out while incubating at 37 ° C. and pH 8.3 for 30 minutes, 250 μl of 1N HCl was added to stop the reaction. As a blind test, 1N HCl was added before the addition of the angiotensin I converting enzyme-containing solution, and the same treatment was performed. Add 1.5 ml of ethyl acetate and add
Shake for 5 seconds to extract hippuric acid produced by the enzymatic reaction,
Centrifuged at 00 rpm for 10 minutes, and ethyl acetate layer 1.0 m
1 was collected in a test tube. After heating ethyl acetate in hot dry bath at 120 ° C for 30 minutes to completely remove it,
It was left at room temperature for 5 minutes. Then, 1.0 ml of water was added, and the amount of hippuric acid produced was determined by measuring the absorbance at 228 nm. The liquid containing angiotensin-converting enzyme used for the enzymatic reaction is rabbit rung acetone powder (manufactured by Sigma) 1
g to 0.1 M borate buffer (pH 8.3) 10 m
Dissolve in 1 liter and stir well, then 40 at 40,000g at 4 ° C.
It was prepared by diluting the supernatant after centrifugation for 0.1 minutes with 0.1 M borate buffer (pH 8.3).

The angiotensin converting enzyme inhibitory activity was determined using the following formula 1.

[0019]

[Equation 1]

A: Absorbance upon addition of distilled water (228 nm) B: Absorbance upon addition of inhibitor (228 nm) a, b: Blinded absorbance for each (228n)
m) IC _{5 0} of the peptide by this method was 4.6MyuM.

[0021]

Example 2 Using a peptide synthesizer 430A from Applied Biosystems,
The peptides shown in the sequence listing were synthesized according to the operation manual of the company. The peptide was extracted and treated with an anion exchange resin column, and then the peptide of the present invention was purified by reverse phase HPLC using an ODS column. As a result of measuring the amino acid composition, it was in good agreement with the expected value.

The obtained peptide was dissolved in water, and Example 1 was used.
When the ACE inhibitory activity was measured in the same manner as above, the same high activity was given.

[0023]

According to the present invention, an angiotensin converting enzyme inhibitory peptide can be isolated from rat blood. Further, by determining the amino acid sequence of the peptide, it became possible to synthesize a peptide having similar activity.

[0024]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Origin Biological name: Rattus norvegicus Sequence: Gly Lys Lys Val Ile Asn 1 5

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C07K 1/18 8318-4H 7/06 Z 8318-4H C12N 9/99

Claims (2)

[Claims] 1. An angiotensin I converting enzyme inhibitory peptide having the following structure. Gly-Lys-Lys-Val-Ile-Asn 2. The method for producing an angiotensin I-converting enzyme inhibitory peptide according to claim 1, which comprises enzymatically decomposing red blood cells, separating and purifying the same.