JPH01110697A - Novel protease inhibitory substance - Google Patents

Abstract

NEW MATERIAL:A compound having an amino acid sequence expressed by the formula. USE:A remedy for congestion, edema, collagen disease, accompanied by inflammation, angiopathy accompanied by fibrinoid degeneration, etc. PREPARATION:For example, the liver of a rat is sufficiently homogenized and trichloroacetic acid is added to adjust the pH to 2.0. Precipitates are then separated by centrifugation to collect a supernatant and 10 N caustic alkali is added to adjust the pH thereof to 8.5. The adjusted supernatant is subsequently passed through a column of immobilized trypsinogen to provide a fraction having trypsin inhibitory activity. The obtained fraction is then concentrated and subjected to gel filtration to collect and concentrate a fraction having a low molecular weight. The resultant concentrate is subsequently subjected to reversed phase high-speed liquid chromatography and eluted with continuous concentration gradient acetonitrile to collect an enzymic inhibitory active fraction. Thereby the aimed serine protease inhibitory substance is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an enzyme inhibitor, and more particularly to a peptidic serine protease inhibitor extracted from animal tissue.

Research on inhibitors of trypsin and trypsin-like serine enzymes has been going on for a long time, and many proteinaceous inhibitors have been isolated from mammalian blood, endocrine fluids, snake venom, and plants such as legumes, and their properties have also been clarified. There is. For example, it is summarized in detail in the following documents.

(1) Methods in Enzymology Volume XLV,
pp. 751-881, edited by Las Uro Roland, Akademik Publishing, 1976 (Methods in En
Zymology, vol XLV.

P, 751-881. ed, by La5zlo Lo
rand, Academic Press, 1976
) (2) Biochemical Data Book ■, pp. 225-231 1 Edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin, 1979 In addition, serine enzymes in blood and tissues such as trypsin and tributase play a role in blood coagulation, kinin formation, etc. It is also well known that it is involved in many pathological conditions such as inflammation and vascular wall damage through enzymatic reactions.

N. Heimburger “Proteinase Inhibitor”
Fritz et al., eds., Sbringerluferlach, Berlin, pp. 14-22, 1974 (N, Hein-
burger'Proteinase 1nhibi
tor″by H, Fr1tz etat, , Sp
ringer-Verlag, Berlin, P.
14-22 (1974)) Therefore, specific inhibitors of these enzymes can prevent or improve pathological conditions such as congestion and edema associated with inflammation, and vascular disorders associated with fibrinoid degeneration due to collagen disease and Behcet's disease. It can be used as a drug.

Problem α that Ming attempts to solve However, none of the trypsin and trypsin-like serine enzyme inhibitors has a high enough specificity for enzymes in tissues to be used as a drug.

In order to solve the above problems, the present inventor searched for a substance in mammals that has a high specific inhibitory activity against serine proteases, especially trypsin or similar enzymes, and as a result of extensive searches, We succeeded in isolating an inhibitory substance with a novel structure from rat liver, lung, tongue, and other tissues.

The inhibitor of the present invention is a novel basic peptide with a molecular weight of about 7.600 and has the following amino acid sequence.

I 1e-A 1a-Al a-Cys-Asn-Le
u-Pro-I 1e-Va 1-G 1n-G ly
-Pro-Cys-Arg-A 1 a-Phe-A
1a-Glu-Leu-Leu-A 1a-Phe-A
sp-A 1a-A 1 a-Gin-G 1y-L
ys-Cys-I 1e-G In-Phe-I 1e
-Tyr-Gly-G 1y-Cys-Lys-Gly
-Asn-Asn-Asn-Lys-G 1y-Tyr
-5er-Glu-Pro-Lys-Cys-Lys-
? rp-Tyr-Cys-G 1y-Val-Pro
-Gly-Asp-G1y4yr All amino acids in the above formula are in the L form.

The enzyme inhibitory effect of the inhibitor of the present invention on trypsin and its related enzymes, such as trypsin, is extremely strong and complete inhibition occurs immediately when mixed at a molar ratio of 1=1. In addition, the effect on chymase, a chymotrypsin-related enzyme, is very weak, approximately 171,000 times the effect on trypsin, and thiol proteases other than serine enzymes, such as catebusin B,
It has no effect on catebusin D, which is an acidic protease, and its inhibitory effect is limited to trypsin-like enzymes.

The inhibitory substance of the present invention is, for example, rat liver or lung.

It can be produced from tissue extracts such as the tongue. That is, the above-mentioned tissue is homogenized in EDTA-containing saline, trichloroacetic acid, etc. are added to adjust p2 to about 2.0, and after appropriate stirring, centrifugation is performed to obtain a supernatant. The precipitate was homogenized again with EDTA-containing saline, and after thorough stirring, the supernatant was collected by centrifugation. Combine both supernatants and adjust the PTT to 8.5 with alkali.
Then, add Tris-HCl buffer ptts 5 to adjust the buffer concentration to about 10 mM. The supernatant is passed through a column such as trypsinogen-Sepharose 4B, and after allowing the inhibitory substances to be eluted with acidic saline, it is concentrated using an ultrafiltration membrane. The concentrated sample is then applied to a Sephadex G-75 column and subjected to gel filtration using sodium acetate buffer as a solvent. Fractions containing the desired inhibitor of the present invention are collected and concentrated using an ultrafiltration membrane or the like. Furthermore, the concentrated sample is passed through a column such as reversed phase HPLC, and acetonitrile series, 11! Elution by concentration gradient method yields the desired inhibitory activity fraction that shows a single band in 5DS-polyacrylamide gel electrophoresis.

The enzyme inhibitory effect of the inhibitor of the present invention on trypsin and its related enzyme, tributase, etc. is extremely strong and complete inhibition occurs immediately when mixed at a molar ratio of 1:1. Furthermore, its effect on chymase, a chymotrypsin-related enzyme, is approximately 1/1.000 of that on trypsin, and is extremely weak. In addition, thiol proteases other than serine enzymes, such as cathepsin B, H,
It has no effect on catebusin D, which is an acidic protease, and its inhibitory effect is limited to trypsin-like enzymes. Therefore, the inhibitor of the present invention inhibits the development of pathological conditions such as congestion and edema associated with inflammation, and vascular disorders associated with fibrinoid degeneration due to collagen disease, Behcet's disease, etc., through its specific trypsin-like enzyme inhibitory action. It can be used as a preventive or ameliorating agent.

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples and Test Examples.

Example 1 500 g of fresh rat liver was added to 10 mM DT
Add to 2.000 mQ of 0.5M saline solution containing A, thoroughly homogenize with an ultra-high speed homogenizer, add trichloroacetic acid to adjust the pH to 2.0, stir at low temperature for 3 hours,
Centrifugation was performed at 4.0 OOX g for 20 minutes. The generated precipitate was diluted with 10mM E D containing no trichloroacetic acid.
Homogenize again with 1.000 mQ of 0.5M saline solution containing TA, stir at room temperature overnight, and then stir at 24.000 x g for 2 hours.
Centrifugation was performed for 0 minutes.

Combine the first and second supernatants and adjust the pH using ION caustic potash.
Return to 8.5 and further IM to a concentration of 100mM.
of Tris-HCl buffer (pH 8.5) was added.

The solution was pre-equilibrated with 40ml of 100mM monohydrochloric acid (pH 8.5) containing 0.5M sodium chloride.
Trypsinogen-Sepharose 4 B (Tryp
Column chromatography was performed by passing it through a Sinogen-5 Epharosa 4B) column at a flow rate of 60 mQ/hr.

After washing the column with 1.000T1111 of 0.5M saline, it was eluted with a 20mM hydrochloric acid solution containing 0.5M saline to obtain a fraction having inhibitory activity against trypsin. The obtained fractions were collected and filtered using an ultrafiltration membrane Amifun YM-2 (Am
iconYM-2). The concentrated sample was 0,
10mM sodium acetate buffer (pH 5.0) containing 5M sodium chloride
15+nQ/hr on a Sephadex G-75 column (3X 1000m) pre-equilibrated with )
Gel filtration was performed at a flow rate of .

The inhibitory activity was separated into three peaks as shown in FIG. Fractions of peak 3, which had the smallest molecular weight, were collected and concentrated using an ultrafiltration membrane.

The concentrated sample was subjected to reverse phase high performance liquid column chromatography (Cosmosil 5C4-300, manufactured by Hanui Chemical Co., Ltd.) and eluted with a continuous concentration gradient of 0 to 90% acetonitrile at a flow rate of 1.0 TnIl/min. Peaks with enzyme inhibitory activity were collected, concentrated using an ultrafiltration membrane, and then reversed-phase high performance liquid column chromatography was performed again under the same conditions. The obtained enzyme inhibitory activity fraction was
7, pp. 680-685, 1970, Laemmle, N. K., (Nature, 227. P, 680-68
5.1970゜Laemmli, U., Cleav
age of 5structural protein
sduring the assembly of t
he head of bacteria-phage
When 5DS-polyacrylamide gel electrophoresis was performed according to the method described in T4), a single band was observed as shown in FIG. 2, and the molecular weight was 7.600±500.

The amino acid sequence of the trypsin inhibitor of the present invention is manufactured by Noah Applied Biosystems (Applied Bio).
-systems) Gas phase protein sequencer
It was determined using model 470A.

Test example 1 Trypsin inhibitory activity was measured as follows.

Sigma crystalline trypsin (derived from pig pancreas, Type
■) 10mM hydrochloric acid solution (IJJg/mQ) 10
7-11 (equivalent to 2.2 m units), sample solution 50-
in 100mM Tris-HCl pH 8.5 buffer 1.92
After reacting for 5 minutes at 25°C, the substrate solution (
20mM Boa-Phe-5er-Arg-M CA
.

The reaction was started by adding 10PQ (dissolved in dimethyl sulfoxide). The amount of liberated MCA (7-amino-4-methylcoumarinamide) was directly measured by connecting a recorder to a fluorescence spectrophotometer (Hitachi, model aso-tos) (emission: 460 nm, excitation: 380 nm).

Inhibitory activity is defined as the activity to inhibit 1 unit of trypsin activity.
Expressed as inhibition units.

[Brief explanation of the drawing]

FIG. 1 is a Sephadex G-75 column chromatogram of a rat liver extract containing the inhibitor of the present invention. FIG. 2 is a 5DS-polyacrylamide gel electropherogram of the inhibitor of the present invention. Patent applicant Taisho Pharmaceutical Co., Ltd.

Claims (1)

[Claims] Peptidic serine protease inhibitor represented by the following structural formula [Gene sequence is available]