JPS58174332A - Novel alpha-amylase inhibiting substance - Google Patents

Abstract

NEW MATERIAL:alpha-Amylase inhibiting substance (WAI-53) which is a protein having the following characteristics. Molecular weitht, 23,000-23,800 by ultracentrifugation, 24,000 by gel filtration, etc.; solubility, soluble in water and dilute salt solution, insoluble in methanol, ethanol, acetone, chloroform and hexane; ultraviolet absorption, E<279>1=12.8 (1cm in aqueous solution) lambdamax 279nm; the degree of migration exhibits single band at 0.53 by polyacrylamide gel electrophoresis by Davis method. The result of the amino acid composition analysis is shown in the table. USE:Fractionation and determination reagent of alpha-amylase isozyme. Useful for the diagnosis of the state of pancreatitis, parotiditis, hepatopathy, etc. The diagnosis can be carried out easily. PROCESS:The water-soluble fraction of wheat seed and wheat flour is extracted, precipitated with ethanol, heat-treated, and purified by the anion exchange chromatography, gel filtration chromatography, cation exchange chromatography, etc. to obtain the objective alpha-amylase inhibiting substance.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel α-an2-ase inhibitor (hereinafter referred to as Wmul-53J in some cases) derived from wheat seeds or wheat flour, a method for producing the same, and a body fluid using the authentic*t- The present invention relates to the fractional quantification of α-7in-2-zeainzyme.

α-Aylase is a hydrolytic enzyme that is widely present in various organisms, and in humans, it is mainly derived from the salivary glands and pancreas, and is associated with various diseases such as pancreatitis, parotitis, liver disease, etc. It is known that the body temperature varies greatly compared to when healthy (depending on the pathological condition such as cancer). Therefore, in order to correctly diagnose these pathological conditions, it is important to understand the accurate sequential trends of α-ylase inozyme derived from the salivary glands and pancreas, rather than simply measuring the total amylase activity. is desired.

Conventionally, electrophoresis has been used to separate and quantify human α-an2-ze isoenzyme in clinical testing, but this method is extremely cumbersome and has drawbacks as a method for rapidly processing specimens. However, as a solution to this problem, an analytical method using an amylase inhibitor derived from wheat flour has been studied by O'Donnell et al. However, it was still unsatisfactory and impractical [J.

011n, oh6m, 23t 560-566
(1977)].

The present inventors have completed the present invention as a result of extensive research in view of the above-mentioned drawbacks of the conventional technology.

The present invention is an α-amylase sourced from wheat flour, which is an inhibitory substance (Wmul-53), and wheat seeds are a water-soluble fraction of wheat flour. A novel α-ami2-ase inhibitor produced by pn through the steps of precipitation treatment, heat treatment, anion exchange chromatography treatment, gel filtration chromatography treatment, and cation conversion chromatography treatment.
The present invention also provides a method for the preparation of α-7<lase in human body fluids using α-amylase inhibitor 'j ((wAx-53)).

The wheat seeds used as the ninth raw material for the TIIT-based α-amylase inhibitor according to the present invention are wheat flour, hard wheat,
Inland wheat, soft wheat, durum wheat or similar
The endosperm of all wheat contains the desired α-amylase inhibitor, regardless of the type and grade of strong wheat flour, medium wheat flour, or thin wheat flour from which it is derived, although the content may vary.

The α-phyllase inhibitor (Wml-53) of the present invention can be obtained by the following steps.

Wheat flour as a raw material is mixed with 3 to 10 times the amount of water, preferably purified water, for a predetermined period of time to extract the W-muko-56 substance by stirring at 1 to 5 hours. The supernatant is collected by appropriate operations such as centrifugation, decantation, and phthalate filtration.

The supernatant of the kernels is subjected to p-treatment by heating under vacuum or normal pressure. ! Heating is usually carried out at about 50 DEG to 70 DEG C. for 10 minutes to 1 hour. Heating at 70° C. for 60 minutes is preferred. If desired, the heat-treated kernel liquid is again subjected to centrifugation, decantation, or p-decay to collect the supernatant.

Here, the heat-treated extract (t or its supernatant) is treated with a water-containing organic solvent such as aqueous acetone, ethanol or methanol [concentration 40-70% (ma/ma)] to remove the generated precipitate. The above solvent was further added to the resulting residual liquid to give a solvent strength of 901 G (V, /'V), and the mixture was left at a low temperature (0 to 10°C) to form a precipitate. Collect the precipitate and dissolve it in water.

Next, the resulting solution was equilibrated with pif&5-a5 Tris-HCl buffer (ion concentration (1005-cL5)) and adsorbed. Elution was carried out at pH 7.0-'18 with N
The eluate is loaded onto a gel gel 27 using Sephadex, Biogel or Ultrogel, preferably Ryufadex D-75, as a carrier, carried out in Tris-HCl buffer enriched with hCJl. . - Slow overspeed part (molecular weight 2CLOOO~301100
), its fireflies, and the resulting product optionally C
The target product is obtained by treatment with an ion exchanger such as M-Ryu 7 allose or OM-Sephadex, and then freeze-drying.

The physical and chemical properties of the thus obtained W-muko-53 substance are as follows.

1) Add water to dilute salt (sodium chloride, potassium chloride,
Ammonium sulfate% Potassium phosphate, Sodium phosphate) #
! ! Insoluble in methanol, ethanol, acetone, chloroform and hexane.

2) Ultraviolet absorption lcT, 12. a (1 steel in aqueous solution) λ
279 nm 5) Molecular weight 24,000 to 25,800 by ultracentrifugation
24,000 by gel filtration method 4) 8D8 polyacrylic acid gel electrophoresis by the method of Mr. Oath et al.
y50'+190-7 (197'3)] gives a single band with a molecular weight of 14,000.

5) The molecular weight is 12.600 as calculated by ultracentrifugation in the presence of a guanidine hydrochloride solution, which is a subunit dissociation agent, and by the sedimentation equilibrium method.

6) The method of Mr. Iwanaga et al. [Protein/nucleic acid/WII element 15°10
37-54 (1970)] When the N-terminus is analyzed, only serine is obtained.

7) Polyacrylamide gel electrophoresis by the method of David et al.
ad@myof 8ci・noe121.404 (19
Reference 64) shows a single band at 1L53.

8) Electrophoresis of 6MR reduced with 2-mercaptoethanol in 1 g solution and then carboxymethylated with monoiodoacetic acid (using M urea solution instead of water according to the method of Mr. Davi et al.) shows that the single electrophoresing WtcL60 Giving a band? ) This substance is a protein, and its composition analysis results are lysine + 455 hisdecine.
+ 111 Arginine + 7.66 Aspartic acid + Melon 97 Threonine 10
542 → hirin + 6294 glutamic acid + 1148 proline
+ 7.71 glycine
+ 9417 ranin + 12
．． 0814 cystine + 9.15 valine + 7.95 methionine
+ 2.19 Inleucine +
2.330 Isine + A18 Tyrosine + 410 Phenylalanine + T61 Tryptophan
Not quantified.

10) The saturation of this substance with each amylase of human 11 fluid glands and pancreas indicates that the required amount ratio of this substance to inhibit each of them by 50 is 12,250 or more (see 1r!iA).

As mentioned above, the α-amylase inhibitor of the present invention is a novel protein having unique properties in terms of molecular weight and electrophoresis.

The α-amylase inhibitor of the present invention (Wml-53) exhibits an extremely 41% inhibitory effect on human salivary gland α-amylase, while its inhibitory effect on human pancreatic α-717-ase is extremely weak. It is. Furthermore, the substance of the present invention inhibits salivary gland α-amylase and pancreatic α-amylase over a wide enzyme concentration range.
I! l! Since the inhibition ratio to α-α2-ze isoenzyme can reach a large value, it is an excellent method for fractionating and quantifying α-α2-ze isoenzyme in various clinical samples including human body fluids. ) It is something that can be used.

S□・.

Already Mr. O'Donnell and others are 1J1j! We reported an α-amylase inhibitor that strongly inhibits pancreatic type amylase (OII) and pancreatic type amylase. This shows the possibility of fractional quantification. (C11nical Ohem
1str723, 560-566 (1977)).

However, in the electrophoretic mobility of the α-2-ase inhibitor reported by Dr. O'Donnell et al.
53, whereas f120 is significantly different. Moreover, compared to the substance of the present invention, the inhibitory ratio for both α-amino-2-zeainzymes is extremely narrow, and the substance of O'Donnell et al. cannot be said to be suitable for differential quantification. Namely.

While the α-amylase inhibitor of Mr. O'Donnell et al. has a 50% inhibition ratio for both amylase einzymes of only 100%, the present substance has a high inhibition ratio of 200% to 300%. - For example, as shown in Figure 1, the required amount of this substance to inhibit 151U of human pancreatic juice α-amylase by 50 μg is about 16 μg, whereas 15 μg of human pancreatic juice α-amylase is 50
% inhibition requires approximately 70 to 80 μg of the α-amylase inhibitor of the present invention, and furthermore, the abundance ratio of human salivary gland type and pancreatic type α-amylase was varied and the amount of the α-amylase inhibitor of the present invention was varied in nine samples. It has been found that when the substance of the invention is applied, the measured value of α-amylase that can be quantified has an extremely high correlation with the amount of pancreatic α-amylase present. Therefore, α-amylase inhibition W% of the present invention! J quality provides an extremely effective and simple method for differentially quantifying amylase.

The method for producing and using the α-amylase inhibitor of the present invention will be described in detail below using Examples.

Example 1 91 parts of purified water was added to 3 # of wheat flour, gently stirred for 1 hour, and then centrifuged to obtain 7 I of purified water. The resulting supernatant was concentrated by 500 dK and the solution was heated at 70° C. for 30 minutes.

The resulting unnatural substances were removed by centrifugation, and anhydrous ethanol was added to the resulting clear supernatant to a final concentration of 60-(ma/ma)K, producing the unnatural substances. After removal by centrifugation, the resulting clear liquid Kj! The concentration is 9
Add absolute ethanol to give os (ma/ma) and leave it overnight to form a precipitate.
99S ethanol, washed with 2gA of 99S ethanol, and dried under vacuum at room temperature, thus obtaining 251 crude α-arynease inhibitors.

Next, the above crude dried product was dissolved in α02M Tris-HCl buffer (pHaoK) at a concentration of 10, and passed through a DIAI-Sepharose column containing an amount of α51 resin to adsorb α-amylase inhibitors. first cjo this column
Wash with sm Tris-H7, 1) 17j, followed by elution of the active substance with a continuous gradient solution of 10M to α2M sodium chloride in the same 17j solution.
The eluate of α21 was obtained, and this solution was concentrated under reduced pressure for 75 d.
A solution containing the α-aminase inhibitor was obtained.

This solution was gel-filtered with Sephadex G-75 using α05M acetate slow-bond buffer solution &0). A solution containing the active fraction of 414IIj was obtained by performing togelafy.

This solution was then passed through 0M-Sepharose, first washed with 151 α05M vinegar solution (11H5,0), and then with a solution containing a continuous gradient of α0M to pα3M sodium chloride in the same buffer. The active fraction was eluted. The obtained active fraction 551d was concentrated under reduced pressure. The obtained chain solution was desalted using Sephadex G-25yl filtration chromatography.
A protein-containing eluate of 609d was obtained. The resulting nine solutions were freeze-dried to obtain 20q of dry matter. As shown in the physical and chemical properties above, this 4 is a novel alpha-alpha-reactive inhibitory substance (W Mu-56), which has unique properties in terms of molecular weight and electrophoresis. The inhibition amount ratio of 50Ls of α-aminase inhibitor (W artificial-53) to human salivary α-7 constructase and human pancreatic juice α-amylase is approximately 25
The inhibitory activity was 150 mu Iυ/q protein.

Example 2 Nine α-aminase inhibitor obtained in Example 1 (Wmul-
53) 1'lF with 10mM sodium chloride
mM Tris-HCl buffer (pB&o) I C15J
Kll explanation nine (exam). Meanwhile, purified nine human salivary gland operculum α
-Amylase (8-muMY) and human pancreatic juice alpha-amylase (P-muMY) were added to 50 mM sodium chloride and alpha 5 mM calcium chloride and 5% calcium chloride, respectively. 5 QrnM Rinshun buffer (pH 7.0) containing bovine serum Alpoon (Klaxon V)
)K] Dilute each 100u/jK, then 0, then P-
AMY and S-AMY t- were mixed at various ratios shown in Table 1, and 30 μl of the sample was applied to vj4.

First sail inferiority 1 2 3 4 5 6 7 8 9-8-
MMY 10101010105 2 1 0P-
MMY 0 1 2 5 1C10101010 B-ffi -α1[12CL5 1 2
5 10 - 10 for each 30μ of mixed samples A1 to 9)
sl reagent solution was added and mixed, and the mixture was kept warm at room temperature for 30 minutes (preliminary reaction). Then, using an automatic analyzer (Model B-100, manufactured by Abbott) using the II bulk method [Anfuuse measurement kit manufactured by Daiichi Kagaku ■]. α-7inlase activity was measured. In addition, as a control, we measured the α-2-ase activity of a sample prepared by adding 10 μj of purified water to the test sample 9.
-MMY and B-AM! A calibration curve was obtained for the differential quantification of both α-anrase isozymes (see Figure 1s2).
1ri.

Using the nine standard curves obtained in this way, the α-anrase isozyme in human serum was determined by fractionation.

A 30μ can of human serum from the 11 cases shown below! Add and mix 10 μl of reagent solution and allow to pre-react at room temperature for 30 minutes. The α-anlase activity is measured using the same method as described above. In addition, as a control, 11 cases of human serum 50μ
10 µm of purified water was added to each sample and mixed, and their α-amylase activity was measured. The results are shown in Table 2.

Table 2 1 41 41 842
30 33 1753 39
36 784 33
32 805
48 45
21 56 23 51
2457 35 37
2028 38
35 1949 2
8 26 14310
5 7
11111 43 41
125 The method for differentially quantifying nine α-amylase using the α-amylase inhibitor of the present invention involves electrophoresis [
"Clinical Chemistry" Vol. 5, p. 118 (1976)
Run.

[Brief explanation of the drawing]

Figure 1 shows the inhibition curve of the YAI-53 substance according to the present invention against salivary α-7 and pancreatic α-amylase, and Figure 2 shows the differential determination of α-amylase according to the present invention. This is the ninth pancreatic amylase calibration curve. Patent applicant Nissin Flour Milling Co., Ltd.

Claims (1)

[Scope of Claims] 1) The following physical and chemical properties (I): Water is soluble in dilute salt solutions and insoluble in methanol, ethanol, acetone, chloroform and hexane; (2) Ultraviolet absorption x ? :'-12,8 (11 in aqueous solution)λ
wax 279 nm (3) Molecular weight 25. ooo~2\80
24,000 by 0y pass-through method (4) 0ath SD8 polyacrylic acid i)' gel electrophoresis by Ujisato's method has a molecular weight of 15,000.
(5) The molecular weight is 12,600 when ultracentrifuged in the presence of a guanidine hydrochloride solution, which is a subunit dissociating agent, and calculated by the sedimentation equilibrium method. (6) Mr. Iwanaga (7) When polyacrylamide gel electrophoresis by the method of Davis et al. shows a single band at CL53, only serine can be obtained. 8) Reduction with 2-mercaptoethanol in a 6M urea solution, followed by carboxymethylation with monoiodoacetic acid, and electrophoresis of the hikimono (according to the method of Mr. Yama et al., instead of water, 6M
(using urea solution) should give a single band with electrophoretic mobility α60. (9) This substance is a protein, and its amino acid combination histidine, t11, and arginine
+ 7.66 aspartic acid +
6.97 threonine + &42
Serine + 494 glutamic acid + 1t48 proline
+ 7.71 glycine
+ 9.41 Annin +
12.08'/S cystine + 91
5 balin +
195 methionine + 2
．． 19 isoleucine + 2.630 isine + a18 tyrosine
+ 4.10 Phenylalanine +
161 Tryptophan 10 Not quantified, and α [WAr − with human salivary gland and pancreatic enzymes]
The saturation curve of the 53 substances is a novel α-7 inhibitor derived from wheat, characterized in that the required amount ratio of the 53 substances inhibiting each by 5° is 1:250 or more. . 2) The following physical and chemical properties are as follows: (1) Suiko-Ku is soluble in dilute salt solutions and insoluble in methanol, ethanol, acetone, chloroform and hexane; (2) Ultraviolet absorption E4%-12,8 (aqueous solution) Middle 151) λ
wax 279 nm (3) Molecular weight: 2,000 to 25,80 by ultracentrifugation
24,000 by gel filtration method (4) Give a single band with a molecular weight of 1000 in F3D8 polyacrylamide gel electrophoresis by 0ath Ujisato's method, (5) Guanidine dibasic acid, a subunit dissociation agent. The molecular weight was calculated to be 12,600 by ultracentrifugation in the presence of a salt solution and the sedimentation equilibrium method. (6) Only serine was obtained when the 9N-terminus was analyzed by the method of Mr. (8) Reduction with 2-mercaptoethanol in a 6M urea solution, followed by 7-mercaptoethanol, Electrophoresis of carboxymethylated with vinegar (using 6M urea S solution instead of water) according to the method of Mr. Ma et al. gives a single band with electrophoresis f: 160. (9) This gtJ substance is a protein, and its anoic acid composition analysis results are lysine + 4.55 histidine + L11 arginine +
7.66 Assu sign lagic acid
Melon 97 Threonine 10 542 Serine + Melon 94 Melta Kenshun + 1t48 Proline 10 Sufu 1 Glycine
+ λ41 alanine +
1L0814 Cystine + [15
Valine + 195 methionine + 2.19 inquisine
+ 2.650 Ishin +
a18 tyrosine + 4.10
Phenylalanine + t61 tryptophan + non-coulostatic, and human - fluid glands and pancreas each attuse and 0vhx -
The saturation line of the 5s substance is 50- inhibiting WAr, respectively.
A reagent for the fractional determination of α-anlase inzyme, characterized in that the active ingredient is a wheat-derived Oα-7 enzyme inhibitor having a required amount ratio of -56 substance of 18,250 or more.