JPS6041498A - Preparation of peptide originated from serum albumin of mammal - Google Patents

Abstract

PURPOSE:To produce a peptide having the activity to promote the action of insulin, by treating a serum albumin of mammal with a specific enzyme. CONSTITUTION:A mammalian serum albumin is decomposed with one or more enzymes selected from tyrpsin, acrosin, koknase and E. coli protease II to produce a peptide having the activity to promote the action of insulin in the reaction liquid. The treatment with the enzyme is carried out preferably under the conditions shown in the table. The peptide can be separated from the reaction liquid e.g. by molecular filtration with a gel-filtration carrier, dialysis, electrophoresis, ion exchange column chromatography, high performance liquid column chromatography, etc. The separated peptide is purified by dissolving the peptide in a weakly acidic solution, carrying out the gel-filtration and freeze-drying, dissolving in a buffer solution, subjecting to the cation exchange column chromatography, and dialyzing and freeze-drying the product.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a method for producing a peptide derived from mammalian blood albumin, specifically, the above-mentioned peptide having insulin action-promoting activity.

Serum albumin in milk ribs accounts for approximately 60% of total plasma protein.
It is the main protein in serum. Although it is known that albumin maintains plasma R3 osmotic pressure and is involved in the fingering of each of the 91 vitamins, hormones, drugs, etc., it does not itself have insulin action-promoting activity.

The present inventors have been conducting intensive research on factors that enhance the action of insulin.
We have discovered a new fact that the above-mentioned peptide having insulin action-promoting activity is present in the decomposed product that is degraded by the action of acrosin, cocnase, and a small amount of coli protease (1).

The present invention was completed based on the above findings, and involves the action of at least one of L-ribusin, acrosin, cocnase, and [:, coli protease ■] on mammalian serum albumin to produce a peptide having insulin action-promoting activity. The present invention relates to a method for producing a peptide derived from bent hole animal serum albumin.

According to the present invention, a peptide having a novel insulin action-promoting activity that has not been previously known can be obtained through in-situ operations.

The above-mentioned peptide obtained by the method of the present invention is characterized by having a unique physiological action of insulin action promoting activity. Here, insulin action-promoting activity is defined as
It refers to the action of insulin, which primarily promotes the uptake of glucose and amino acids into muscle and fat II and the conversion of glucose into glycogen and fat in the body, thereby lowering blood sugar. Therefore, the above-mentioned peptide obtained according to the present invention is useful for the treatment of diabetes, which is caused by an abnormal condition of groin opening caused by insufficient insulin action.

-sugar 'ij'A in U'2 @ is broadly divided into industrial type diabetes (insulin dependent stoliotic disease) which is caused by an absolute lack of insulin secretion due to pancreatic starvation, and type ■ which is mainly due to decreased sensitivity to insulin. Diabetes mellitus is classified as non-insulin dependent (1i). The peptide (q) according to the present invention can improve insulin sensitivity by promoting insulin action, and is particularly effective in treating type 1 diabetes.

In addition, by using the peptide obtained by the present invention in conjunction with insulin therapy, whether type ■ or type ■ churiuresis, the use of insulin preparations can be greatly saved, and at the same time, it can be used for long-term or high-dose insulin therapy. The development of insulin shock and insulin resistance caused by the production of insulin antibodies can be prevented.

The mammalian serum albumin used in the method of the present invention may be any commercially available serum albumin isolated and purified from mammals such as cows, horses, sheep, dogs, humans, monkeys, and mice by various methods. good. Alternatively, the monotjiU4 may be produced from the above-mentioned various mammals according to a conventional method. In addition, 1 which acts on serum albumin
The enzyme harmful to helipsin may be either a commercially available enzyme@agent or one isolated and prepared according to a conventional method.

In the method of the present invention, the serum albumin is treated with at least one enzyme selected from the group consisting of lipsin, acrosin, cocnase, and E, Co11 protease (2) to decompose the peptide bond at a specific site. The decomposition reaction of peptide bonds by each of the enzymes described above is carried out under conditions of optimal water temperature and pH for the action of the enzyme used. Although the 2nd condition differs slightly depending on the r4 type of enzyme, it is generally appropriate to set the temperature range to about 20 to 40°C and the I)H range of 5.0 to 9.5. ) It is used in a range of about 1/10 to 1/250 of the weight, and the reaction is completed in about 15 minutes to 36R. Examples of the decomposition reaction conditions for each enzyme are shown in Table 1 below.

See Table 1 ■ The reaction is stopped according to the conventional method, and if necessary, an enzyme inhibitor such as soybean I/lipsin inhibitor (5oybeantryps) is used.
This is done by using a method such as in 1nhtbitor). Due to the decomposition reaction by trypsin, the raw serum albumin is decomposed into arginine (Arg> or lysine (Lys) at a site without steric hindrance.
), the peptide bond is cleaved by the carboxyl group, and thus the desired peptide can be obtained as an active fraction having insulin action-promoting activity.

The above-mentioned peptide can be purified from the reaction solution by a conventional peptide method, and can be stored by a conventional trapping and drying method.

As the above-mentioned distillation purification means, for example, Sephadex G-
50, Sephadex G-100 (manufactured by Pharmacia)
Molecular filtration using gel filtration carriers such as dialysis, electrophoresis, ion exchange column chromatography such as SP-Sephadex C-25 (manufactured by Pharmacia), DEAE-hirulose (manufactured by Whatman), or high-performance liquid Examples include methods such as column chromatography and combinations of these methods. A preferable example of the above purification means is as follows. That is, the reaction solution or its dry product is dissolved in a weakly acidic solution such as ammonium acetate or acetic acid, gel-filtered, and freeze-dried. At this time, if desalting is insufficient, suspend in water and freeze-dry again.

The active fraction obtained by the above gel filtration is then dissolved in a temporary solution of sodium acetate, phosphoric acid, citric acid, etc., and then subjected to cation exchange column chromatography.
Contains less than 0.1M of salt after washing with acetate buffer! Wash with 0.1-2.0 M1 preferably 0.1-0
．． Elute with a linear 0 degree gradient of approximately 8M sodium chloride, and collect the maximum activity peak. After dialysis, a purified product is obtained by freeze-drying. This can be further purified by high performance liquid chromatography or the like, if necessary.

Thus, by the method of the present invention, a purified mammalian serum albumin-derived peptide having insulin action-promoting activity is obtained.

The desired peptide product obtained by the above method has A east or Lys at its C-terminus, an amino acid bonded to the carboxyl group of Arg or 1jls of raw albumin at its N-terminus, and is derived from raw albumin. Contains a specific amino acid sequence.

The peptide of the present invention obtained as described above can form a salt with a pharmacologically acceptable acidic or basic compound. Such acidic compounds include, for example,
1 Silent acids such as sulfuric acid, phosphoric acid, hydrobromic acid, fumaric acid,
Examples of basic compounds include sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium hydrogen carbonate, etc. I can do it.

The above-mentioned peptide or salt thereof obtained according to the present invention can be used as an antidiabetic agent alone or in combination with insulin. In that case, the active ingredient is usually processed together with a pharmaceutical carrier and used in the form of a pharmaceutical composition.
Among these, it is preferable to use it as an injection. When manufactured as an injectable, the resulting formulation is preferably sterile and isotonic with blood. When molding into an injection form, diluents commonly used in this field can be used. For example, water, physiological saline, etc. can be mentioned. In this case, the preparation in the form of an injection may contain enough sodium chloride or glycerin to prepare an isotonic solution. In addition, the above preparations contain ordinary buffering agents, non-toxic agents, preservatives, etc., and if necessary, coloring agents, preservatives, fragrances, seasoning agents, preservatives, etc., and other pharmaceuticals. It is something that Moreover, the above-mentioned active ingredients can be dissolved in distilled water for injection at the time of use, and can also be used as a solubilizing agent.

The preparation prepared as described above can be administered in a manner depending on its form. In the case of an injection, it is administered intravenously alone or mixed with a normal replacement fluid such as an amino acid.

Examples will be given below to explain the method of the present invention in more detail. Insulin action promoting activity of samples in Examples 11
was measured by the following method.

Insulin action-promoting activity> Adipose tissue was excised from an attic Wistar rat (weighing 150-300 g), and E
Preparation of xplant (fat crude suke). 10 E's
xplant (7.5-10m as wet weight!+>
, D-(U-"C) glucose 0.05μCi/Tsuru, baking soda 1.5111g/l+112, penicillin 35U
/IQ and streptomycin 0.11n! +/111
M-199 including 2 [“Age of Medicine” Vol. 62, No. 6
issue, p. 435, published on August 5, 1960] a u (To
pper, Y.

J., et al., Methods in Enzy.
molovy, 39°443 (1975)) and cultured at 37°C for 20 hours in a gas phase containing 3% carbon dioxide. After culturing, measure the moisture content of the E Xplant, and the total m
Hydrolyze in a 50% ethanol solution of -KOH at 100° C. for 2 hours. Fatty acids were extracted with 3mG of petroleum ether from the melted ice acidified with 6N-sulfuric acid and 5mff, and the release capacity was measured using a conventional method. i Radioactivity J: Extract.

The insulin action-promoting activity of a sample is calculated by adding the above specific radioactivity value obtained by adding the sample and insulin (0.2 m LJ/mQ) to the above reaction system, and by adding the sample alone (the same calculation obtained). Expressed as a percentage of the value.

Example 1 Crystalline bovine serum albumin (manufactured by Miles) was dissolved in 0.1M ammonium bicarbonate (pH 8,1) for 12 hours using TPCKt-lipsin (manufactured by Worthington) while remaining undenatured.
time digested (substrate 'I 11 degrees 1%, WI element: substrate -1
:100 weight ratio). Immediately after the digestion, the peptide was freeze-dried (the peptide of the present invention manufactured by Seibu used in the pharmacological tests described later was obtained at this stage).

1 g of dry peroxide was dissolved in 0.1 M ammonium acetate (pH
5,6) Dissolved in 14.5-, Sophadex G-50
Gel filtration (flow rate 20 mQ/hour, 4°C, fraction 8.
7 mQ/tube), and the active fraction (fraction No.
, 87-130) were collected and lyophilized.

The elution activity pattern in gel filtration using Sephadex G-50 is shown in FIG. Horizontal 0 in Figure 1
indicates fraction NO, and 11 indicates insulin action promoting activity. In the figure, (1) shows the absorbance curve at 230 nm, (2) shows the insulin action promoting activity pattern, and (3) shows the absorbance curve at 280 nm.

The dried active product obtained above was further incubated at 50°C for 1 hour under suction, suspended in water, and freeze-dried again. The active fractional 7201110 collected by performing the same gel 8 filtration operation three times as above was dissolved in 60 pieces of 0.1M sodium acetate (pH 4,3) and added to SP-Sephadex C-25. Ion exchange column L1 mudgraphy (flow rate 60 prints/hour, 4° C., 8 fractions/tube) was carried out using a column (manufactured by Pharmacia, 3×20 Cm).

160m217)0. IMlffffff class 1 liquid (D H
After washing with 4,3), further washing with 500 Tsuru of 0.1M acetate buffer (pH 4,3) containing 0.1M sodium chloride, and 2000
Elution was performed using a 7f4m gradient of mQ from 0.1M to 0.8M sodium chloride.

The above elution pattern is shown in FIG. In Figure 2, the horizontal axis is the fraction NO0, the vertical axis is the absorbance at 230 nm,
Shows insulin action promoting activity and electrical conductivity. In the figure, (1) shows the absorbance curve at 230 nm, (2) shows the insulin action promoting activity pattern, and (3) shows the electrical conductivity curve in the column.

It can be seen from FIG. 2 that the maximum activity peak appears near 0.53 MNa C12.

Collect the maximum activity peaks mentioned above and use Spectra Ball 3 (manufactured by Spectrum Medical Co., Ltd., MWcutoff3500).
) Dialysis using dialysis tubing (5 times with 50x n0 distilled water,
70 hours) and freeze-dried.

The lyophilized product obtained above was dissolved in 0.1% trifluoroacetic acid, and purified by high-performance liquid chromatography using a reversed phase column under the following conditions.

<High performance liquid chromatography conditions> Device: Waters, ALC/GPC model 244
, M-body Roma I-graphy system, M-660 solvent programmer, Column: Ultrapol RPSC (C3, Beckman Artex, 4.5 mm x 7.5 cm) Flow rate: 11r, Q/min, 25°C Chart speed: 6 (Mm/hour injection amount: 300μg/30μQ Elution: 0-60% ace-1-nitrile (straight cotton mouth gradient,
30 minutes) The results are shown in Figure 3. In Figure 3, the horizontal axis is the retention time (
The vertical axis shows the absorbance at 220 nm and 280 nm, and (1) in the figure shows the absorption variation at 22On11 and (
2) shows the absorbance curve at 280 nm. Approximately 2 from the figure
It can be seen that a peak showing maximum activity appears at 8 minutes.

The above activity peak was lyophilized to obtain the desired bovine serum albumin-derived peptide product. The yield was 590 μq per gram of raw bovine serum albumin (peptide A
).

The peptide of the present invention obtained above has the following physical properties.

1) SDS polyacrylamide gel electrophoresis (
The method of ItOK, ) et al. (J, Biol.

CherQ, 255.2123 (1980)), 20.86% acrylamide, 0.0969f,
Bisacrylamide, 6M urea, 0.033M salt, 0
．． A separation gel of 34M Tris-HCl (+) H8,7> was used. The stacking gel, running buffer and sample spinneret were prepared using the method of Laemmli et al. (NatUre).

227.680 (1970)).

As a molecular weight marker, a kit manufactured by BDH Chemicals (
Molecule If) 16949.14404.8159.621
4.2512-) was used.

Figure 4 shows the results of measuring the molecular weights of the above-mentioned peptides and those obtained by reducing them with [beta]-mercap].ethanol. From FIG. 4, the above peptide shows two bands,
Each band appeared between molecular weight markers 6214 and 2512, and their respective molecular weights were approximately 4900 and 3500.

2) Amino acid analysis The peptide (dried product) obtained above was analyzed by Simpson et al.
0■,, et al) method (J.

Biol, CC11e, 251.193 (197
6) Hydrolysis was carried out at 110°C for 24118 hours according to ). After neutralizing the hydrolyzate, it was analyzed using a Hitachi Model 835 amino acid automatic analyzer. The results are shown in Table 2 below. In the table, each amino acid is indicated by the abbreviation ILIPAC, and the composition of each amino acid is expressed in moles of each amino acid per mol of sample. Also, is HaH-cys excessive? Shown as cysteine after oxidation.

Table 2 Amino acid composition (mol) Measured value Theoretical value Asp+Asn 6.7 7 Thr　1.8　2 ser o.

GIu 10 GIn s, 7 9 pro 4.8 5 Gly 2.9 3 A18 5.6 6 Val 1.2 1 Half -Cys 3.6 4 Met O, 61 11e 1.9 2 Ij314 6.8 7 Tyr 6 .1 6 Phe 4,04 Trp* 0.1 1 LV'S 7.7 '8 His 1.0 1 Aro 2.8 3 Total 70 M, W, 8333 * Measured value because Trp is decomposed during hydrolysis The value is much lower than the theoretical value.

3) N-terminal amino acid sequence peptide screening was subjected to Edman degradation and measurements were performed up to three steps (IwanaaaS,, et at
.

Eur, J. Biochem, 8.189 (1
969) ).

Identification of phenylthiohydantoin derivatives of amino acids
CIFI reverse phase column (TSK1LS410K.

ODS, manufactured by Toyo Soda Co., Ltd.) (0mlcbi K, et at, J, Bio
chem.

87.483 (1980)).

As a result, Ar(+ and leu were identified in the M1 stage, HIS and LIS in the second stage, and Pro in the third stage.

4) The isofrost point peptide sample of the present invention was subjected to the method of Orrell (Q'), P, H,) (J
, Biol.

Chcm, 250.4007 (1975)), the isoelectric point was about 5.
It was 1.

From the above-mentioned physicochemical properties and graphical characteristics, the peptide obtained in this example has leu, the 115th amino acid of bovine serum albumin, as the N-terminus, and Ar as the 143rd amino acid.
A peptide consisting of 29 amino acids with g at the C-terminus, ΔrO at position 144 at the N-terminus, and Ar at position 184
A peptide consisting of 4111Fl amino acids with g as the C-terminal is cross-linked between Cys at position 123 and CyS at position 167 with an S-8 bond, as shown by the following primary structural formula. Presumed.

Primary structural formula [1] S Glu 1 1840 terminal Example 2 In Example 1, instead of ion exchange chromatography using SP-Sephadex C-25, DEAE cellulose (Whatman DE52, manufactured by Whatman Inc., 3.
0.01 per g of Gelmil superactive fraction dried smoked product 500II using Sephadex G-50.
M ammonium bicarbonate 50 times as a solution (pH 8,0), ammonium bicarbonate 11 degree gradient (0,1-0.4M
) method using ion exchange chromatography (flow rate 43 m
1/hour, fraction 10 mQ/tube).

The same operations as in Example 1 were then repeated for the active fraction obtained above to obtain the desired bovine serum albumin-derived peptide. The yield is 1350μ per gram of raw material albumin.
It was g. Moreover, its physical properties were consistent with those obtained in Example 1.

Example 3 The same procedure as in Example 1 was repeated using human I-blood albumin (fraction v1 fatty acids, manufactured by Seikagaku Corporation) in place of bovine serum albumin to obtain a human serum albumin-derived peptide (peptide B). Ta.

The human serum albumin-derived peptide obtained above has the following physical properties.

1) SDS polyacrylamide gel electrophoresis Example 1
FIG. 5 shows the results measured by the same method as in 1).

From FIG. 5, the above peptide showed two bands, each band appearing between molecular good markers 6214 and 2512, and their respective molecular peaks were approximately 5,000 and 3,500.

2) Amino acid analysis The results of analysis performed in the same manner as in Example 1, 2) are shown in Table 3 below.

Table 3 Amino acids Composition (moles) Measured value Theoretical value Asp + Asn 4.7 5 Thr 2.7 3 3er Q 0 Glu + GIn 7.8 8 Pro 4.0 4 Gly Q □ Ala 9.6 10 Vat 3. 1 3 Half-Cys 3. 5 4 Met O,81 1181,11 Leu 8.9 9 Tyr 4.7 5 Ph0 5.9 6 Tri) O0 Lys 5.7 6 His 2.2 2 ArO5,05 Total 72 M, W, 8538 3) N - Terminal amino acid sequence As a result of measurement using the same method as in Example 1, 3), Aro and leu were detected in the first step, and His was detected in the second step.
and V'a I, Pro and Arc, respectively, were identified in the third step.

From the above physical and chemical properties and artificial characteristics, this example
The resulting peptide has lcu, which is the 115th amino acid of human self-exhausting albumin, as the N-terminus, and A as the 144th amino acid.
A peptide consisting of 30 amino acids with rg at the C-terminus, Arg at position 145 at the N-terminus, and A at position 186
A peptide consisting of 42 amino acids with rg at the C-terminus is cross-linked with an S-8 bond between Cys at position 124 and Cys at position 169, as shown by the following primary creation formula. It is estimated that

Primary drawing formula [2] C-terminus 144 H〇-Δ'g 351 Leu -1ys-Lys-Tyr-Leu-Tyr-
Glu-1le-Δ1a12G H-Aro-His-Pro-Tyr-Pl+e-Ty
r-Ala 3159 l l Δro Tyr Lys Ala Ala PhQ T
l1r-G! u Cys CVS Glu-Δ1a10
91 S Ala I Aru-0) 1 186G end Example 4 In Example 1, rat serum albumin (fraction v1 manufactured by Seikagaku Kotai Co., Ltd.) was used instead of noble albumin (bovine blood), and the same procedure was repeated to prepare rat serum. An albumin-derived peptide (peptide C) was obtained.

The rat serum albumin-derived peptide obtained above has the following physical properties.

1) SDS polyacrylamide gel electrophoresis real muscle example 1
FIG. 6 shows the results measured by the same method as in 1).

From FIG. 6, the above peptide showed two bands, each band was located between molecular weight markers 6214 and 2512, and the molecular weights were approximately 5,700 and 3,500, respectively.

2) Aminoro analysis The results of analysis conducted in the same manner as in Example 1, 2) are shown in Table 4 below.

Table 4 Aminotrines Composition (moles) Measured value Theoretical value ASI) + ASn 7.5 8 Thr 4.6 5 Ser 2.7 3 GIu + GIn 10.8 11 Pro 8.2 8 Gly 1.0 1 Ala 8.0 8 Vat 3.2 3 Half -Cys 3.6 4 Met O,71 11e O0 Lcu 7.8 8 Tyr 6. '0 6 Phe 3.8 4 Trp O0 LyS3. ．． 8 4 +ts 2.9 3 28- Arg 2.9 3 Total 80 M, W, -9243 3> N-terminal amino acid sequence As a result of measurement using the same method as in Example 1, 3), in the first step Ar(+ and ASI) were identified in the second step, HIS and ASI1 in the second step, and pro and Asn in the third step.

From the above physicochemical properties and 476 structural characteristics, the peptides obtained in this example and 476
Asp, which is the 7th amino acid rη, is the N-terminus, and 144
A peptide consisting of 38 amino acids with the 145th Ar(] as the N-terminus and 18
A peptide consisting of 42 amino acids with Lys at the 6th position as the C-terminus, Cys at the 124th position and c at the 169th position.
It is estimated that the following primary (Yoshizo formula) is cross-linked with S-8 bond between ys and ys.

Primary 478 formula [3] 107 N-terminus C-terminus 144 Leu-Asn-Pro-Asn-Asp-Asp-H
HO-Ar.

+ 135 1 Pro Leu-ally His Tyr Leu-H
is-Glu vat-AIa1 1 12G Pro Phe-8er-Thr-Pro-Asn-G
lu-Gln-Pl+e-3er+ 124 l Pl+e-G In-Ar0- Pro-G Iu-A
1a-G 1u-A Ia-Met-Cys-Tbr
N-terminus 145 151 1 H-Arg-nee 1is-Pro-Tyr-Pt+e-
Tyr-Ala 5159 1 1 Gltl-AIa-'ryr'-Tyr-Leu-Le
u-Glu-pro Sl 1681 171 LVS Tyr Asn Glu Val LeLI
Thr Gln CVS, CVS Tl1r Glu
1t391 3 3er I S Asp 185 1 1 Val-△Ia-ASp-1eu-1ys-pro-T
hr-1-eu-Cys-AIa-ΔIa-LyS1
177 174 ys-OH 186 C-terminus Sample samples of the insulin action-promoting activity of the peptide samples obtained in the above examples are given below.

Trial Example 1 Regarding the trypsin treatment of bovine blood slaked albumin carried out in Actual Example 1, the lubrication period with trypsin, that is, 15 minutes, 3
D-(U-1' C
) It was measured by the method described above using the poor conversion of glucose to fatty acids as an index. The results are shown in Figure 7.

In FIG. 7, (1) shows the case of the digested product + 0.2 IllU/Tsuru insulin, and (2) shows the case of the digested product alone.

Figure 7 shows that when using the digestate alone, there is no increase in conversion to fat [t] for any treatment time, whereas in the presence of insulin, the number of members converted to fatty acids increases as the treatment time increases. That is, insulin action-promoting activity appears, and the maximum activity (approximately 50096>) is obtained for 4 hours or more.

Sample Sample 2 For each mammalian serum albumin, D-(U- ＋A
C) Insulin action promoting activity was measured by the method described above using the conversion of glucose to fatty acids as an indicator. The results are shown in Table 5.1゜From Table 5 above, under conditions without the addition of insulin, no increase in conversion R to fatty acids was observed for any serum albumin, regardless of trypsin treatment. In contrast, in the presence of insulin and without trypsin treatment, no increase in the number of fatty acid conversion sites was observed in the precious albumin of any blood (blood), but with trypsin treatment, no increase in the number of fatty acid conversion sites was observed in serum of bovines, humans, and rats. It can be seen that albumin also showed a significant increase in conversion to fatty acids compared to the control group, and insulin action-promoting activity.

Test Example 3 The insulin action-promoting activity of the serum albumin-derived peptides (peptides A, B, and C) of each of the dairy animals obtained in Actual Examples 1.3 and 4 at various degrees a was measured by the method described above. The results are shown in FIG.

In Figure 8, B-1 is bovine serum albumin-derived peptide + 〇, 2m U/mQ insulin, B-0 is bovine serum albumin-derived peptide alone, H-1 is human serum albumin-derived peptide + 〇, 2i U /mα insulin, H-0 is human serum albumin-derived peptide alone, R-1 is rough 1-serum albumin-derived peptide + 0.
2 mU/mQ insulin, and R-0 represents rat surface serum albumin-derived peptide alone.

From Figure 8, a concentration-dependent increase in insulin action-promoting activity was observed from 10-7M for all peptides derived from cows, humans, and rats; It can be seen that C shows a promoting activity of about 400%.

Test Example 4 The insulin action promoting activity of the fixed amount (2X10''''M) of the peptide derived from bovine serum albumin (Peptide A) in Example 1 was determined by changing the concentration of insulin added stepwise. The aforementioned D-(U=A C)
The amount of conversion from glucose to fatty acids was measured as an index. The results are shown in FIG.

In Figure 9, (1) shows peptide A + insulin, (
2) shows the case of insulin alone.

Figure 9 shows that peptide A exhibits activity depending on the degree of insulin added;
~1. It can be seen that the OI increases markedly at [lU/ground] and remains constant above that level.

Test Example 5 As another indication that the bovine serum albumin-derived peptide (peptide A) obtained in Example 1 has insulin action-promoting activity, D-(U-1' C
) An experiment was conducted to determine whether the above-mentioned peptides had a similar promoting activity on the action of insulin on CO2 production from glucose.

That is, the subcircle adipose tissue of the rat was removed, and the rod bell (
Rodbell, M.'s method (J, Biol.

Chem,, 239. 375-380 <1964>
), 1% bovine serum albumin and 1%
Krebs Ringer (Kreb-
Rinoer) HEPESEA buffer solution (pH 7,4)
suspended in it. The *0.5μCi Tsuru's D-(U-I
A C) Approximately 1.2 x 105 cells/pure cells were placed in the same buffer as above containing glucose, 100% air, 3
The cells were cultured at 7°C for 3 hours.

” CO2 collection and measurement is done by O/ (One, M
) and other methods ([31ochti, B 1ophys,
Acta, 284.

285-297 (1972)).

The results are shown in Table 6.

Table 6 Effect on CO2 production + 16.53 ± 1.29 10 - 28.15 + 2.06 + 38.71 ± 2.23 30 - 51.51 + 3.04 + 66.51 ± 3.11 (n = 4 ) (Peptide A) is D-(U-” in the presence of insulin)
C) It has been demonstrated that it promotes glucose internalization and increases CO2 production, and it is suggested that there is an optimal hydration level at the added concentration of insulin for this effect.

[Brief explanation of drawings]

Figure 1 shows the elution pattern by gel filtration using Sephadex G-50 according to the method of the present invention, Figure 2 shows the elution pattern by ion exchange column chroma 1 to graphie, and Figure 3 shows the elution pattern by high performance liquid chroma. The elution pattern is shown below. Figures 4 to 6 are embodiments 1.3 of the present invention.
and 1 by 4! The results of molecular weight measurement by SDS polyacrylic acid gel electrophoresis of each peptide to be 'P' are shown. FIGS. 7 to 9 are graphs showing the insulin action promoting activity of the peptides obtained according to the present invention. (Above) Agent: Patent Attorney Eiji Saegusa, Figure 4', 龜, Figure 5, Figure 61ヅ[~2, -1, Uta, Eishigeko Time (time) fj'g 8 I left the red shirt and went to wammin+i+Af 0→-1'
5. LJi (M) Procedures Ministry of Procedure (Method) Held January-February 23, 1980;) Director-General Kazuo Wakasugi 1 Indication of the case 1981 Special Edition) Fragment No. 148552 2 Name of the invention Mammal self Process for producing peptides derived from depleted albumin 3 Relationship with the 8 amendments Patent applicant 115 Akutahara, Tateiwa, Shipiyo-cho, Naruto-shi Representative of Otsuka Pharmaceutical Factory Co., Ltd. Yoshitsune Otsuka 4th agent Mitsuru Otsuka Hirano-cho, Higashi-ku, Osaka-shi 2-10 Sawanotsuru Building 1. -17:
. (6521) Patent attorney Hide Saegusa -1'. 5 Date of amendment order ゛-゛・-゛November 8, 1982 (Shipping date: November 29, 1982)
6) Sections ``Detailed Description of the Invention'' and ``Brief Explanation of Drawings'' in the clear line of the amendment, and Contents of the 1. The phrase "shown in Figure 4" should be corrected to "shown in reference photo 1. From reference photo 1." 2 “Figure 5” on page 23, lines 7 and 8, respectively.
Correct each text to ``Reference Photo 2''. 3 In page 27, line 11 and line 12 of the specification, “No. 6
Correct each "Figure" to "Reference Photo 3". 4. On page 31 of the specification, lines 10, 11, and 14, the words "Fig. 7" are corrected to "No. 40." 5. In the specification, page 35, line 16, line 18, and page 36, line 7, the words ``Figure 8'' are corrected to ``Figure 5.'' 6. In Mei 1, page 36, lines 19-20, page 37, lines 1 and 3, the words ``Figure 9'' are corrected to ``Figure 6.'' 7. On page 40 of the Book of Memories, lines 12 to 17, “Figure 4...
It is... ” is corrected as follows. ``Figures 4 to 6 are graphs showing the insulin action promoting activity of the peptide 1q according to the present invention.'' 8 Figures 4 to 9 were deleted, and attached figures 4 to 6 and reference photographs were used. Add reference photos 1 to 3. Figure 4) Ushisho Ihito Hour [s'i (Time Crystal H) 51λj Ushikashi, % Alshi Fu”min ()r Half'4JW-(f
) Procedural Amendment (Voluntary) February 14, 1980, a11□ ↑, Director General of the Y License Office, Kazuo Wakasugi, 1, Indication of the case Relationship to the 1981 Patent Fragment No. 148552 case Patent Applicant Co., Ltd. Otsuka Mulberry Factory 4, Agent 10 Sawa-no-Tsuru Hill, 2 Hirano-cho, Higashi-ku, Osaka Phone: 06-203
-0941 (Main) Voluntary Amendment as attached to attached sheet Key 7 Is the statement in the specification as shown in the errata below? I correct it. 2 Akira, I'llll j'' from the bottom of the 16th shell in lines 3 and 2, where it says ``Results...''2'' and ``Results are shown in photo l.Reference to i:'' From Photo 1 (right)”-”
I am corrected. 3 Myotsumugi 1′? Page 23 rl's 11t1 in lines 7-8
``Results are shown in reference photo 2.Reference qL,) From true 2 (left)'' is written as ``2''.
I am corrected. 4 Ming #! tl horoscope page 27 lines 11-12 says “results.
・・・・・・・・・"2 Notes" [Results are not shown in Photo 3.] Photo 2. 'c3 (right) Corrected to -1 above. 5 Details! 1 Page 38, lines 1-4 [Then...
``Insert ...,'' it says. (Correct as it says. J, +-IJi+I 0.4
yvt (, ili [fig 'i: (approximately 5 x 10'''
), add 10 μl (0
, 1ltci) and ``5 clear flu j'! ; i' Written on page 539%'Q t
y), qS6 Table K I-'):, / It'll F
1't Alzusan derived Pezuchi l;, 4 (2X lO"
A (it,': aru o "bovine blood) I"j Pezug l derived from Alp Sanshi: B (2X IO-"I'f) l and tears j
Do E. 6. Correct section 2 and 4 in attached sheet 9. 7. Correct Figure 3 as shown in the attached sheet. 8. Correct Figure 5 as shown in the attached sheet. (10 more)

Claims (1)

[Claims] ■ A mammal Ifll characterized in that a peptide having insulin action-promoting activity is obtained by causing at least 10 of trypsin, acrosin, cocnase and E, coii protease ■ to act on mammalian albumin
Method for producing FFJ albumin-derived peptide.