JPH0542264B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a peptide derived from mammalian serum albumin, and more particularly to a method for producing the above-mentioned peptide having insulin action-promoting activity. Mammalian serum albumin accounts for approximately 60% of total plasma protein and is the major protein in serum. Although the serum albumin is known to maintain plasma colloid osmotic pressure and to be involved in the transport of various vitamins, hormones, drugs, etc., it does not itself have insulin action-promoting activity. The present inventors have been conducting intensive research on factors that promote and enhance the action of insulin, and in the process, trypsin, acrosin, cocnase, and E.
The present inventors have discovered a new fact that the above-mentioned peptide having insulin action-promoting activity is present in a decomposed product that is degraded by the action of at least one type of coli protease. The present invention was completed based on the above findings, and is characterized in that a peptide having insulin action-promoting activity is obtained by reacting mammalian serum albumin with at least one of trypsin, acrosin, cocnase, and E. coli protease. The present invention relates to a method for producing a peptide derived from mammalian serum albumin. According to the present invention, it is possible to obtain a peptide having a novel insulin action-promoting activity that has not been previously known through simple operations. The above-mentioned peptide obtained by the method of the present invention is characterized in that it has a unique physiological action of insulin action promoting activity. Insulin action-promoting activity here refers to the action of insulin, that is, the action that promotes the uptake of glycose and amino acids into muscle and adipose tissue and the conversion of glucose into glycogen and fat in the body, thereby lowering blood sugar. say. Therefore, the above-mentioned peptides obtained according to the present invention are useful for the treatment of diabetes, which is defined as a metabolic disorder due to insufficient insulin action. Generally, diabetes is broadly classified into type diabetes mellitus (insulin-dependent diabetes mellitus), which is caused by an absolute deficiency in insulin secretion in the pancreas, and type diabetes mellitus (non-insulin-dependent diabetes mellitus), which is mainly caused by decreased sensitivity to insulin. The peptides obtained according to the present invention can improve insulin sensitivity by promoting insulin action, and are particularly effective in treating type diabetes.
In addition, regardless of type or type of diabetes, the use of the peptide obtained by the present invention in combination with insulin therapy can significantly reduce the amount of insulin preparations used, and at the same time reduce the amount of insulin antibodies that occur with long-term or high-dose insulin therapy. It is possible to prevent the development of insulin shock and insulin resistance due to insulin production. The mammalian serum albumin used in the method of the present invention includes bovine, equine, ovine, canine,
It may be any commercially available serum albumin isolated from mammals such as humans, monkeys, and mice by various methods. It may also be isolated and prepared from the above-mentioned various mammals according to conventional methods. Furthermore, the enzyme such as trypsin that acts on the serum albumin may be either a commercially available enzyme preparation 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 trypsin, acrosin, cocnase, and E. coli protease to degrade the peptide bonds at specific sites. The decomposition reaction of peptide bonds by each of the enzymes described above is carried out under optimal temperature and pH conditions for the enzyme used. These conditions vary slightly depending on the type of enzyme, but in general, the temperature range is about 20 to 40℃, and the temperature range is from 5.0 to
The appropriate pH range is 9.5, the enzyme is usually used in an amount of about 1/10 to 1/250 of the weight of the raw serum albumin (substrate), and the reaction is completed in about 15 minutes to 36 hours. Examples of the decomposition reaction conditions for each enzyme are shown in Table 1 below. [Table] If necessary, the reaction may be stopped using an enzyme inhibitor such as soybean trypsin inhibitor. Due to the above decomposition reaction with trypsin, the raw serum albumin is
In the sterically unhindered site, the peptide bond on the carboxyl group side of the arginine (Arg) or lysine (Lys) constituting this is cleaved, and thus the desired peptide can be obtained as an active fraction having insulin action-promoting activity. The above-mentioned peptide can be separated and purified from the reaction solution by a conventional peptide separation method, and can be stored by a conventional freeze-drying method.
The above separation and purification means include, for example, molecular filtration using a gel carrier such as Cephadex G-50 and Cephadex G-100 (manufactured by Pharmacia), dialysis, electrophoresis, and SP-Sephadex C-25.
Examples include ion exchange column chromatography (manufactured by Pharmacia), DEAE-cellulose (manufactured by Whatman), high performance liquid column chromatography, and methods that combine these methods. A preferable example of the above purification means is as follows. That is, the reaction solution or its lyophilized product is dissolved in a weakly acidic solution such as ammonium acetate or acetic acid, gel-filtered, and lyophilized. At this time, if desalting is insufficient, suspend in water and freeze-dry again. The active fraction obtained by the above gel filtration was then treated with sodium acetate,
After dissolving in a buffer such as phosphoric acid or citric acid, perform cation exchange column chromatography. If necessary, wash with an acetate buffer, then wash with a buffer containing the following saline for 0.1M and 0.1M. Elute with a linear salt concentration gradient of ~2.0M, preferably about 0.1-0.8M, 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 obtained by the above method has Arg or Lys at its C-terminus, an amino acid bonded to the carboxyl group of Arg or Lys of raw material albumin at its N-terminus, and a specific peptide derived from raw albumin. Contains an 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. Examples of such acidic compounds include inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, and hydrobromic acid, fumaric acid, maleic acid, acetic acid,
Examples of organic acids such as oxalic acid, malic acid, and citric acid, and basic compounds include sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, and potassium hydrogen carbonate. 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 this case, the active ingredient is usually processed into a pharmaceutical composition together with a pharmaceutical carrier, and is preferably used as an injection. When prepared as an injectable solution, 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, a sufficient amount of common salt or glycerin to prepare an isotonic solution may be included in the preparation in the form of an injection. The above preparation may also contain conventional buffering agents, soothing agents, preservatives, etc., as well as colorants, fragrances, flavors, sweeteners, etc., and other pharmaceuticals, if necessary. Furthermore, the above-mentioned active ingredients can be dissolved in distilled water for injection at the time of use, and used as a solubilizing agent at the time of use. 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. The insulin action promoting activity of the samples in the Examples was measured by the following method. <Insulin action promoting activity> Male Wistar rat (body weight 150
Remove epididymal adipose tissue from ~300g),
Prepare Explant (fat tissue mass). 10 pieces
Explant (7.5-10 mg as wet weight) was added to D-[U-
¹⁴ C) Glucose 0.05μCi/ml, baking soda 1.5mg/ml,
Penicillin 35U/ml and streptomycin 0.1
M-199 containing mg/ml [“Medical History” Vol. 62,
No. 6, p. 435, published on August 5, 1960] Placed on siliconized lens paper floating on a medium [Topper.YJ, et al, Methods in
Enzymology. 39 , 443 (1975)] and cultured at 37°C for 20 hours in a gas phase containing 3% carbon dioxide. After culturing
Measure the wet weight of the Explant and calculate the total amount by 2N−
in 1 ml of a 50% ethanol solution of KOH at 100°C.
time to hydrolyze. Extract fatty acids with 3 ml of petroleum ether from the hydrolyzate acidified with 0.5 ml of 6N-sulfuric acid, measure the radioactivity by a conventional method, and calculate the amount of conversion from glucose to fatty acids from the specific radioactivity in the medium. . The insulin action-promoting activity of a sample is expressed as the percentage of the specific radioactivity calculation value obtained by adding the sample and insulin (0.2 mU/ml) into the reaction system to the same calculation value obtained by adding insulin alone. represent Example 1 Crystalline bovine serum albumin (manufactured by Miles) was
0.1M ammonium bicarbonate (PH8.1) using undenatured TPCK trypsin (Worthington)
Digestion was performed for 12 hours in a medium (substrate concentration 1%, enzyme:substrate = 1:100 weight ratio). Immediately after digestion, the peptide was freeze-dried (the unpurified peptide of the present invention used in the pharmacological tests described below was obtained at this stage). Dissolve 1 g of the dry digest in 14.5 ml of 0.1 M ammonium acetate (PH5.6) and add it to Cephadex G-50.
Gel filtration (flow rate 20ml/hour, 4℃, fraction
8.7 ml/tube), and active fractions (fractions No. 87 to 130) were collected and lyophilized. The elution activity pattern in gel filtration using the above-mentioned Sephadex G-50 is shown in FIG. In FIG. 1, the horizontal axis shows the fraction number, and the vertical axis shows the insulin action promoting activity. Also, 1 in the figure is 230nm.
2 shows the insulin action promoting activity pattern, and 3 shows the absorbance curve at 280 nm. The dried active fraction obtained above was further dried for desalting.
The mixture was sucked for 1 hour while being kept warm at 50°C, suspended in water, and freeze-dried again. Approximately 720 mg of the active fraction collected by performing the same gel filtration procedure as above three times was added to 60 ml of
Dissolved in 0.1M sodium acetate buffer (PH4.3),
SP-Sephadex C-25 (manufactured by Pharmacia,
Ion exchange column chromatography (flow rate 60 ml/hour, 4°C, fraction 8 ml/tube) was performed using a 3 x 20 cm) column. 160ml
After washing with 0.1M acetate buffer (PH4.3), add 500ml
The cells were washed with 0.1M acetate buffer (PH4.3) containing 0.1M NaCl, and eluted with a 2000 ml linear gradient of 0.1M-0.8M NaCl. The above elution pattern is shown in FIG. In FIG. 2, the horizontal axis shows the fraction number, and the vertical axis shows the absorbance at 230 nm, insulin action promoting activity, and electrical conductivity. In addition, 1 in the figure is the absorbance curve at 230 nm,
2 shows the insulin action-promoting activity pattern, and 3
shows the electrical conductivity curve. It can be seen from Figure 2 that the maximum activity peak appears near 0.53M NaCl. Collect the maximum activity peaks above and use Spectrapore 3.
(Manufactured by Spectrum Medical,
Dialysis using a dialysis tube (MWcutoff3500) (50
(double volume of distilled water 5 times for 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> Equipment: Waters, ALC/GPC model 244,
Liquid chromatography system, M-660 solvent programmer, Column: Ultra Ball RPSC (C ₃ , Beckman Artex, 4.6 mm x 7.5 cm) Flow rate: 1 ml/min, 25°C Chart speed: 60 cm/hour injection rate; 3000μg/30μ elution; 0-60% acetonitrile (linear concentration gradient,
30 minutes) The results are shown in Figure 3. In Figure 3, the horizontal axis shows the retention time (minutes), and the vertical axis shows the absorbance at 220 nm and 280 nm and the insulin action promoting activity (%), where 1 is the absorbance curve at 220 nm and 2 is the absorbance curve at 280 nm.
Absorbance curves at m and 3 indicate insulin action promoting activity, respectively. It can be seen from the figure that a peak showing maximum activity appears at about 28 minutes. The above activity peak was lyophilized to obtain the desired bovine serum albumin-derived peptide. The yield was 590 μg per gram of raw material bovine serum albumin (peptide B). The peptide of the present invention obtained above has the following physical properties. (1) SDS polyacrylamide gel electrophoresis method by Ito K. et al. [J.Biol.Chem., 255, 2123
(1980)], 20.86% acrylamide,
0.096% bisacrylamide, 6M urea,
A separation gel containing 0.033M common salt and 0.34M Tris-HCl (PH8.7) was used. A concentrated gel, running buffer, and sample buffer were prepared according to the method of Laemmli et al. [Nature, 227, 680 (1970)]. As molecular weight markers, BDH Chemicals KIT (molecular weight 16949, 14404, 8159,
6214, 2512) were used. Reference Photo 1 shows the results of measuring the molecular weight of the above peptide reduced with β-mercaptoethanol. From the reference photograph 1 (right), the above peptide showed two bands, each band appearing 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 using the method of Simpson et al. using 4N methanesulfonic acid (containing 0.2% 3-(2-aminoethyl)indole). [J.Biol.
Chem., 251, 193 (1976)] at 110°C.
Hydrolyzed for hours. After neutralizing the hydrolyzate, Hitachi
The analysis was performed using a model 835 amino acid automatic analyzer.
The results are shown in Table 2 below. Furthermore, each amino acid in the table is
It is indicated by the abbreviation IUPAC, and the composition of each amino acid is expressed as the molar amount of each amino acid per mol of sample. Half-Cys is shown as cysteic acid after performic acid oxidation. [Table] (3) N-terminal amino acid sequence Peptide samples were subjected to Edman degradation and measured up to three stages [Iwanaga S., et al.
Eur. J. Biochem., 8, 189 (1969)]. Identification of phenylthiohydantoin derivatives of amino acids
Performed by HPLC using a C ₁₈ reverse phase column (TSK LS410K, ODS, manufactured by Toyo Soda Co., Ltd.) [Omichi K., et al, J.Biochem., 87, 483
(1980)]. As a result, Arg and Leu were identified in the first step, His and Lys in the second step, and Pro in the third step. (4) The peptide sample of the present invention was prepared using the method of O'Farrell (PH) [J. Biol. Chem.
250, 4007 (1975)], the isoelectric point was approximately 5.1. Based on the above physicochemical properties and structural characteristics, the peptide obtained in this example has Leu, the 115th amino acid of bovine serum albumin, as its N-terminus, and
A peptide consisting of 29 amino acids with the 143rd Arg as the C-terminus, and the 144th Arg as the N-terminus,
A peptide consisting of 41 amino acids with Arg at position 184 as the C-terminus, Cys at position 123 and Cys at position 167
It is estimated that it is cross-linked with Cys through an S--S bond and is represented by the following primary structural formula. Primary structural formula [1] [Table]
｜ ｜

S Asp
183
｜ ｜
Met−Thr−Glu−Ile−Lys−Pro−Le
u−Leu−Cys−Ala−Gly−Lys
｜
175 172
Arg−OH

184 C-terminus

Example 2 In Example 1, SP-Sephadex C-
Instead of ion exchange chromatography using 25
DEAE cellulose (Watsuman DE52, manufactured by Watzman, 3.2 x 60 cm) was used. 500 mg of dried gel hyperactive fraction was obtained using Sephadex G-50.
Dissolve in 50 ml of 0.01M ammonium bicarbonate solution (PH8.0) and add ammonium bicarbonate concentration gradient (0.1~
0.4M) by ion exchange chromatography (flow rate 43ml/hour, fraction 10ml/tube)
I did this. 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 was 1350 μg per 1 g of raw material albumin. Moreover, its physical properties were consistent with those obtained in Example 1. Example 3 In place of bovine serum albumin in Example 1,
The same operation was repeated using human serum albumin (Fraction V, free of fatty acids, manufactured by Seikagaku Corporation) to obtain a human serum albumin-derived peptide (peptide H). The human serum albumin-derived peptide obtained above has the following physical properties. (1) SDS polyacrylamide gel electrophoresis The results of the measurement performed in the same manner as in (1) of Example 1 are shown in Reference Photo 2. From reference photo 2 (left), the above peptide shows two bands, and each band is a molecular weight marker.
They appeared between 6214 and 2512, and their respective molecular weights were approximately 5000 and 3500. (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] [Table] (3) N-terminal amino acid sequence As a result of measurement in the same manner as in Example 1 (3), Arg and Leu were
His and Val in the third stage, Pro and Arg in the third stage
were identified respectively. Based on the above physicochemical properties and structural characteristics, the peptide obtained in this example consists of 30 amino acids, with Leu, the 115th amino acid of human serum albumin, as the N-terminus and Arg as the 144th amino acid as the C-terminus. A peptide consisting of 42 with Arg 145 as the N-terminus and Arg 186 as the C-terminus
It is estimated that the peptide consists of 124 amino acids and Cys 124th and 169th Cys are cross-linked by an S-S bond, as shown by the following primary structural formula. Primary structural formula [2] [Table]
｜ ｜

S Asp
185
｜ ｜
Leu−Glu−Asp−Leu−Lys−Pro−
Leu−Leu−Cys−Ala−Ala−Lys
｜
177 174
Arg−OH
186 C-terminal Example 4 In place of bovine serum albumin in Example 1,
The same operation was repeated using rat serum albumin (Fraction V, manufactured by Seikagaku Corporation) to obtain a rat serum albumin-derived peptide (Peptide R). The rat serum albumin-derived peptide obtained above has the following physical properties. (1) SDS polyacrylamide gel electrophoresis The results of measurements performed in the same manner as in Example 1 (1) are shown in Reference Photo 3. From reference photo 3 (right), the above peptide shows two bands, and each band is a molecular weight marker.
It appeared between 6214 and 2512, and the molecular weights were about 5700 and 3500, respectively. (2) Amino acid analysis The results of analysis performed in the same manner as in Example 1 (2) are shown in Table 4 below. [Table] [Table] (3) N-terminal amino acid sequence As a result of measurement in the same manner as in (3) of Example 1, Arg and Asp were
His and Asp in the third stage, Pro and Asn in the third stage
were identified respectively. Based on the above physicochemical properties and structural characteristics, the peptide obtained in this example has Asp, which is the 107th amino acid of rat serum albumin, as its N-terminus.
A peptide consisting of 38 amino acids with the 144th Arg as the C-terminus, and the 145th Arg as the N-terminus,
A peptide consisting of 42 amino acids with Lys at position 186 as the C-terminus, Cys at position 124 and Cys at position 169
It is presumed to be represented by the following primary structural formula in which it is cross-linked with Cys through an S--S bond. Primary structural formula [3] [Table]
｜ ｜

S Asp
185
｜ ｜
Val−Ala−Asp−Leu−Lys−Pro−
Thr−Leu−Cys−Ala−Ala−Lys
｜
177 174
Lys−OH

186 C-terminus

Examples of testing the insulin action promoting activity of the peptide samples obtained in the above examples will be given below. Test Example 1 Regarding the trypsin treatment of bovine serum albumin performed in Example 1, the digestion time with trypsin,
That is, the insulin action-promoting activity of the digested product was measured at 15 minutes, 30 minutes, 1 hour, 2 hours, and 14 hours by the method described above using the amount of D-[U- ^14C ] glucose converted to fatty acids as an indicator. The results are shown in Figure 4. In FIG. 4, 1 indicates the case of the digested product + 0.2 mU/ml insulin, and 2 indicates the case of the digested product alone. Figure 4 shows that when using the digestate alone, there is no increase in the amount converted to fatty acids for any treatment time, whereas in the presence of insulin, as the treatment time becomes longer, the amount converted to fatty acids increases. That is, it can be seen that insulin action-promoting activity appears, and the maximum activity (approximately 500%) is obtained for 4 hours or more. Test Example 2 Regarding the serum albumin of each mammal, the values of untreated trypsin and trypsin treated (non-sugar) obtained in Examples 1, 3, and 4, D
-[U- ^14C ] Insulin action-promoting activity was measured by the method described above using the amount of conversion from glucose to fatty acid as an index. The results are shown in Table 5. [Table] From Table 5 above, under conditions without the addition of insulin, no increase in the amount of fatty acid conversion was observed regardless of whether or not trypsin treatment was used, and no increase in the amount of fatty acid conversion was observed even in the presence of insulin. In the untreated state, no increase in the conversion amount of any serum albumin to fatty acids was observed. On the other hand, by trypsin treatment, cattle,
It can be seen that both human and rat serum albumin showed a significant increase in the amount of fatty acid conversion compared to the control group, that is, insulin action-promoting activity. Test Example 3 The insulin action promoting activity of each mammalian serum albumin-derived peptide (peptide B, H, R) obtained in Examples 1, 3, and 4 at various concentrations was measured by the method described above. The result is shown in FIG. In Fig. 5, B-1 contains bovine serum albumin-derived peptide + 0.2 mU/ml insulin;
0 is bovine serum albumin-derived peptide alone, H
-1 is serum albumin-derived peptide + 0.2 mU/
ml insulin, H-0 contains human serum albumin-derived peptide alone, R-1 contains rat serum albumin-derived peptide + 0.2 mU/ml insulin, and R-1 contains rat serum albumin-derived peptide + 0.2 mU/ml insulin.
and R-0 each represent a rat serum albumin-derived peptide alone. As can be seen from Figure 5, a concentration-dependent increase in insulin action-promoting activity was observed from 10 ^-7 M for all peptides derived from cows, humans, and rats.
At 10 ^-5 M, approximately 500 for peptide B and peptide H
%, and Peptide R shows approximately 400% promoting activity. Test Example 4 For a fixed amount (2×10 ^-6 M) of the bovine serum albumin-derived peptide (peptide B) obtained in Example 1, the insulin action promoting activity was determined when the concentration of added insulin was changed stepwise. The above-described amount of D-[U- ^14C ]glucose converted to fatty acid was measured as an index. The results are shown in FIG. In FIG. 6, 1 shows the case of peptide B+insulin, and 2 shows the case of insulin alone. From FIG. 6, it can be seen that peptide B exhibits activity depending on the insulin concentration added, and its value increases markedly at an insulin concentration of about 0.10 to 1.0 mU/ml, and remains constant above that level. Test Example 5 As another indicator showing that the bovine serum albumin-derived peptide ⁽ peptide B) obtained in Example 1 has insulin action-promoting activity, Experiments were conducted to determine whether the above peptides had similar promoting activity on the effect of insulin on CO ₂ production. That is, the epididymal adipose tissue of rats was removed and the method of Rodbell (M) [J. Biol. Chem.
239, 375-380 (1964)] were treated with 1% bovine serum albumin and 1 mM.
Krebs Ringer (Krebs−) containing glucose
Ringer) HEPES buffer (PH7.4). 0.4ml of this fat cell suspension (number of cells approximately 5×
¹⁰⁴ ), D-[U- ^14C ]glucose 10μ
(0.2 μCi) was added and cultured with shaking at 37°C in 100% air for 3 hours. ¹⁴ Collection and measurement of CO ₂ was performed using the method of Ono et al. [Biochim. Biophys. Acta.
284, 285-297 (1972)]. The results are shown in Table 6. [Table] [Table] From Table 6, bovine serum albumin-derived peptide (peptide B) was
- ¹⁴ C] Promotes intracellular transfer of glucose and CO ₂
It has been demonstrated that insulin increases insulin production, and it is suggested that there is an optimal concentration of insulin added for its effect.

[Brief explanation of drawings]

FIG. 1 shows a SEPHADEX G obtained by the method of the present invention.
Figure 2 shows the elution pattern of gel filtration using -50, Figure 2 shows the elution pattern of ion exchange column chromatography, and Figure 3 shows the elution pattern of high performance liquid chromatography. Figures 4 to 6 are graphs showing the insulin action promoting activity of the peptides obtained according to the present invention.

Claims (1)

[Claims] 1. A method for producing a peptide derived from mammalian serum albumin, which comprises reacting mammalian serum albumin with at least one of trypsin, acrosin, cocnase, and E. coli protease to obtain a peptide having insulin action-promoting activity.