JPH0617310B2 - Manufacturing method of human urogastrone - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing human urogastrone (hereinafter abbreviated as human UG).

[Background of the Invention]

Human UG has been known to exist in urine since 1939, based on clinical observation that pregnant women have few peptic ulcers. 1975, H. Gregory
Has undergone a long purification process, and has two types (β-type, γ-type) from urine.
Was successfully isolated, and its amino acid sequence was determined (H. Gregory; Nature 257 , 3).
25-327, 1975). Gregory has these β
The -type and γ-type UGs were identified as follows, respectively.
Total amino acids 53 and 52 (both are single-chain polypeptides consisting of 16 amino acids), isoelectric points 4.5 and 4.
3. Relative mobility 0.54 and 0.6 relative to Bromphenol for acrylamide gel electrophoresis at pH 8.9
6, Rf of filter paper chromatography; 0.59 and 0.6
5, and γ-type UG lacks the C-terminal arginine residue of β-type UG. This UG is Cohen
Are isolated from the submandibular line of the mouse [(Epidermal Growth Factor); hereinafter abbreviated as EGF; S. Cohen et al .; The Journal of Bioc
hemistry), 237 , pp. 1555-1562, 196.
2 years)], and thus EG derived from human urine
It is considered to be the same as F and has a gastric acid secretion inhibitory effect and a growth promoting effect on epithelial cells and other cells, and is therefore useful as an additive used in medicines and tissue culture.

Savage et al. Applied a mouse EGF purification method to isolate two types of human EGF from pregnant woman urine [CR Savage et al.
al); Analytical Biochemistry (Analytic)
al Biochemistry) 111 , 195-200, 19
1981]. This human EGF has a molecular weight of about 5,500 and is composed of 16 kinds of 49 amino acids, but its sequence order and purity are not clear.

Human UG is known to exist in body fluids such as saliva, blood, and urine, as well as in the submandibular gland and the digestive tract. The content of urine, which is said to be relatively high, is extremely low and is 50-100 μg /
It is not so much and it is difficult to recover economically. As a method for recovering human UG in human urine, a method of treating acidic urine with a protein precipitating agent is known. Benzoic acid as a precipitant [Endocrinology, Vol. 30 , 12
9 (1942)], tannic acid [Hoppe-Seyler's Z. Physiol. Che.
m.) 356 , p. 1765 (1975)] and the like. These lack the specificity of human UG by common precipitation methods for proteins. Other adsorption method using ion exchange resin [Journal of Clinical Endocrinolo
gy and Metabolism) 48 : 667 (1979)
Year)] is also known.

As a method for further purifying them, Gregory uses a method such as a fractional extraction method using an organic solvent, an ion exchange method, or a gel filtration method.
The process was repeated, and Savage et al. Combined the ion exchange method and the gel filtration method to shorten the process to 5 steps for purification.

However, it is difficult to say that the gastric acid secretion inhibitor obtained by the above method is sufficient in terms of purity and yield.

[Object of the Invention]

The present invention provides a purification step which is useful for solving such problems and obtaining a highly accurate human UG in a high yield, and can be combined with other steps.

[Structure of Invention]

 That is, the present invention provides the following 1. ~ 6. Regarding

1. Human urogastrone was separated into multiple components from a sample containing human urogastrone by reversed-phase partition liquid chromatography using an aqueous solution having a neutral pH of 18 to 28% by volume as an eluent, and A method for producing human urogastrone for collecting.

2. 1. Use of porous silica gel having a hydrophobic functional group as a column agent for reversed-phase partitioning liquid chromatography. Of human urogastrone.

3. The sample containing human urogastrone is derived from human urine. Of human urogastrone.

4. For a sample containing human urogastrone, (1) a step of subjecting to reverse phase partitioning liquid chromatography using an aqueous solution having a neutral pH and 18 to 28% by volume of acetonitrile as an eluent; and (2) having a pH of Reversed-phase partition liquid chromatography using an acidic aqueous solution containing 18-28% by volume of acetonitrile as an eluent; a combination of both steps, that is, step (1) then step (2), or , Step (2) and step (2) are carried out in this order to separate human urogastrone into multi-components and collect each of them.

5. 3. Using porous silica gel having a hydrophobic functional group as a column agent for reversed-phase partition liquid chromatography. Of human urogastrone.

6. 3. The sample containing human urogastrone is derived from human urine. Of human urogastrone.

In the present invention, the sample containing human UG means urine itself, a filtrate obtained by filtering urine with Celite, an extract from a tissue or cell producing human UG, a culture solution of the tissue or cell, a human by genetic recombination. From an extract from a UG-producing bacterium or yeast, a culture broth of the bacterium or yeast, and the like, usually, various methods as shown in, for example, (1) to (9) below, partially purified humans It is a liquid containing UG. However, if the impurities and interfering substances contained in the extract and the like are small in amount, they may be omitted even if they are not partially purified.

(1) Weakly acidic adsorbent (polyacrylic acid type ion exchange resin,
(Polymethacrylic acid-based ion exchange resin, etc.)
A sample containing human UG after being adsorbed to 3 to 4 and then washed under weakly acidic conditions of pH 3 to 5 and eluted with weak alkalinity such as ammonium hydroxide to obtain a sample containing human UG (adsorption-elution method).

(2) A giant reticulated polymer (styrene-divinylbenzene-based copolymer, acrylic acid ester-based polymer, methacrylic acid ester-based polymer, etc.) which is a neutral synthetic adsorbent is used as the adsorbent, and the above sample itself, pH 3 to 8 is used. After adsorption using the above sample buffered between or the above sample to which salt, salt such as sodium sulfate is added, water-soluble organic solvent (for example,
A sample containing UG by elution with an aqueous solution containing methanol, acetonitrile, n-propanol, etc. (adsorption-elution method).

(3) A human obtained by subjecting the above-mentioned sample to celite filtration to remove the precipitate, and subjecting the filtrate to ultrafiltration with a molecular weight cutoff of 30,000, and further concentrating the filtrate by ultrafiltration with a molecular weight cutoff of 1,000. UG fractionation (ultrafiltration method).

(4) A human UG fraction obtained by partial purification by combining two or more steps among the steps (1), (2) and (3).

(5) The partially purified product obtained in (1), (2), (3) or (4) above is subjected to gel filtration (a hydrophilic gel such as crosslinked dextran gel or crosslinked polyacrylamide gel is preferable as a carrier). ,
Human U obtained by developing with a buffer solution (acetate buffer solution, phosphate buffer solution, etc.) having a pH of 5 to 9 preferably at 0.02 M or more
G fraction (gel filtration method).

(6) Weakly basic ion exchange resins such as diethylaminoethyl cellulose and diethylaminoethyl agarose were packed in a column and equilibrated with a buffer solution (acetic acid buffer solution, phosphate buffer solution, etc.) of pH 5 to 8.5 at 0.05 M or less. Later, above
(1), (2), (3), (4) or (4) or (5) loaded with a desalting solution of the human UG fraction to adsorb human UG to the above resin, the same buffer solution as above A human UG fraction (anion exchange chromatography) obtained by elution by a salt concentration gradient method (sodium chloride, ammonium acetate, etc. are used as a salt).

(7) A column packed with a weakly acidic ion exchange resin such as carboxymethyl cellulose or carboxymethyl agarose, which contains the human UG fraction obtained in (1), (2), (3), (4) or (5) above. Load with desalting solution 0.01-0.05M pH 3.5-
4.0 buffer (eg acetate buffer, formate buffer, etc.)
At a salt concentration gradient (for example, sodium chloride as salt,
Human UG fraction (anion exchange chromatography) obtained by elution with (eg, ammonium acetate).

(8) The human UG fraction obtained in (1), (2), (3), (4) or (5) above was acidified with hydrochloric acid or the like (pH 1-2) and then acidified with hydrochloric acid or the like (pH 1- A human UG fraction (adsorption chromatography) obtained by loading and developing the crosslinked polyacrylamide gel equilibrated in 2).

(9) The desalted solution of the human UG fraction obtained in (1), (2), (3), (4) or (5) above is used in the steps (6), (7) and (8) above. A human UG fraction obtained by partially purifying by combining two or more steps.

The column agent packed in the reversed phase partition type column used in the reversed phase partitioning type liquid chromatography of the present invention includes porous styrene / divinylbenzene copolymer particles, crosslinked acrylic acid ester polymer particles, and crosslinked particles. Polymerized methacrylic acid ester polymer particles, porous silica gel chemically bound with a hydrophobic functional group, and the like can be used. In particular, porous silica gel having a hydrophobic functional group,
It has excellent resolution and recovery. Examples of the hydrophobic functional group introduced into silica gel include methyl group, ethyl group, propyl group, octyl group, alkyl group such as octadecyl group, cyanopropyl group, phenyl group and the like. It is preferably contained in an amount of 8 to 20% by weight. The pore size is 60Å ~
Those having a hole diameter of 300Å are preferable. Further, it is desirable that the particle size is small, but practically about 5 to 70 μm is preferable.

The reversed-phase partitioning liquid chromatography of the present invention is as follows.
An aqueous solution containing no organic solvent is used as a sample, and the aqueous solution is loaded on a column to adsorb human UG on the resin, followed by elution with an eluent. The eluate of human UG is obtained by mixing an aqueous solution containing acetonitrile with an acid such as acetic acid, formic acid, hydrochloric acid, phosphoric acid, trifluoroacetic acid, or methylsulfuric acid or a mixture of these acids with an alkali salt such as ammonia or caustic soda. pH
Buffers in the range of 1.5-7 are used.

Here, the concentration of acetonitrile in the eluate must be adjusted to 18 to 28% by volume so as to be suitable for elution.

In the present invention, the aqueous solution containing 18 to 28% by volume of acetonitrile is an aqueous solution containing a constant concentration of acetonitrile in the range of 18 to 28% by volume, or the acetonitrile concentration of the eluate when human UG is eluted. But 18-2
It means an acetonitrile concentration gradient liquid set to have a concentration in the range of 8% by volume.

In the former case, the more preferable acetonitrile concentration in the case of an aqueous solution containing a constant concentration of acetonitrile in the range of 18 to 28% by volume is in the range of 20 to 25% by volume. When the concentration of acetonitrile is less than 18% by volume, human UG is not eluted at all or the elution time becomes long, and when it exceeds 28% by volume, it is eluted near the elution tip and the purification effect is reduced.

In the latter case, when the acetonitrile concentration of the eluate when human UG is eluted is an acetonitrile concentration gradient liquid set to a concentration in the range of 18 to 28% by volume,
The initial concentration of acetonitrile in the eluate may be 0 to 16% by volume, and the final concentration may be 20 to 30% by volume. In addition,
The final concentration of acetonitrile can exceed 30% by volume. Human UG is eluted when the acetonitrile concentration of the acetonitrile concentration gradient solution is 18 to 28% by volume, particularly 20 to 25% by volume. The above elution may be performed under normal pressure or under high pressure.

The human UG fraction obtained by the present invention can be desalted by a gel filtration method, an ultrafiltration method or the like after distilling off an organic solvent by concentration under reduced pressure. When a volatile salt such as ammonium acetate or ammonium formate is used as the salt in the eluate and a volatile salt is used as the acid, the salt-free dried product can be obtained by freeze-drying as it is.

After the step according to the present invention, if necessary, the above (1)
The steps (8) to (8) may be appropriately applied, and the steps according to the present invention may be repeated twice or more.

The step according to the present invention is preferably combined with the step (4), (5), (6) or (8). In this case, in the reversed-phase partition chromatography of the present invention, octadecylated silica gel is used as a column agent, and the eluent is neutral (for example, ammonium acetate, pH 7) and has an acetonitrile concentration of 20 to 25% by volume. Human UG in
Are first separated into three types. Let these be UG-1, UG-2, and UG-3 in the order of elution, respectively. When these are further subjected to the reversed phase chromatography of the present invention under acidic conditions (for example, 0.5 vol% of acetic acid is added to the above eluate), UG-1 is separated into UG-1-1 and UG-1-2 in the elution order. Two
UG-2 is classified into two types, UG-2-1 and UG-2-2, and UG-3 is classified into UG-3-1 and UG in the order of elution.
-3-2 and UG-3-3 are separated. That is, a total of 7 types of novel human UGs can be separated by such a method.

UG-1-1 and UG-2 among the above 7 types of human UGs
5 types of human UG except 2 types of -2 were recognized as single bands when analyzed by SDS-urea-polyacrylamide gel disk electrophoresis (monomer concentration 13.8%), and their estimated molecular weights were myoglobin. Partial hydrolyzate (Molecular weight 16949, 14404, 8159, 6214, 2512,
It is located at about 6000 when the molecular weight marker using the mixture of 1360) as a standard is used. Also, a pH gradient (pH 4 to pH 6.
When isoelectric focusing was performed using the polyacrylamide gel plate having 5), it was recognized as a single band at different positions. These isoelectric points measured using a surface electrode are 4.45 for UG-1-2 and UG-2-1.
Is 4.40, UG-3-1 is 4.65, UG-3-2 is 4.60 and UG-3-3 is 4.45. Each of these was given histamine stimulation to the dog every 15 minutes and then administered by intravenous injection of 0.25 μg / kg of human UG. Human UG was administered to the total amount of gastric acid secretion 1 hour before administration of human UG. The total amount of gastric acid secretion in the next 1 hour is 50%
It was as follows.

In the present invention, the confirmation of the human UG fraction is measured by utilizing the property that mouse EGF obtained from the mouse submandibular line competitively binds to human UG to the EGF receptor on the mouse hepatocyte membrane. That is, human UG or a sample containing human UG, an EGF receptor and mouse EGF labeled with radioactive iodine are mixed in an aqueous solution and reacted to give EGF.
A binding product of the receptor and mouse EGF or human UG is produced, and this is separated, the radioactivity thereof is measured, and the human UG concentration is determined by observing the calibration curve [hereinafter, this method is referred to as Radio Receptor Assay]. (Abbreviated as RRA)].

(Example) Next, a preparation agent for a sample containing human UG, and then an example will be shown. In the following, the amount of human UG is the above RRA.
The specific activity is the amount of human UG per product of the absorbance at 280 nm and the liquid volume (ml) of the sample.

Reference Example 1 Male urine 3 kl (human UG amount 160 mg, specific activity 1.6 x 10
^-3 ) was adjusted to pH 3.0 by adding 4N hydrochloric acid. Acrylic acid type cation exchange resin (WK-20, trade name of Mitsubishi Kasei Kogyo Co., Ltd.) which has been converted to a carboxylic acid type with 4N hydrochloric acid in advance.
The column (40) filled with the mixture was flowed at a flow rate of 200 / hour. The column is then washed with 100 water and 100
Ammonium acetate buffer (50 kg of sodium chloride,
Ammonium acetate 7.7 kg and ammonia water 15.5
Was added to water to make 100) and then eluted with 48 4N caustic soda solution and further 90 water. The pH after elution increases with elution time. Of these, after elution
Fractions with a pH below 11 were collected. Human UG in this eluate
Was 60.4 mg (recovery rate 38%, specific activity 0.05).

Reference Example 2 800 ml of a methacrylate-based neutral adsorption resin (HP20: Mitsubishi Kasei Co., Ltd. trade name) was added to 20 (adjusted to pH 4.5) of the eluate obtained in Reference Example 1 and stirred for 2 hours to give human U
G is adsorbed, and then a hydrochloric acid-methanol mixture (6N hydrochloric acid:
Elution was carried out with methanol = 1: 12.5, volume ratio 8).
After neutralizing this, it was concentrated under reduced pressure to 500 ml and loaded on a cross-linked dextran gel (Sefadex G-25, Fumarmacia Huaine Chemical AB trade name), developed with 0.01 M ammonium acetate and the salt-free portion was developed. It was collected. Then, it was adsorbed on a column of diethylaminoethyl (DEAE) cellulose (DE-32, trade name of Wattman Chemical Separation Co., Ltd.) equilibrated with a 0.01 M ammonium acetate solution of pH 5.3. After washing with a 0.01 M ammonium acetate solution, elution was performed with a 0.3 M ammonium acetate solution. The amount of UG in this eluate was 14.4 mg (recovery rate 38
%, Specific activity 1.2).

Reference Example 3 The eluate obtained in Reference Example 2 was subjected to ultrafiltration (molecular weight cutoff of 100).
0) desalting and concentrating to 60 ml, cross-linked dextran gel (Sephadex G-50, Pharmacia.
Gel filtration was performed using Huine Chemical AB product name). The developing solution was developed with 0.01M ammonium acetate solution, and RRA
The active fraction was collected. The amount of human UG in this eluate is 10
It was mg (recovery rate 70%, specific activity 5.6).

Reference Example 4 In this Reference Example, the eluate containing the human UG obtained in Reference Example 3 was equipped with a column packed with octadecylsilanized silica gel to perform high performance liquid chromatography (HPLC).
An example purified by

The eluate obtained in Reference Example 3 was treated with ocdecylsilylated silica gel (Ricrosolve RP-
18, column (8 mmφ × 250 mm) packed with Merck's trade name, carbon content 19.8% by volume (this is HPLC
Equipment Hitachi 635A type, installed in Hitachi, Ltd.)
And the mixture was developed by a gradient method by changing the mixing ratio of the first liquid and the second liquid of the developing liquid. The first liquid is a solution in which water and acetonitrile are mixed at a ratio of 9: 1 (volume ratio), and the second liquid is a 0.2M sodium chloride aqueous solution containing 0.01M hydrochloric acid and acetonitrile in a ratio of 2: 3 (volume ratio). Solution. Mixing the first liquid and the second liquid is the first liquid 100
The second liquid was added to ml at a rate of 2 ml / min, and the mixed liquid of the first liquid and the second liquid was fed into the column at a flow rate of 2 ml / min. The chromatogram is shown in FIG. Retention time on the horizontal axis and 280 nm on the vertical axis
The absorbance of is shown. Human UG was eluted during a retention time of 42-52 minutes. The amount of human UG in this eluate is 0.75 mg
(Yield 75%, specific activity 260). This is SDS
-Four bands were detected on polyacrylamide gel electrophoresis. Further, FIG. 1 shows the RRA activity value of each fraction in the above-mentioned chromatography in a shaded bar graph. R
RA activity appeared in the 42-46 and 46-52 minute fractions, but not in the other fractions.

The eluate eluted at the retention time of 42 to 52 minutes was concentrated under reduced pressure, and then the ultrafiltration membrane (YM-
2. Ultrafiltered with Amicon (trade name), desalted, and freeze-dried to obtain a dried human UG.

Reference Example 5 The eluate obtained in Reference Example 3 was human UG amount of 1.2 mg, and octadecylsilylated silica gel having a carbon content of 20% by weight (Develodil ODS, Nomura Chemical Co., Ltd. trade name) was used. It was used and eluted in the same manner as in Reference Example 4. However, the first solution of the developing solution is an aqueous solution of 0.05 M ammonium acetate, and the second solution is an aqueous solution of acetonitrile [water: acetonitrile = 1: 1 (volume ratio)] in which 0.05 M of ammonium acetate is dissolved. Using. First
The mixing of the liquid and the second liquid is adjusted so that the second liquid flows at a rate of 2 ml / min into 100 ml of the first liquid, and the mixture of the first liquid and the second liquid is fed into the column at a flow rate of 2 ml / min. The flow rate was adjusted so that The chromatogram is shown in FIG. Human UG eluted at a retention time of 38-46 minutes. The amount of human UG in this eluate is 0.9 mg as measured by the RRA method.
(Yield 75%, specific activity 32). This eluate is
Four bands could be detected by SDS-polyacrylamide gel electrophoresis. Further, FIG. 2 shows the RRA activity value of each fraction in a shaded bar graph. RRA activity has a retention time of 38-42
It appeared in the fractions and in the 42-46 minute fraction, but not in the other fractions. A human UG dried product was obtained in the same manner as in Example 1 except that the eluate having a retention time of 38 to 46 minutes was used.

Reference Example 6 Separately, the eluate (specific activity 7.6) obtained by the same method as in Reference Example 3 was used to obtain 19 mg of human UG and develodil ODS.
(Nomura Chemical Co., Ltd. trade name) packed column (36.7)
mmφ × 500 mm) was used and eluted in the same manner as in Reference Example 4. However, the first solution of the developing solution used was a 0.05M ammonium acetate aqueous solution, and the second solution used was a solution of 0.05M ammonium acetate dissolved in an aqueous acetonitrile solution (water: acetonitrile = 3: 2, volume ratio). . The first liquid and the second liquid were mixed by adding 600 ml of the first liquid to the second liquid at 25 ml / min and allowing the mixed liquid to flow into the column at a flow rate of 25 ml / min. The resulting chromatogram is shown in FIG. Human UG was eluted at a retention time of 25 to 45 minutes, of which a fraction with a retention time of 30 to 40 minutes was collected. The amount of UG in this eluate was 16.9 mg (yield 89
%, Specific activity 80). When this was subjected to SDS-polyacrylamide gel electrophoresis, three bands were detected.
Further, in FIG. 3, the RRA activity value of each fraction is shown by a shaded bar graph. RRA activity has a retention time of 25 to 30 minutes, 30 to
It appeared at 35 minutes, 35-40 minutes and 40-45 minutes, and not in the other fractions.

Example 1 In this example, the holding time 30-40 obtained in Reference Example 6 was used.
Minute eluate was further purified to separate three UGs.

The eluate obtained in Reference Example 6 was injected into a column (20 mmφ × 500 mm, Hitachi 635A type apparatus) packed with 2.5 mg of develodil ODS (trade name of Nomura Chemical Co., Ltd.) in a UG amount, and ammonium acetate of 0. Acetonitrile aqueous solution (acetonitrile: water = 140: 500, volume ratio) containing 05M was used as a developing solution at a flow rate of 5 ml / min to elute. The chromatogram at this time is shown in FIG. The horizontal axis shows the retention time and the vertical axis shows the absorbance at 280 nm. Human UG
Has a retention time of 26 to 30 minutes, 34 to 42 minutes and 44 to
It eluted at 50 minutes. When the eluate was collected at each retention time, the specific activities of UG were 1200, 1750,
2200, the amount of human UG was 1: 1: 2 in the order of elution, and the total amount of these UGs was 2.0 mg. Also,
FIG. 4 shows the RRA activity value of each fraction in a shaded bar graph.
RRA activity has a retention time of 26 to 28 minutes, 28 to 30 minutes,
30-32 minutes, 34-36 minutes, 36-38 minutes, 38-
It appeared in the 40, 44-46, 46-48 and 48-50 minute fractions, but not in the other fractions.

The above-mentioned three kinds of eluates were concentrated under reduced pressure and then freeze-dried, and the obtained dried products were dissolved in physiological saline. When these solutions were intravenously injected into intact Fistula dogs at a dose of 0.25 μg of human UG per 1 kg of dog body weight, they all showed the same gastric acid secretion inhibitory effect.

Example 2 Separately, an eluate (specific activity 4.3) obtained by the same method as in Reference Example 3 was used as human UG in an amount of 12.3 mg and Deverodil O.
Column (3) filled with DS (trade name of Nomura Chemical Co., Ltd.)
(6.7 mmφ × 500 mm) was used and eluted in the same manner as in Reference Example 4. However, the first solution of the developing solution was an aqueous acetonitrile solution containing ammonium acetate 0.05M (water: acetonitrile = 5: 1, volume ratio), and the second solution was an aqueous acetonitrile solution containing ammonium acetate 0.05M (water:
Acetonitrile = 5: 1.6, volume ratio) was used. Second
The liquid was allowed to flow into 100 ml of the first liquid at 25 ml / min, and the mixed liquid of the first liquid and the second liquid was allowed to flow into the column at 25 ml / min. As a result, the obtained chromatogram is
Shown in the figure.

When each elution fraction was measured by the RRA method, three active fractions appeared, and human UG in each fraction was found to have a gastric acid secretion inhibitory action. Each of the active fractions has a retention time of 4
Elute at 8-52 minutes, 54-62 minutes and 62-68 minutes,
The total amount of human UG was 10.5 mg, and the amount of human UG in each fraction was 2.4 mg, 1.9 mg and 6.2 in the order of elution.
mg, and the specific activity of each human UG was 130, 24 in the order of elution.
0 and 290. Each fraction was concentrated under reduced pressure and freeze-dried to obtain a dried product, which was designated as UG-1, UG-2, and UG-3 in the above elution order. SDS-urea-polyacrylamide gel electrophoresis (monomer concentration 13.8%, standard substance: partial hydrolysis product of myoglobin, molecular weight 16949, 14404, 815
9, 6214, 2512 and 1360) for UG-1, a niband having a molecular weight of about 6000 and about 7400,
Two bands with molecular weights of about 6000 and about 7,000 were detected for UG-2, and one band with a molecular weight of about 6000 was detected for UG-3.

These UG-1, UG-2 and UG-3 corresponded to the respective three elution fractions obtained in Example 1 in the order of elution.

FIG. 5 shows the RRA activity value for each fraction in the above elution in a shaded bar graph. RRA activity is 46-48 minutes, 4
8-50 minutes, 50-52 minutes, 54-56 minutes, 56-58
Minutes, 58-60 minutes, 62-64 minutes, 64-66 minutes and 6
It appeared in the 6-68 minute fraction and not in the other fractions.

Example 3 This example is based on UG-1, UG-2 obtained in Example 2.
And UG-3 are again purified by reverse phase partition chromatography.

UG-1, UG-2 and UG-3 are each 500 in UG amount.
A column (8 mmφ × 250 mm, Hitachi 635A type device) loaded with μg and Develodil ODS (trade name of Nomura Chemical Co., Ltd.) was loaded, and an aqueous acetonitrile solution containing 0.05 M ammonium acetate (acetonitrile: water = 140: 5) was loaded.
(00, volume ratio) to 200 ml, and 0.1 ml of acetic acid was added as an eluent to elute at a flow rate of 1 ml / min. Obtained chromatogram (retention time on the horizontal axis, 280 nm on the vertical axis)
Absorbance) is shown in FIG. 6 for UG-1 and FIG. 7 for UG-2. 6 to 7, the RRA activity value is shown as a shaded bar graph. RRA activity is U for UG-1 at retention times of 26-30 minutes and 46-58 minutes.
The G fraction (fraction showing RRA activity) appeared, and for UG-2, the UG fraction appeared at 48 to 60 minutes and 65 to 77 minutes. For UG-3, it was not eluted by the retention time of 80 minutes.

UG-3 was eluted in the same manner as above using 200 ml of an aqueous acetonitrile solution containing 0.05 M ammonium acetate (acetonitrile: water = 170: 500) to which 1 ml of acetic acid was added as a developing solution. The chromatogram and RRA activity value at this time are shown in FIG. As a result, as for RRA activity, for UG-3, UG fractions with retention times of 22 to 26 minutes, 27 to 30 minutes and 49 to 55 minutes appeared. For UG-3, the absorbance peak at 280 nm corresponded to the RRA active fraction.

For UG-1, the separated UGs are UG-1 in the order of elution.
-1 and UG-1-2, UG-2 as elution order UG-2-1 and UG-2-2, and UG-3 as elution order UG-3-1, UG-3-2 and UG-3. -3
And

The yield (UG amount) ratio of UG-1-1 to UG-3-3 is 0.5: 4: 4: 0.5 in order for the total UG amount (10.5 mg) obtained in Example 2. It was 8: 2: 2. Of these, UG-1-1 and UG-2-2 were in small amounts. UG-1-2, UG-2-1, UG-3-1, UG
-3-2 and UG-3-3 have specific activities of 2030,
2200, 2450, 2300 and 2400, and the isoelectric points by the isoelectric focusing method are 4.45 and 4.4, respectively.
The values were 0, 4.65, 4.60 and 4.45. Also SD
S-urea-polyacrylamide disk electrophoresis showed a single band, and when the myoglobin hydrolyzate (mixture of molecular weights 16949, 14404, 8159, 6214, 2512 and 1360) was used as a standard substance, the molecular weights were all About 60
It is estimated to be 00.

The eluate containing UG-1-1 to UG-3-3 is concentrated under reduced pressure, freeze-dried, and UG-1-1 to UG-3-3.
Got

Test Example The gastric acid secretion inhibitory action of UG-1-1 to UG-3-3 obtained in Example 3 is shown.

An intact fistula dog was prepared by attaching a gastrostomy tube to the body of a beagle dog (male) weighing 13 kg, about 5 cm away from the pylorus, and then subcutaneously injecting histamine 4 to 6 μg / kg body weight every 15 minutes. Gastric juice was collected from the gastrostomy tube at intervals of 15 minutes. When the gastric juice volume becomes almost constant, UG-
Each of 1-1 to UG-3-3 is dissolved in physiological saline to make U
The G dose was 0.25 μg / kg body weight injected intravenously. In any case, the gastric juice volume decreases after 15 to 30 minutes,
After 60 minutes, the volume of gastric juice became the smallest. Of these results, bar graphs showing the relationship between the amount of gastric acid secretion by administration of histamine for UG-1-2, UG-2-1, and UG-3-2 are shown in FIGS. 9, 10, and 11, respectively. .

In addition, the ratio of gastric acid secretion (B) suppressed during 30 to 90 minutes after UG administration to the gastric acid secretion (A) secreted during 60 to 0 minutes before UG administration [gastric acid secretion inhibitory rate: ( B / A)
× 100%] is shown in Table 1.

(Effects of the Invention) As is clear from the above, by the process according to the present invention, human UG can be obtained with high yield and high specific activity from a sample containing human UG, and 7 kinds of novel UG amounts can be collected. You can also

[Brief description of drawings]

FIG. 1 is a chromatogram and RRA activity bar graph showing the result of chromatographie in Reference Example 4, FIG. 2 is a chromatogram and RRA activity bar graph showing the result of chromatographie in Reference Example 5, and FIG. 3 is a reference example. 6
Chromatogram and RRA activity bar graph showing the result of chromatograph in FIG. 4, and FIG. 4 is a chromatogram and R showing the result of chromatograph in Example 1.
RA activity bar graph, FIG. 5 is a chromatogram showing the result of chromatography in Example 2, and RRA activity bar graph, FIG. 6 is a chromatogram showing the result of chromatography for UG-1 in Example 3, and R
RA activity bar graph, FIG. 7 shows UG-2 in Example 3.
Chromatograms and RRA activity bar graphs showing the results of chromatograms for UG-3 in Example 3, FIG. 8 is a chromatogram and RRA activity bar graphs showing the results of chromatography for UG-3 in Example 3, and FIG. A bar graph showing gastric acid secretion for 1-2,
FIG. 10 is a bar graph showing gastric acid secretion relating to UG-2-1 in the test example, and FIG. 11 is UG- in the test example.
3 is a bar graph showing gastric acid secretion for 3-2.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Kohei Hirano 4-13-1, Higashimachi, Hitachi City, Ibaraki Prefecture, Ibaraki Laboratory, Hitachi Chemical Co., Ltd. (72) Daisuke Irie 4-13, Higashimachi, Hitachi City, Ibaraki Prefecture No. 1 Hitachi Chemical Co., Ltd. Ibaraki Research Laboratory (56) References "Protein, Nucleic Acid and Enzyme" Vol. 27, No. 7, P.I. 1056-1068 (1982) Published by Kyoritsu Shuppan Co., Ltd.

Claims (6)

[Claims] 1. A multi-component human urogastrone from a sample containing human urogastrone by reverse phase partitioning liquid chromatography using an aqueous solution having a neutral pH of 18 to 28 vol% acetonitrile as an eluent. A method for producing human urogastrone, which is separated into two and collected. 2. The method for producing human urogastrone according to claim 1, wherein porous silica gel having a hydrophobic functional group is used as a column agent for reversed-phase partitioning liquid chromatography. 3. The method for producing human urogastrone according to claim 1, wherein the sample containing human urogastrone is derived from human urine. 4. A step of subjecting a sample containing human urogastrone to (1) reverse phase partitioning liquid chromatography using an aqueous solution having a neutral pH and 18 to 28% by volume of acetonitrile as an eluent; and (2) Reversed-phase partitioning liquid chromatography using an aqueous solution having an acidic pH of 18 to 28% by volume as an eluent; The two steps are combined to separate human urogastrone into multiple components, A method for producing human urogastrone for collecting. 5. The method for producing human urogastrone according to claim 4, wherein porous silica gel having a hydrophobic functional group is used as a column agent for reversed-phase partitioning liquid chromatography. 6. The method for producing human urogastrone according to claim 4, wherein the sample containing human urogastron is derived from human urine.