JPS6256430A - Antitumor acidic glycoprotein - Google Patents

Abstract

PURPOSE:To obtain two kinds of alpha1-AG compound useful as antitumor agent, by administering a mammal with antitumor lichen polysaccharide to produce a large amount of acidic glycoprotein alpha1-AG in body liquid, and separating and purifying the compound. CONSTITUTION:A mammal, preferably a mammal affected with ascites carcino ma is administered with antitumor lichen polysaccharide (abbreviated as ALP) to produce a large amount of 'alpha-AG in ascites, and two kinds of alpha1-AG compounds, i.e. alpha1-AG-1 and alpha1-AG-2 are separated and purified by a combina tion of conventional separation and purification processes. Fractional isoelectric electrophoresis is most effective as the final purification means. The isoelectric point of alpha1-AG-1 is 3.2 and that of alpha1-AG-2 is 3.4 on a Model 1404 electrophore sis cell of Bio-Rad Inc. using Ampholin pH2.5-4.5R. The alpha1-AG of mouse exhibits strong antitumor activity against leukemia cell L-1210, Sarcoma 180, etc. The alpha1-AG-1 has an antitumor activity of about 10 times as high as that of the alpha1-AG-2.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention provides an acidic glycoprotein with antitumor activity, a method for producing the same,
The present invention also relates to antitumor agent compositions.

(Prior Art) It has been reported that an acidic glycoprotein α,-AG exists in the body fluids of mammals, and that this α,-AG has the ability to control liver cell division [for example, cancer (Gann)].

+39 (1974) and 66.227 (1975)
), Journal of Theoretical Biology (J, theor, Biol, ), 77, 3
67 (+979): Hoppesailers Zeitschrift Fair Physiologist Hemi (Ilo
ppe-3eyler's Z, Physiol,
Chem,, 361.

1567 (1980)]. However, in general α1-A
Since the content of G is very small and is a mixture of two or more substances, there has been no example of α,-AG being obtained in a large quantity in an isolated and purified form. Therefore, the effects of α,-AG other than the hepatocyte division regulating action and the physicochemical properties of α,-AG are unknown, and only the estimated molecular weight of α,-AG has been reported.

(Problems to be solved by the invention) As a result of searching for a system that produces a large amount of α, -AG, the present inventors found that antitumor lichen polysaccharide (hereinafter abbreviated as ALP)
It has been found that a large amount of α,-AG is produced in the ascites of a mammal, preferably an ascites cancer-bearing mammal, by administering the following. The present inventors also obtained α, -AG from ascites containing a large amount of α, -AG obtained by the above method.
We succeeded in isolating and purifying 2N α,-AG, which is the main constituent of
It was discovered that the antitumor effect is based on the antitumor effect of α,-AG that is secondarily produced by the administration of LP, and the present invention was completed after further investigation.

(Means for solving the problem) The present invention provides (1) substantially isolated and purified α,-Ac;
(2) α, characterized by administering ALP to a mammal and isolating and purifying α, -AG produced in body fluids;
The present invention relates to a method for producing -AG, and (3) an antitumor composition containing α, -AG.

There are two types of αrAG isolated and purified in the present invention, and each is hereinafter referred to as α, -AG-1 and α, -AG-2. α, -AG-1 and α, -A(, -2 each have the following physicochemical properties.

α, -AG-1 (compound according to claim 2): ■ Isoelectric point 3,2 "Bio-Rad model ■404
On the electrophoresis cell, Amphorin ■ 1) Use R2.5-4.5 (manufactured by LKB Products) ■ Molecule ff 140,000 ± 4,000 [Bovine serum albumin and chymotrypso as indicator proteins Nogen A, ovalbumin and myoglobin, (7A7.V, A molecular weight?
Molecular weight: 40,000 ± 4,000 [as indicator proteins: bovine serum albumin, chymotrypsinogen A, ovalbumin, and myoglobin (Falcia molecular weight marker 0)]
comolecule ff139,500±4. Q OO[N-7-t?
Calculated from analytical values using chirneuraminic acid as an indicator] ■Molecular composition Sialic acid 12.5% (approximately 16 elements in 1 molecule) [by cresol-periodic acid method] Hexose 12.2% (approximately 27 elements in 1 molecule) [By phenol-sulfuric acid method] Amino sugar 11.4% (approximately 25 per molecule) [3-methyl-2-benzothiazolone hydrazone hydrochloride (MBTH
) Method Coprotein 64.0% [Microbiuret method] ■ Amino acid composition of the above protein ASP about 12 THR about 18 5Er (about 10 GLU about 29 GLY about 8) [ALA about 8 V A L about 8 cys approx. 1 MET approx. 3 [LEU approx. 10 LEU approx. 18 TYR approx. 5 PHE approx. 13 LYS approx. 18 His approx. 6 ARG approx. 9 PRO approx. 6 Pharmalite pH 2,5-5 , 0■ (manufactured by Pharmacia Fine Chemicals) to give a single spot in polyacrylamide gel analysis isoelectric focusing electrophoresis ■ To give a single spot in polyacrylamide gel electrophoresis using sodium dodecyl sulfate (1) 9q'i-bu'J!JT',ytobu)Iy
Color development α+-Aa 2 (compound according to claim 3) at -R250° (manufactured by Gyudon Kagaku): ■ Isoelectric point 3.4 [Bio-Rad model 1404
On the electrophoresis cell, amphorin pH 2.5-4.5o (
r-zl, = -K, H-, [a'1 Shoes Co., Ltd.)] ■ Molecular weight 35,300 ± 3,500 [Bovine serum albumin, chymotrypsinogen A, ovalbumin and myoglobin, (7 A2. Molecular weight by sucrose gradient centrifugation using -car)] Molecular weight 38,000 ± 3,800 [as indicator proteins bovine serum albumin, chymotrypsinogen A, ovalbumin and myoglobin (Pharmacia molecular weight marker 0)
By polyacrylamide gel electrophoresis using sodium dodecyl sulfate] Molecule ff 134,200 ± 3.400 [Calculated from analytical values using N-acetylneuraminic acid as an indicator] ■Molecular composition Sialic acid 11.4 % (approx. 13 per molecule) [by cresol-periodic acid method Cohexose 15.1% (approx. 30 per molecule) [by phenol-sulfuric acid method] Amino sugar 12.0% (approx. 23 per molecule) ) [3-Methyl-2-benzothiazolone hydrazone hydrochloride (MBTH
) Method Coprotein 61.6% [Microbiuret method] ■ Amino acid composition of the above protein ASP about 12 THR about 14 SER about 9 GLU about 16 GLY about 12 ALA about 7 VAL about 6 CYS about 1 MET about 4 pieces I LEU about 8 pieces LEU about 17 pieces TYR about 4 pieces P HE about 13 pieces LYS about 17 pieces HIS about 5 pieces AR [about 9 pieces Pr1O about 5 pieces ■ ■ Pharmalite pH 2,5~5 .0 (manufactured by Pharmacia Fine Chemicals) to give a single spot in polyacrylamide gel analysis Isoelectric focusing electrophoresis using sodium dodecyl sulfate gives a single spot ( 1) ')? U-7"lJ'J7:/) 2Jl/
-R250'' (manufactured by Gyudon Kagaku) α, -AG-1 and α, -AG-2 of the present invention can be produced by the following procedure.

(1) Cancer cells that induce ascites cancer are inoculated into a mammal. Examples of cancer cells that induce ascites cancer include Ehrlich's carcinoma cells and Sarcoma 180, and Ehrlich's carcinoma cells are frequently used in the present invention. Examples of the mammals used include mice, rats, rabbits, and dogs, with mice and rats being more preferred. It is most preferable to use a mouse in terms of ease of use and cost. The amount of cancer cell inoculation is determined by the type of cancer cells and the type of mammal. For example, in the combination of mouse and Ehrlich cancer cells, mouse 1
It is common to use 104 to 10'Ehrlich's cancer cells per animal. Inoculation of cancer cells can be performed according to known methods. For example, when inoculating mice with Ehrlich cancer cells, an aqueous solution containing the cancer cells is intraperitoneally administered. Cancer cells may be administered at once, or may be administered in 2 to 5 divided doses. For example, mice to which Ehrlich's cancer cells are administered become ascites-bearing cancer mice after about 2 to 7 days.

(2) ALP is administered to the ascitic fluid-bearing mammal.

Examples of ALP used here include β(!→6) glucan 1β(1-3)(l→4) glucan, α-glucan, and peptidoglycan obtained from various lichens. More specifically, ALP is one in which puttran, a type of β(1→6) glucan, is partially acetylated (hereinafter abbreviated as GE-3), and GE-3 treated with urea. (hereinafter abbreviated as UGE-3), β(
1-3) (1-4) Lichenan (hereinafter abbreviated as L), which is a type of glucan, is exemplified.

ALP is usually administered by subcutaneous injection of ALP dissolved in physiological saline, at a dose of 30 to 30 mg/day.
100 mg/kg is divided into 1 to 5 doses and administered continuously for 2 to 14 days. In the case of mice, the best method is to administer 40 to 80 mg/kg of ALP per day in 1 to 2 divided doses for 3 to 10 days.

Todenμ nosu melted down ★di・noshuiki m++thing 2koA
When I P is given at t0, α, -A in ascites and serum
G level increases rapidly.

(3) Collect the entire amount of ascites 10 to 30 hours after the final administration of ALP. In the case of mice, on average 1 per mouse
Zero ascites was obtained, and when α1-AG in the ascites was quantified according to the quantitative method described later in Reference Example 3, the ascites contained an average of 800 μg of α,-AG.

(4) The ascites and serum containing a large amount of αrAG obtained in the previous section are treated to separate, isolate, and purify α,-AG. Separation, isolation, and purification are performed using a combination of conventional chemical methods. That is, extraction, liquid conversion, centrifugation, concentration, freeze-drying, various column chromatography, liquid chromatography, electrophoresis, etc. are used. For example, when mouse α, -AG is isolated and purified, the above-mentioned α, -AG-1 and α are obtained.

Although -AG-2 can be obtained in pure form and in large amounts, preparative isoelectric focusing is the most effective method for final purification.

α, -AG obtained by the above production method may be crude α+AG before being completely purified, or purified α, -AG-1 and α, -AG-2. has excellent antitumor activity as shown in the Examples. For example, mouse α, -AG (α+AG
1. α, -AG-2) are leukemia cells L-1210,
Shows activity against tumors such as Sarcoma 180. αI
Comparing -AG-1 and α1-AG-2, the former shows about 10 times more antitumor activity than the latter.

The antitumor agent composition containing α,-AG of the present invention is α1-AG.
or isolated and purified α, -AG-1 or α, -A
It can be obtained by dissolving G-2 in a solvent such as physiological saline or physiological saline containing a buffer solution. The concentration of the active ingredient is usually 0.01-30% (W/V), preferably 0.1-10% (W/V), and is suitable for humans or warm-blooded animals (e.g. mice, rats, rabbits, dogs, etc.). ), usually 1 to 3 times a day, 0.01 to lod/kg
Administer intravenously or locally. Other antitumor agents may also be added to the composition.

(Examples) Hereinafter, the present invention will be explained in more detail in the form of Examples and Reference Examples, but the present invention is not limited to these Examples. The mice used in this example were 100 female mice belonging to the ddY species, 6 to 7 weeks old, weighing 22 to 26 g.
I chose the one.

Example I Preparation of ALP aqueous solution (1) GE-3 Umbilicaria esculenta (Miyoshi) mink,
(Umbilicaria esculenta (
mjyoshi) Mink,.

Iwatake thallus L OOg is heated in 80% ethanol IC at 80° C. for 3 hours and the solution is removed by filtration. The residue is heated again at 80° C. in 80% ethanol IQ for 3 hours and the solution is removed by filtration. 80% residue
Heat at 80° C. for 3 hours in ethanol IQ three times and remove the solution by filtration. Next, the residue is heated in water [σ] at 90°C for 6 hours and filtered. The residue is heated again in 1f2 of water at 90° C. for 6 hours and filtered. The residue is heated in 1Q water at 90° C. for 3 times for 6 hours and filtered.

The filtrates are combined, ethanol of the same fi & (312) is added, and the resulting precipitate is collected by filtration. Add 500 ml of water to the filtered solid, heat it to 80°C, filter off insoluble matter, and then freeze the solution. Subsequently, the temperature is returned to room temperature, insoluble matter is filtered off, and the mixture is dried to obtain 36 g of GE-3.

(2) UGE-3 Add 100 am of 8M urea aqueous solution to 1 g of GE-3 and heat at 60°
Heat at C for 6 hours. After cooling, the solution is placed in a dialysis tube, and the tube is immersed in 512-g. distilled water to perform dialysis for 24 hours. UGE-30.7g is obtained by freeze-drying the aqueous solution from which urea has been removed by dialysis.

(3) From 100 g of L, 6 g of L can be obtained by performing the same process as in the case of GE-3.

If you perform exactly the same process as in the case of GE-3,
6g is obtained.

The above ALP is dissolved in physiological saline to 30%
(W/V) Millipore filter as physiological saline solution (
0.45 μm), and diluted with physiological saline to adjust to a predetermined concentration before use.

Example 2 Production of mouse α, -AG (1) Ehrlich's carcinoma cells, on average 2XIOB per mouse, are inoculated intraperitoneally into mice. Ehrlich's cancer cells obtained as in Reference Example 1 are inoculated using a syringe. UGE-3 is administered by subcutaneous injection for 4 days starting 7 days later (ie, day 8). The dose and concentration per mouse was 60 mg/kg/O, 1me/day;
Administer once a day. Twenty hours after the final administration of UGE-3, the entire amount of ascites is collected using a syringe. 3.00Orp
centrifugation for 30 minutes to remove the precipitate (.The ascitic fluid obtained in this way from which the cells were removed has an average of 10 TnIl per mouse. In other words, in this example, α+
100d of ascitic fluid containing AG is obtained. It can be determined that this ascites contains 800 gg/d of α,-AG by the simple immunodiffusion method described in Reference Example 3 using rabbit antiserum.

Solid ammonium sulfate was added to the obtained 1 000 d of ascites to bring the concentration of ammonium sulfate to 60%, and the precipitate formed was removed. Solid ammonium sulfate is further added to the solution to bring the concentration of ammonium sulfate to 90%.

The precipitate is placed in a dialysis tube and dialyzed for 24 hours in distilled water containing I(N2).

After removing ammonium sulfate by dialysis, DE
AE cellulose (diethylamine ethylcellulose,
Apply to a Braun Co.'lA) column.

DEAE cellulose is equilibrated by soaking it in 0.05 M acetate i buffer (pH 5,0) for one day, and then packed into a column with a diameter of 20 mm to a height of 50 cm. The column is emptied using a salt gradient method.

That is, initially 0.05M acetate buffer (pH 5,
0) alone, then the acetate buffer and 0.5M
0.05M acetate buffer containing saline (p115.
0) and (1:l V/V) (gradually increasing the ratio of the latter), and finally only 0.05M acetate buffer (pH 5,0) containing 0.5M salt. Flow.

Both flows are collected with a buffer solution containing 0.27-037M sodium chloride and dialyzed against 10% distilled water for 24 hours, followed by preparative isoelectric focusing. Electrophoresis is performed using Bio-Rad model +404. ?Ii pneumophoresis cell, amphorin p
82.5 to 4.5 (manufactured by LKB Products).

At the isoelectric point 3.2, α, -AG-1. ．． Isoelectric point 3,4
α,-AG-2 is obtained. 20 each corresponding portion
Rinse each separately with distilled water and use the dialysis tube described above! Dialysis is performed against distilled water of (2)
After 4 hours, freeze-dry separately.

The yield of α, -AG-1, α, -AG-2 was 35% each.
mg, 25 mg.

Example 3 Production of Mouse α,-AG (2) The same experiment as in Example 2 was performed by replacing UGE-3 with GE-3. The administration period of GE-3 will be 6 days. The conditions other than these are exactly the same as in Example 2. α+ AG 1
40 mg, at AG-227 mg is obtained.

Example 4 Generation of Mouse α,-AG (3) The same experiment as in Example 2 was performed except that UGE-3 was replaced with L. The administration period of L will be 10 days.

The conditions other than these are exactly the same as in Example 2.

α, AG 1 33mg, α+ AG 2
24 mg is obtained.

Example 5 Purity of α1-AG-1 and αI-AG-2 α,-AG-1 and α,-A obtained in Examples 2 to 4
The purity of G-2 is determined by analytical isoelectric focusing.

Fluorite I) Analytical isoelectric focusing on a polyacrylamide gel plate using N2.5-5.00 (Pharmacia Fine Chemicals) (B) and polyfluorescence using sodium dodenyl sulfate α1-AG-1 in acrylamide gel electrophoresis (A)
(a), αt-AG-2 (b) gives a single spot on the tiger.

■ Based on R250 (manufactured by Hirai Chemical). (See drawing) Example 6
Molecular composition of α, -AG-1 and αI-AG-2 The content of each component was analyzed by the following method.

Sialic acid---Cresol-periodic acid method [Methods in Enzymology, Volume 6, Academic Press, New York, 459
Page (1963)] Hexose--Phenol-Flow Acid Method [Method.
In Carbohydrate Chemistry = (Metho)
ds in Carbohydrate Chemi
stry), Volume 1, Academic Press.

[Chem, Pharm,
Bull, ), I 7.

1505 (1969)] i White ------- Microbiuret method [Analytical Biochemistry (Anal, Bioc
hem, ), 9.401 (1964)] The composition of each component obtained by the above method is as follows. The numbers in parentheses are the estimated numbers in the α,-AG1 molecule.

α, -AG-1α, -AG-2 Sialic acid I2,5% (16) 11.4%
(13) Hexose 12.2% (27) 1
5.1% (30) Amino sugar 11.4% (25)
12.0% (23) Protein 64.0%
61.6% Next, amino acid analysis of the protein was performed. First, 1mg of protein was added to 1Tn1. 11 in a sealed tube with 6N hydrochloric acid.
After hydrolysis at 0'c for 20 hours, hydrochloric acid was completely distilled off under reduced pressure, and the residue was directly subjected to amino acid analysis using a high-speed amino acid analyzer (Hitachi Model 835). The results of amino acid analysis are as follows. The numbers represent the estimated number of each amino acid contained in one protein molecule constituting α1-AG.

αI -A C-1α, -A G -2 Aspartic acid (ASP) +2 12 Threonine
(THR) 18 14 serine (
SER) +0 9 Glutamic acid (GL
U) 19 16 glycine (GLY)
11 12 Alanine (ALA)
8 7 Valin (VAL) 8
6 Cysteine (CYS) 1
1 Methionine (MET) 3
4 Isoleucine (ILEU) 10
80 Ishin (LEU) 18 17
Tyrosine (TYR) 5 4 Phenylalanine (PHE) 13 13 Lysine
(LYS) 18 17 Histidine ()IIs) 6 5 Arginine (ARG) 9 9 Proline
(PRO) 6 5 Example 7 Molecular weight measurement (1) Sucrose gradient ultracentrifugation - 1 - As indicator proteins, bovine serum albumin, chymotrypsinogen A, ovalbumin and myoglobin (Pharmacia molecular weight = - car
) to calculate the estimated molecular weight by a known method.

(2) Polyacrylamide gel electrophoresis using sodium dodecyl sulfate - 11 - Using the same molecular weight marker as in (1) as the indicator protein, the estimated molecular weight is calculated by a known method.

(3) Molecular weight calculated from the estimated composition value of the molecule in Example 6---The molecular weight of each composition is added using N-acetylneuraminic acid as an indicator as sialic acid.

The molecular weight estimated by the above method is as follows.

α, -AG-1α, -AG-2 (1) 40,000±4.000 35,3
00±3,50.

[2] 40,000±4.000 38,0
00±3,800 Example 8 α1-AG-1 and α+
After the L 1210 proliferation inhibitory effect of AG2, the L 1210 proliferation inhibitory effect was examined by the method described in Reference Example 4. The results were as follows.

α, -AG μg/d Proliferation rate (%) (1+
AG I 0 (= lntrow 11/)
100too 10g, 6200
62.6 400 19.7 800 15.6 α1-AG-22,00053,2 Example 9 10 mg of antitumor agent composition U+ AG 1 is dissolved in 1 mM physiological saline and sealed in an ampoule.

Reference Example 1 Ehrlich's cancer cells 1 d of an aqueous solution containing 5XIO"Ehrlich's cancer cells is inoculated into ddY mice (female) with an average weight of 25 g, and ascites is collected after 7 days. The ascites contains an average of 10" of Ehrlich's cancer cells.
Contains individual/preceptive Ehrlich cancer cells. The ascites is diluted with physiological saline to prepare an aqueous solution containing a predetermined number of ascites.

Reference Example 2 Production of antiserum of mouse α, -AG α, -AG
Dissolve 500 μg in 1 d of physiological saline and add 1 ml of liquid paraffin containing Aracel to make an emulsion. The entire amount of the emulsion is injected into the calf and back of a white rabbit weighing 3 kg.

This operation is repeated five times at intervals of IO days. 10 days after the final administration, all blood was collected from the carotid artery and kept at 37°C.
After 2 to 3 hours, leave it in the refrigerator for 24 hours. Remove the separated supernatant with a pipette. The supernatant is mouse α
, -AG antiserum.

Reference Example 3 Quantification of α, -AG by simple immunodiffusion method 5 μρ of a standard α, -AG aqueous solution prepared to a predetermined concentration was injected into a well on an agar plate containing the antiserum obtained in Reference Example 2. , leave for 48 hours. Wash the agar plate with physiological saline and stain with Kuman Buriri Ant Blue R250 (manufactured by Hirai Chemical).

A calibration curve can be obtained by plotting α, -A(1; fi on the horizontal axis and the square of the diameter of the settling ring on the vertical axis. α, -A
The amount of G can be easily determined by assaying this sample according to the method described above and using the calibration curve described above from the value of the sedimentation ring.

Reference Example 4 L 1210 proliferation inhibitory effect of α, -AG (
(in vitro) A test tube is filled with RTMI-1640 medium and 5% concentration of fetal bovine serum. On top of this were sequentially 5 μmol of 2-hydroxydiethyl disulfide, IO μg of streptomycin.
/d aqueous solution + d, penicillin 6100 units /d aqueous solution 1
Then, add 5XIO' of L-1210 cells/1 drop of aqueous solution.

1 d of physiological saline (control) or 1 d of α,-AG aqueous solution of a predetermined concentration is added to the above test tube, and cultured at 35° C. for 48 hours in a carbon dioxide incubator. Number of control cells (C), number of cells whose proliferation was inhibited by α-AG (
S), and let S × 100 (%) be the proliferation rate.

[Brief explanation of the drawing]

FIG. 1 shows an analytical diagram obtained by analytical isoelectric focusing. (2) B shows the results of analytical isoelectric focusing on a polyacrylamide gel plate using Pharmalite I) R2,5-5.0 (manufactured by Pharmacia Fine Chemicals). A shows the results of polyacrylamide gel electrophoresis using sodium dodecyl sulfate. a represents α, -AG-1. b represents α, -AG-2. The color is Coomassie Brilliant Blue R250.
' (manufactured by Hirai Chemical Co., Ltd.).

Claims (1)

[Scope of Claims] [1] Substantially isolated and purified α_1-AG. [2] α_1-AG according to claim 1 having the following properties: (1) Isoelectric point 3.2 [Bio-Rad Model 1404
On the electrophoresis cell, Amphorin pH 2.5-4.5^■ (manufactured by LK B Products) was used] (2) Molecular weight 40,000 ± 4,000 [Bovine serum albumin and chymotrypsinogen were used as indicator proteins] A, by sucrose concentration gradient ultracentrifugation using ovalbumin and myoglobin (Pharmacia molecular weight marker ^■)] Molecular weight 40,000 ± 4,000 [as indicator protein bovine serum albumin, chymotrypsinogen A, ovalbumin and myoglobin (Pharmacia molecular weight marker By polyacrylamide gel electrophoresis using sodium dodecyl sulfate using ^■)] Molecular weight 39,500 ± 4,000 [Calculated from analytical values using N-acetylneuraminic acid as an indicator] (3) Molecular composition Sialic acid 12.5% (approximately 16 in one molecule) [By cresol-periodic acid method] Hexose 12.2% (approximately 27 in one molecule) [By phenol-sulfuric acid method] Amino sugar 11.4% ( (about 25 per molecule) [3-Methyl-2-benzothiazolone hydrazone hydrochloride (MBTH) method] Protein 64.0% [Microbiuret method] (4) Amino acid composition of the above protein ASP about 12 THR about 18 SER about 10 pieces GLU about 29 pieces GLY about 11 pieces ALA about 8 pieces VAL about 8 pieces CYS about 1 piece MET about 3 pieces ILEU about 10 pieces LEU about 18 pieces TYR about 5 pieces PHE about 13 pieces LYS about 18 pieces HIS Approximately 6 pieces ARG Approximately 9 pieces PRO Approximately 6 pieces (5) Single by isoelectric focusing for polyacrylamide gel analysis using Pharmalite pH 2.5-5.0^■ (manufactured by Pharmacia Fine Chemicals) (6) Polyacrylamide gel electrophoresis using sodium dodecyl sulfate gives a single spot (7) Coomassie Brilliant Blue R250＾■
(manufactured by Hanui Chemical Co., Ltd.) [3] α_1-AG according to claim 1 having the following properties: (1) Isoelectric point 3.4 [Bio-Rad model 1404]
On the electrophoresis cell, use Amphorin pH 2.5-4.5^■ (manufactured by LK B Products)] (2) Molecular weight 35,300 ± 3,500 [Bovine serum albumin and chymotrypsinogen as indicator proteins] A, by sucrose concentration gradient centrifugation using ovalbumin and myoglobin (Pharmacia molecular weight marker ^■)] Molecular weight 38,000 ± 3,800 [bovine serum albumin, chymotrypsinogen A, ovalbumin and myoglobin (Pharmacia molecular weight marker ^ (2) By polyacrylamide gel electrophoresis using sodium dodecyl sulfate] Molecular weight 34,200 ± 3,400 [Calculated from analytical values using N-acetylneuraminic acid as an indicator] (3) Molecular composition Acid 11.4% (approx. 13 per molecule) [by cresol-periodic acid method] Hexose 15.1% (approx. 30 per molecule) [by phenol-sulfuric acid method] Amino sugar 12.0% (1 (about 23 in molecule) [3-Methyl-2-benzothiazolone hydrazone hydrochloride (MBTH) method] Protein 61.6% [Microbiuret method] (4) Amino acid composition of the above protein ASP about 12 THR about 14 SER about 9 pieces GLU about 16 pieces GLY about 12 pieces ALA about 7 pieces VAL about 6 pieces CYS about 1 piece MET about 4 pieces ILEU about 8 pieces LEU about 17 pieces TYR about 4 pieces PHE about 13 pieces LYS about 17 pieces HIS about 5 pieces ARG about 9 pieces PRO about 5 pieces (5) Single spot by isoelectric focusing for polyacrylamide gel analysis using Pharmalite pH 2.5-5.0^■ (manufactured by Pharmacia Fine Chemicals) (6) Gives a single spot in polyacrylamide gel electrophoresis using sodium dodecyl sulfate (7) Coomassie Brilliant Blue R250＾■
(manufactured by Hanui Kagaku) [4] A method for producing α_1-AG, which comprises administering an antitumor lichen polysaccharide to a mammal and isolating and purifying α_1-AG produced in body fluids. (5) An antitumor agent composition containing α_1-AG.