JPS60158115A - Plasminogen-activator kym and its preparation - Google Patents

Abstract

NEW MATERIAL:Plasminogen activator KYM produced by the mutant KYM-A of human rhabdomyosarcoma cell. Appearance, white powder; molecular weight, two bands between 56,000 and 62,000 (nonreduced type) and between 32,000 and 36,000 (reduced type) by SDS-polyacrylamide gel electrophoresis; solubility, 50mug/ ml in water and salt solution, insoluble in ethanol and ether. USE:Thrombolytic agent. It is a weakly basic glycoprotein having stronger fibrin- bonding capability than urokinase. It is prepared preferably in the form administrable by intravenous injection. PREPARATION:The cell KYM-I separated from human rhabdomyosarcoma (presented by Dr. Morimasa Sekiguchi, Medical Science Laboratory, Tokyo University) is cultured, and the mutant KYM-A prepared by the repeated separation, selection and culture of the cell is subjected to the suspension culture under agitation to obtain the titled substance on an industrial scale.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a plasminogen activator KYM that activates plasminogen, a method for producing the same, and a pharmaceutical composition having thrombolytic activity.

In recent years, diseases caused by blood clots have been highlighted as an important problem, and streptokinase, urokinase, etc. have been used for their treatment. However, none of these methods achieve the intended purpose of thrombolytic dissolution, and it is known that they also degrade coagulation factors in circulating blood, resulting in a bleeding tendency. The reason for this is that streptokinase, urokinase, etc. have poor affinity with the thrombus to be lysed, and therefore
It is thought that it breaks down blood coagulation factors, etc.

Therefore, there was a need to develop a thrombolytic agent that has a high affinity for blood clots and has fewer side effects such as bleeding tendency.

Therefore, research has been conducted on plasminogen activators (hereinafter referred to as PA), which are said to dissolve blood clots and have few side effects, and presentations regarding this have also been made. Regarding human-derived PA, detailed studies have been conducted on enzymes obtained from endothelial cells and normal tissues such as the uterus, as well as tumor tissues and cultured cells thereof. (References include Wilson et al. K., Cancer Research, Sat. 0933-938 (1980), and Ritsken and Fran, Journal of Biological Chemistry 256, 7035-7041 (1981).)

It has been known that normal tissue cells such as human endothelial cells and human uterine cells, and tumor cells such as human melanoma and breast cancer cells produce PA.

However, since normal tissue cells eventually die, they cannot be used for industrial production.

In addition, conventionally known culture of tumor cells such as human melanoma and breast cancer cells is only by adhesion culture in bottles, etc., and the productivity of PA is extremely low, and it has not been possible to mass-produce it industrially.

Therefore, the present inventors first conducted research on tumor cells that can be cultured in suspended agitation, and found that KYM'-1 cells isolated from human transverse strangulation myoma (granted from Morimasa Sekiguchi, Institute of Medical Science, University of Tokyo) They succeeded in discovering and isolating a mutant strain that can float and proliferate from culture. This mutant strain was named mutant strain KYM-A.

As a result of further research, it was found that when this mutant strain KYM-A is cultured, it produces and accumulates a significant amount of PA in the culture solution. This PA was named FA-KYM.

It is generally believed that there are mainly two types of PA in humans. That is, one type is a urokinase type that has poor affinity for fibrin, and the remaining type is a so-called tissue plasminogen activator (TPA) type that shows a high affinity for fibrin.

Melanoma cells are well known as tumor cells that produce TPA-type PA. Melanoma cells have attracted attention because they mainly produce only TPA type PA.

On the other hand, as an exception, breast cancer cells have only been reported as other types of tumor cells that produce only TPA-type FA. Furthermore, in previous searches, it has been thought that lateral strangulation myoma cells produce only urokinase-type PA.

Urokinase-type FA is present in the culture solution of mutant strain KYM-A.
cannot be detected.

Therefore, PA-KYM of the present invention is novel in its origin since it is produced from human strangulated myoma cells, and is recognized as a novel TPA-type FA from its physicochemical properties.

Mutant strain KYM-A used for production of FA-KYM of the present invention
is a mutant strain that was discovered through repeated isolation and selective culture from a culture of KYM-I cells isolated from human lateral strangulation myoma, and is highly distinctive by producing a significant amount of FA-KYM through suspension agitation culture. be. This mutant strain KYM-A can be subcultured by floating agitation culture, but
The deposit at the Institute of Fine Technology was not accepted.

The properties of mutant strain KYM-A are as follows.

1. Individual cells exhibit a refractive spherical shape.

2. Cells may exist singly as floating cells, but may also form chains or spherical aggregates.

The number of cells contained in the aggregate is about 2 to 100.

3. When cultured in a plastic Petri dish or stirred culture in a tank, this mutant strain can mostly grow as floating cells.

4. This mutant strain can be cultured using floating agitation culture.

5. This mutant strain changes into adherent cells when conditioned medium is used.

The altered cells exhibit epithelial cell-like morphology.

The conditioned medium is a culture supernatant of cells other than this mutant strain, which is obtained by culturing appropriate cells in an appropriate basal medium for 1 to 100 hours, and then removing cells and their fragments from the culture medium. be. Examples of cells other than this mutant strain include human liver cancer cells (HuH6-075 strain, HuH7 strain) (Nakamura tt.
Cancer Research, vo142 3858-38
65 1982), etc. can be used.

In order to make this mutant strain adhesive, in addition to using the above-mentioned conditioned medium, it is necessary to use a medium containing fibronectin, or to use collagen, gelatin, poly-L-lysine, or egg white on the adhesive surface of a petri dish. Adhesive culture of this mutant strain becomes possible by treatment with basic proteins such as lysozyme.

6. When 10' cells of this mutant strain are transplanted subcutaneously into a nude mouse or into the anoid sac of an ALS-treated hamster, a tumor is formed.

When the number of Z chromosome chromosomes was determined according to the conventional method of cytogenetics, the most frequent chromosome numbers were 46 and 47, and the distribution showed a slight width around these. Therefore, the chromosome number of mutant strain KYM-A is
Although these cells were derived from a tumor, they were found to be relatively similar to normal human diploid cells (46 chromosomes).

8. This mutant strain produces a significant amount of FA-KYM.

The culture for producing PA-KYM of the present invention may be either adherent culture or floating agitation culture.

Mutant iKYM-A adheres to plastic petri dishes and glass petri dishes and proliferates, so it is possible to perform adhesive culture using roller bottles or microcarriers for the purpose of producing PA-KYM.

However, floating agitation culture is suitable for industrial moth production.

For the production of PA-KYM, it is preferable to culture the mutant KYM-A cells in a serum-supplemented medium, and to change the medium to a serum-free medium when the cells enter the stationary phase of growth. Serum-supplemented media include RPMI (manufactured by Flow) MHM (
Flow Inc.) or a mixed medium of both, to which about 10% fetal bovine serum is added.

In addition, serum-free media include RPMI and ME as basic media.
It is preferable to use a mixed medium of M or both, and to which insulin, human transferrin, monoethanolamine, and selenite are added (this four-component supplemented medium is hereinafter referred to as ITFi8 supplemented medium). For the purpose of producing PA-KYM from cell line KYM-A, the cell line was cultured in suspension in a plastic Petri dish.
When an appropriate number of cells is reached, floating agitation culture using a spinner flask is started.

The spinner flask preferably has a capacity of 100 m to 8000 m/. In addition, the inoculated cell density was 104 to 1 o'
a cell/+lLh>tut L, <, 5×10s to 2
Stationary phase is reached at a density of X106 cells/ml. Although the culture temperature is not particularly limited, it is suitably about 60 to 40°C, and particularly preferably about 57°C. The gas phase is preferably 100% air, air containing 5 to 10% returned acid gas, or air containing 5 to 100% oxygen.

Batch culture may be used, but if the cells grow sufficiently, 1~
By continuously replacing the medium at intervals of 4 days, a culture medium containing FA-KYM can be obtained for about one month after the start of culture.

FA-KYM of mutant KYM-A cells is an enzyme that activates plasminogen to plasmin and belongs to proteins, so any general method applied to protein purification can be applied. In general, salting out, adsorption, affinity chromatography, ion exchange chromatography, molecular sieves, etc., and appropriate combinations thereof can be considered. Further, these purification processes can be carried out either continuously or batchwise, and the method may be selected in consideration of efficiency, operability, etc. in purification.

The FA-KYM-containing serum-free medium obtained by culturing was mixed with Tris-HCl buffer containing sodium chloride (Tween 80
and sodium nitride (also added to all buffers below)). After loading, wash the column using the same buffer. Thereafter, gradient elution is performed using the same buffer and a buffer containing imidazole. FA-KYM activity is observed in the imidazole gradient portion. After collecting the active fractions, they are concentrated using polyethylene glycol and dialyzed against a phosphate buffer at 4° C. for 24 hours while exchanging the external solution.

PA-KYM activity is observed in the dialysate fluid. The dialyzed fluid thus obtained is loaded onto a concanavalin A Sepharose column sufficiently buffered with the same phosphate buffer.

After loading, wash the column with the same buffer. Subsequently, gradient elution is performed using a buffer solution containing potassium thiocyanate and α-methylmannoside. F
A-KYM activity is observed in the gradient elution portion of potassium thiocyanate and α-methylmannoside.

After collecting the active fractions, they were concentrated to an appropriate amount using polyethylene glycol, and then added to physiological saline containing Tween 80 and sodium nitride for 4 hours.
Dialysis is performed while exchanging the external dialysis fluid. After dialysis, a cloudy precipitate is formed in the dialysis solution, and this precipitate is collected by centrifugation. FA-KYM activity is mainly observed in the recovered precipitate.

This precipitate is dissolved in a small amount of phosphate buffer containing potassium thiocyanate and centrifuged to remove insoluble matter, thereby obtaining a supernatant containing PA-KYM activity.

The supernatant obtained here was loaded onto a Sephadex (G-200) column sufficiently buffered with a phosphate buffer containing potassium thiocyanate and developed with the same buffer, resulting in FA- An effluent fraction is obtained which contains KYM activity.

Next, the physicochemical properties of purified PA-KYM will be shown.

a, Product of human transverse strangulation myoma cell mutant KYM-A.

b1 Molecular weight: The molecular weight of this substance in non-reduced form measured by 5DS-polyacrylamide vir electrophoresis is approximately 56.00 CI to 62.
．． It has two bands close to each other between 000 and 000. In addition, the reduced form of this substance measured in a similar manner has a molecular weight of approximately 3
It has two bands at 2,000 and approximately 56,000.

C1 action and substrate specificity This substance is an enzyme protein that causes a fibrin dissolution reaction in the presence of plasminogen, and since this reaction requires the presence of plasminogen, it is not a typical plasminogen actin. It belongs to a group of substances called beta, and when compared to urokinase, it shows a much stronger binding ability to fibrin than urokinase.

In addition, K when using the synthetic substrate S-2444 as a substrate
m is 1.52 x 10-'M and Vmax is 0
,'046 IU/min-ml.

d, optimal- The optimum is about 8-10, and the working curve is shown in FIG.

e. Stable pH The stable pH is about 5-11, and the residual activity is shown in FIG.

Appropriate temperature range for f1 action The working temperature curve at 30-45°C is shown in FIG.

g. Temperature resistance When heated for 90 minutes, there is almost no deactivation up to 50°C, but at temperatures above 60°C, the residual activity decreases to 60% or less. The residual activity is shown in FIG.

h, Inhibition The residual activity of each inhibitor was examined at 0.1 mfi, 1 mM, and IDmM, and the results in Table 1 were obtained.

Table 1 Inhibition and activation were investigated using the activity of the sample without the addition of drugs as 100.

1. Amino acid composition This substance, which is an enzyme protein, was hydrolyzed with 6N hydrochloric acid and subjected to amino acid analysis, and the results shown in Table 2 were obtained.

Amino acid composition is shown in terms of total number of amino acid residues.

Table 20 Cystine and tryptophan were not quantified.

The ultraviolet absorption spectrum of an aqueous solution of this substance is as shown in FIG.

k, solubility in solvents Solubility in salt solutions such as water and phosphate buffer is approximately 5
When preparing solutions with concentrations above 0 μg/d, the presence of a solubility promoter, such as 1.6 M potassium thiocyanate, is required.

It is insoluble in organic solvents such as ethanol and ether.

! , properties of substances The freeze-dried specimen is a white powder.

m. Color reaction When PAS reaction was performed after 5DS-polyacrylamide gel electrophoresis, the product showed a pink color characteristic of glycoproteins, indicating that it was a glycoprotein. Furthermore, the fact that this substance showed affinity for concanavalin A-agarose resin suggested that it was a glycoprotein.

n6 isoelectric point When this substance was analyzed by chromatofocusing method in the presence of 8M urea, the main component, isoelectric point pl (Z5 to 8.
0, and the isoelectric pH of the subcomponent was 7.0 to Z5, indicating that it was a mixture of weakly basic proteins.

Next, we will show a method for measuring the activity of FA-KYM.
The method for measuring the activity of A is the same.

Medium Fibrin plate method (according to the method of Astrup et al.) (Arch, Biochem, Bioph
ys.

40 346-451 (1952)) 67 in an agarose suspension containing fibrinogen and plasminogen.
A fibrin plate containing plasminogen is prepared by reacting with thrombin at .degree. The fibrin plate has three diameter holes for adding samples for activity measurement. A sample volume of 5-10 μl is added to this sample hole, the added fibrin plate is placed in an incubator button at approximately 67°C, and after a certain period of time, the sample is removed and the diameter of the distinct fibrin lysis zone is measured. The activity in the fibrinolytic zone is reflected in the diameter of the fibrinolytic zone, so the activity can be determined by comparing the diameters.

(11) FA-KYM activity measurement method that utilizes a two-step reaction using a synthetic substrate (8-2251).

50mM) containing 0.15M sodium chloride
Plasminogen dissolved in hydrochloric acid buffer (pH 7,4) and FA-KYM solution [50 mM containing 0,15M sodium chloride] Lis-hydrochloric acid buffer solution (pH 14)]
and held at 57°C for 10 minutes (first stage reaction). After holding for exactly 10 minutes, immediately add 0.15M 1l of hydrochloric acid buffer containing 0.33M lysine solution (0.15M sodium chloride, Stop the first stage reaction.

Next, using a synthetic substrate solution (5 my/ml of synthetic substrate 8-2251 dissolved in a 50 mM Lis-HCl buffer containing 0.15 M sodium chloride (p) 47.4), the mixture was incubated at 67°C for 60 minutes. The content of plasmin produced in the first stage reaction is determined colorimetrically (wavelength 405 nm).
step reaction). The second stage reaction is stopped by immediately adding a 2.5M acetic acid solution after holding it for exactly 30 minutes. FA-KYM activity at 405 nm
It is measured by the absorption of

(iiD FA-KYM activity measurement method using a one-step reaction using a synthetic substrate (S-2444). 50 mM Tris-HCl buffer containing 0.15 M sodium chloride (
PA-KYM solution [50 mM phosphate buffer containing 1,6N potassium thiocyanate (pHs, s)]
pH 7.5)), synthetic substrate solution [synthetic substrate S-244
A solution of 4 dissolved at 3 mp/mA' in 50mM Lis-HCl buffer (pH 8,8) containing 0.15M sodium chloride] was added and kept at 37°C for exactly 90 minutes.
．． The reaction is stopped with a 5M acetic acid solution, and PA-KYM activity is determined colorimetrically (wavelength: 405 nm).

The PA-KYM of the present invention can be effectively used to dissolve thrombi generated in the blood.

The 2-KYM of the present invention is preferably in a dosage form that can be administered intravenously. Additives for the composition include stabilizers such as mannitol, albumin, gelatin, sodium hydrogen sulfite, etc.; regulators such as sodium hydroxide, sodium phosphate; tonicity agents such as sodium chloride, mannitol, grape lint, etc. etc. can be mentioned.

The dosage and administration method for clinical use of FA-KYM of the present invention are as follows: IDO-100,000 IU of this product is prepared as a solution containing the above additives, and the dose and method are adjusted as appropriate according to age, body weight, symptoms, progress, etc. It is used by intravenous injection, intravenous drip injection, intravenous drip injection, subconjunctival or bulbar injection.

Next, examples of the present invention will be shown.

Example 1. Culture For culturing the mutant strain KYM-A, add 2.2 ml of sodium bicarbonate per 11 ml of medium. Heat-inactivated beef tallow serum (KC Biological, USA) containing 1.192 y of C1,N-2-hydroxyethylpyhalazine-N'-2-ethanesulfonic acid and 60 μl of kanamycin sulfate was added to a final concentration of 10 % plus 9 RPMI-1640 medium (Flow Laboratories, USA) or 2 parts sodium bicarbonate per 11 parts of the medium.
．． MEM medium (Flow Laboratory, USA) containing 0g of Kanamai sulfate and 7760 da of Kanamai sulfate and heat-inactivated beef tallow serum added to a final concentration of 10%.The above two types of media were mixed in a 1:1 ratio. use something

KYM-A cells were inoculated into a plastic petri dish,
When a cell suspension of about 100 cells was obtained as a result of proliferation,
Start floating agitation culture using a spinner flask. By progressively increasing the size of the spinner flask, floating agitation culture of 81 is possible. The initial seeding density for each stage was 104 to 105 cells/-, with 5X10
When the number of cells reaches 2 x 106 cells/go, grafting is performed.

Example 2. When the harvested cell density reaches 5 x 1011 to 2.10' cells/ml, change to serum-free medium. The medium is 2.0 g of sodium bicarbonate per 11, and N-hydroxyethylpi is 1.192.9 g of lazine-N'-2-ethanesulfonic acid.
Namycin sulfate 60m9, insulin (3,5II?
y, 1 d of human transferrin (manufactured by Sigma), 4.6 Ing of ethanolamine, and 16 μg of arsenic selenate;
This is a solid PMI-1640 medium containing 20 KIU/ml of aprotinin (Sigma). Suspension agitation culture was carried out in this serum-free medium for 1 to 4 days, and this culture was carried out for 4 to 10 days.
The filtration is repeated several times, and the obtained recovered liquid is subjected to purification.

Example 6. The recovered solutions obtained in Purification Example 2 were combined to contain aprotinin, Tween 80, and sodium nitride, each with a final concentration of 20 KIU/mA! , 0.01 and 0
．． Add so that it becomes 0.2%. (These six chemicals are added to all buffer solutions below at these concentrations.) This sample was purified by the following method.

fal Porath et al.'s method (Nature, Uo
Zinc chelate agarose column (9cInφX 21 (
m) with 20 mM Tris-HCl buffer (pHZ05) containing 1M NaCl, culture solution 5
Load Dl.

After loading, add 3000ml of the same buffer! Rinse and wash the column. No FA-KYM activity was observed in the culture p fluid that passed through and the column washing solution.

Subsequently, gradient elution is performed using 1500m of the same buffer and a buffer containing 150mM imiguzol (pH 7,3'), and fractionation is carried out at a ratio of 17 textures 15a. When the FA-KYM activity was measured for each of the 72 fractions, the FA-KYM activity was remarkable mainly in fraction number 125. This active fraction is collected in a dialysis tube, sprinkled with polyethylene glycol 20,000 powder and concentrated at 4°C. After concentration, 0.01M phosphate buffer (
Dialysis is performed against pH 6, 7) at 4° C. for 24 hours while exchanging the external solution. After dialysis, the solution in the tube is centrifuged (
10,00 Orpm, 10 m1n) to obtain a supernatant.

fbl 130 mA of the solution containing Pt-KYM thus obtained! in advance with 0.01M phosphate buffer (pHH
Concanavalin A Sepharose column (I X 2 Qc++t) well buffered with I3 (Pharmaci
a Fine (manufactured by Chemicals). After loading, wash with 100 ml of the same buffer. Elution is then carried out with a direct gradient formed from the same buffer (150d) and a buffer containing 0.6M potassium thionanate and 5M α-methylmannoside (150+++l). Fractions were collected in 6.5d increments, fraction number 32.
FA-KYM activity was observed mainly in No.

After collecting both parts, polyethylene glycol 20,
000 to 5d, and dialyzed against physiological saline at 40° C. for 24 hours while exchanging the external dialysis fluid.

During dialysis, the enzyme solution became cloudy and was centrifuged (15,00 Or
pm, 50 minutes), FA-KYM activity is recovered in the precipitate fraction. At this stage, in the Merlot method, two people - K
It was possible to remove mixed proteins, mainly proteins recognized as YM polymers. The precipitate obtained by centrifugation was diluted with 2d of 1.6N potassium thiocyanate.
Dissolve in 01M heptacid buffer (p) 16.7). After removing residues that cannot be completely dissolved by centrifugation, P
A supernatant containing A-KYM activity is obtained.

The resulting solution was diluted with 1.6N potassium thiocyanate. Sephadex G-200 (Pharmac) sufficiently buffered with OIMIJ acid buffer (PH6,7)
ia Fine Chemicals) column (1
, 6 x 85 CIIL) and developed with the same buffer.

When 2.4 d of fractions were collected, PA-KYM activity was observed mainly in fraction number 35.

As mentioned above, PA is processed using a four-step method including the fractional precipitation method.
A purified sample of -KYM was obtained, and the degree of purification at each stage is shown in Table 6.

■ Protein concentration was determined by the Lowry method and expressed in μg/d.

(2) A color reaction using synthetic substrate S-2444 is carried out at 67 DEG C. for 90 minutes using an appropriate volume of 11 (aIIL1) enzyme solution. Color development degree OD'° at 405 nm in that case
6-b, the displayed enzyme concentration C is expressed as c=bx1.

■ Specific activity is ■/■.

(2) The degree of purification indicates the increase in specific activity at each stage, assuming that the condition of the medium is 1.0.

Example 40 Molecular Weight and Purity Regarding the purified sample obtained in Example 6, the molecular weight was determined and the purity was verified at the same time.

First, a non-reduced preparation was prepared using the Laemmli method (Nat
Electrophoresis on SD8 polyacrylamide containing 10% acrylamide was carried out at room temperature under a constant current of 5 m for 18 hours according to URE VOI 227 680 (1970). The gel thus obtained was heated to 2.5 t
Shake for 1 hour in riton X-100 aqueous solution, and
Remove n2. This was applied to an agarose plate containing fibrin and plasminogen (Granelli
Piperno and Re1ch, J.

Exp, Med vol 148 225-234.
(1978)) and maintained at 67°C for 1 to 5 hours and observed, the estimated molecular weight was 5.
Two dissolution zones close to each other were observed in the vicinity of 6.000 to 62,000, and no dissolution zones were observed in other locations. On the other hand, when plasminogen was not added to the agarose gel using a similar analytical method in which fibrin was layered on a plate, no dissolution zone could be observed, indicating that fibrin dissolution is dependent on plasminogen. did. Furthermore, when 20 μg/Tnl of the IgG fraction of anti-urokinase antiserum was added to the fibrin plate, the lysis zone was comparable to that of the fibrin plate without IgG. Therefore, the molecular weight is 56.
The two bands present in the vicinity of 000 to 62,000 were shown to belong to TPA-type PA rather than urokinase-type plasminodan activator.

Purified sample (g) of FA-KYM obtained in Example 3 0.
1 μg) was separated by 5D8-polyacrylamide gel electrophoresis in the same manner as above, either unreduced or reduced by addition of 5% 2-mercaptoethanol. The gel thus obtained was processed using the method of Qakly et al. (Analytical Biochem Vol.
105 561-365 (1980)), the contained proteins were stained.

From the results obtained in this way, the non-reduced purified sample shows two bands around the molecular weight of 56,000 to 62,000, and the reduced sample shows two bands with a molecular weight of about 32,000.
Two bands were seen at 0 and about 66,000. Furthermore, since no other notable bands were observed other than these bands, it was shown that the purified sample was purified in an almost pure Ki state.

The standard proteins with known molecular weights used in the molecule-i measurement were 7-osphorylase b (94,000), bovine serum albumin (67,000), and ovalbumin (43,000).
) and carbonic acid ambatrase (30,000).

Example 5. Examination of affinity for fibrinCNBr
-activated 8epharose 4B (
Pharmacia) 10g to 1mM HCA2
After swelling with 1 and washing, add 1 g of fibrinogen (Kabi)
0,5 M NaClj/a I M NaHC
Coupling buffer of Os (pH, 3) 200
d for about 10 hours at room temperature to allow adsorption. Next, this resin was mixed with a 0.05M phosphate buffer solution (pi-17,5) containing thrombin (2 units, bovine thrombin, Kawasaki Pharmaceutical Co., Ltd.) for 10 minutes.
Place the fibrin in a vacuum cleaner and keep it warm at 37°C for 10 minutes.
-5 epharose resin was obtained. The resin thus obtained was packed into a minicolumn (6+117)0.
Equilibration was performed with 05M phosphate buffer (containing 0.01% Tween 80, pil 7.5).

The column was loaded with 111 Ll of PA-KYM solution or urokinase solution. First, 0.05M! The fraction was washed with 15+A' of 7-acid buffer (pH 7,5), and the fraction was divided into 1
It was collected every d. It was then developed with the same phosphate buffer 251R1 containing 1.6M potassium thiocyanate, and
It was separated into fraction d. The enzyme activity contained in the washing solution and the potassium thiocyanate solution was measured using synthetic substrate S-2444 to determine the recovery rate.

As shown in Table 4, PA-KYM is not included in the cleaning solution;
Since potassium thiocyanate is required for its elution, it is evaluated to have a high affinity for fibrin.
Urokinase has poor affinity and its activity is recovered in the wash solution.

Table 4 [Stock] Appropriate volume of enzyme solution (usually 0.001 to 0.
Using 3d), a color reaction using the synthetic substrate S-2444 is carried out at 37°C for 90 minutes. 4 measured at that time
This value was obtained by calculating the degree of color development at 405 nm based on the absorbance at 05 nm, assuming that the entire enzyme solution was used.

For example, when a reaction is performed at 67°C for 90 minutes using 0.1 mJ of the loaded sample (1d), the color development is 0D4.
If 0'=0.1218, the displayed numerical value will be 1.0 0.1218 x o, i = 1-218.

[Brief explanation of the drawing]

Figure 1 is a diagram showing the action curve of each time of PA-KYM, Figure 2 is a diagram showing the residual activity of each -, Figure 6 is a diagram showing the action temperature curve, and Figure 4 is a diagram showing the action temperature curve at each temperature. FIG. 5 is a diagram showing the residual activity, and FIG. 5 is a diagram showing the ultraviolet absorption spectrum. Agent: Patent attorney 1) Parent: Procedural amendment April 26, 1985 Dear Commissioner of the Japan Patent Office 1, Indication of the case, Patent Application No. 013397, filed in 1982, 2, Name of the invention: Plasminogen activator KYM and its manufacture Law 6, Relationship with the amended person's case Patent applicant address: 2-3-6 Kyobashi, Chuo-ku, Tokyo Name: Meiji Dairies Co., Ltd. Representative: Tozo Shimamura, 44, agent address: 105 Minato-ku, Tokyo Toranomon-chome 19-14 No. 5, Number of inventions increased by amendment None 6, Specification subject to amendment Z Contents of amendment (1) The scope of claims will be amended as a separate sheet. (2) In line 6 from page 8 F of the specification, the phrase “lateral strangulation muscle” should be replaced with “
"Lateral constriction muscle" is corrected. +3) In the 6th line from the bottom of page 9 of the 8JJ specifications, the text "Lateral strangular myoma" has been corrected to "Lateral strangular myoma." (4) In the second line of page 10 of the specification, the phrase "lateral strangulation myoma" should be corrected to "transverse strangulation myoma." (5) On page 10, line 7 of the specification, it says "lateral strangulation fibroid." Corrected to "lateral strangulation myoma." (6) On page 17 of the 8A specification, line 4, it says "lateral strangulation fibroid." Corrected to "lateral strangulation myoma." 2. Claims (1) Plasminogen activator KYM0 a, which has the following physical and chemical properties, is a product of the human lateral strangulosarcoma cell mutant KYM-A. b9 Molecular weight 5D8-polyacrylamide gel electricity The molecular weight of the non-reduced substance measured by electrophoresis is approximately 56, and the ODD is 62.
It has two bands close to each other between 000 and 000. In addition, the reduced form of this substance measured in a similar manner was found to be about 5 per molecule.
It has two bands at 2.000 and approximately 36.000. CO action and substrate specificity This substance is an enzyme protein that causes a fibrin dissolution reaction in the presence of plasminogen, and since this reaction requires the presence of zolasminogen, it is a typical plasminogen activator. It belongs to a group of substances called
When compared to urokinase, urokinase exhibits a much stronger binding ability to fibrin. In addition, when synthetic substrate 8-2444 was used as a substrate, h
is 1.52x10-”M, sb, Vmax is 0,046
IU/m1n-111/. d, Optimum - Optimum - is about 8-10 and the action curve is shown in FIG. e, Stable - Stable - is about 5-11, and the remaining activity is shown in Figure 2. The action temperature curve in the f2 action temperature range of 50 to 45°C is shown in FIG. g. Temperature Resistance After heat treatment for 90 minutes, it hardly loses its activity up to 50°C, but the residual activity becomes 60% or less at temperatures above 60°C. The residual activity is shown in FIG. h, inhibition of each inhibitor 0.1 rr? Ji, 1 rrM and 10mM
The residual activity was investigated and the results shown in Table 1 were obtained. Table 1 Inhibition and activation were investigated with the activity of the sample without any drug added as 100%. i. Amino acid composition This substance, which is an enzyme protein, was hydrolyzed with 6N hydrochloric acid and subjected to amino acid analysis, and the results shown in Table 1 and 2 were obtained. The amino acid composition is indicated by - relative to the total number of amino acid residues. Table 2 ◎ Cystine and tryptophan were not quantified. j. Ultraviolet absorption spectrum The ultraviolet absorption spectrum of an aqueous solution of this substance is as shown in FIG. k, solubility in solvents Solubility in salt solutions such as water and phosphate buffer is approximately 5
At 0 μg/ml, the preparation of solutions with higher concentrations requires the presence of a solubilizer, such as 1.6 M potassium thiocyanate. It is insoluble in organic solvents such as ethanol and ether. 1. Properties of the substance The freeze-dried specimen is a white powder. m. Color reaction After performing 5DS-polyacrylamide gel electrophoresis, l) A, S reaction was performed to show the pink coloration characteristic of glycoproteins, indicating that it was a glycoprotein. Furthermore, the fact that this substance showed affinity for concanavalin octa-agarose resin suggested that it was a glycoprotein. 10 Isoelectric Point When this substance was analyzed by chromatofocusing method in the presence of 8M urea, the isoelectric point of the main component ranged from pH 7.5 to 8.
It was shown that it was a mixture of weakly basic proteins at pH 7.0 to 7.5. (2) Cultivating human lateral strangulation cell carcinoma cell mutant KYM-A,
Plasminogen activator K was extracted from the obtained culture solution.
A method for producing plasminogen activator KYM, which comprises collecting YM. (3) A pharmaceutical composition containing an effective amount of plasminogen activator KYM and having thrombolytic action. (4) Zolazminogen activator KYM according to claim 2, wherein the culture is floating agitation culture or adhesive culture.
manufacturing method. ”

Claims (4)

[Claims] (1) Plasminogen activator KYM0a, which has the following physicochemical properties, is a product of human strangulated myoma cell mutant KYM-A. b8 Molecular Weight The non-reduced substance has a molecular weight of about 56,000 to 62. O
It has two bands close to each other between DD. Also,
The reduced form of this substance measured in a similar manner has a molecular weight of approximately 32
,000 and two bands at approximately 36,000. C1 action and substrate specificity This substance is a binding protein that induces a fibrin dissolution reaction in the presence of zolasminogen, and since this reaction requires the presence of plasminogen, it is not a typical plasminokin activator. Belongs to a group of substances called beta,
When compared to urokinase, urokinase exhibits a much stronger binding ability to fibrin. In addition, when synthetic substrate S-2444 is used as a substrate, the fat is 1.52X10"-"M, and Vmax is 0,0
46 IU 7ml/nlL/. d. Optimum pH Optimum -1 is about 8-10 and the action curve is shown in FIG. e, stable pH Stable- is about 5-11, and the residual activity is shown in FIG. The operating temperature curve in the range of 30 to 45° C., which is the optimum temperature for 10 operations, is shown in FIG. g. Temperature resistance When heated for 90 minutes, tl hardly deactivates up to 50°C, but at temperatures above 60°C, the residual activity becomes 60tl or less. The residual activity is shown in FIG. h, Inhibition The residual activity of each inhibitor was examined at 0.1 mM, 1 mM, and 10 mM, and the results shown in Table 1 were obtained. Table 1 Inhibition and activation were investigated using the activity of the sample without the addition of drugs as 100. i. Amino acid composition When this substance, which is an enzyme protein, was hydrolyzed with 6N hydrochloric acid and subjected to amino acid analysis, the results shown in Table 2 were obtained. Amino acid composition is shown in terms of total number of amino acid residues. Table 2 ◎ Cystine and tricytophan were not quantified. The ultraviolet absorption spectrum of an aqueous solution of this substance is shown in FIG. k, solubility in solvents Solubility in salt solutions such as water and phosphate buffer is approximately 5
When preparing solutions with higher concentrations than 0 μI/1 l of Ti, the presence of a solubility promoter, such as 1.6 M potassium thiocyanate, is required. It is insoluble in organic solvents such as ethanol and ether. 16 Properties of the substance The lyophilized specimen is a white powder. m. Color reaction 8 After performing DS-polyacrylamide gel electrophoresis, a PAS reaction was performed, and a pink coloration characteristic of glycoproteins was exhibited, indicating that it was a glycoprotein. Furthermore, the fact that this substance showed affinity for concanavalin A-agarose resin suggested that it was a glycoprotein. When this substance was analyzed by chromatography in the presence of 8M urea, the isoelectric point of the main component was Z5 to 8.0, and the isoelectric point of the subcomponent was 7, 0 to Z5,
It was shown to be a mixture of weakly basic protein threads. (2) Culture human lateral strangulation fibroid cell mutant KYM-A, and extract plasminogen activator KY from the resulting culture solution.
A method for producing plasminogen activator KYM, which comprises collecting M. (3) A pharmaceutical composition containing an effective amount of plasmaminogen activator KYM and having a thrombolytic effect. (4) The plasminogen activator KYM according to claim 2, wherein the culture is floating agitation culture or adhesive culture.
manufacturing method.