CA1275062A - Plasminogen activator kym - Google Patents

Abstract

Abstract of the Disclosure:
A plasminogen activator PA-KYM available as a thrombolytic drug is prepared from a suspension culture medium of human rhabdomyosarcoma cells KYM-A. The PA-KYM
is a plasminogen activator of the tissue plasminogen activator type, which has been never prepared from human rhabdomyosarcoma cells hitherto.

Description

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P~ASMINOGEN ACTIVATO~ ~YM
Field of the Invention:
This invention relates to plasminogen activator KYM which activates plasminogen, a process for the preparation thereof and a pharmaceutica:L composition having a thrombolytic activity.
Background of the Invention:
It has been a serious problem that thrombi would result in various diseases. Conventionai therapeutical agents therefor include streptokinase and urokinase.
However it has been known that either of these enzymes would not only effec-t the aimed thrombolysis but decompose clotting factors in circula-~ng blood to thereby bring about hemDrrhage since these enzymes have poor affinities for the thrombi to be lysed and are li~ble to decompose clotting factors in circulating blood. m erefore it has been an urgent problem to develop a thrombolytic , drug which has a strong affinity for thrombi with little side effects including hemorrhage~
Under these circumstances, there have been many studies and reports on plasminogen activators (which will be referred to as PA hereinaftèrj capable of lysing thrombi with little side effects. With reference to PA originating from human tissues, detailed studies on enzymes obtained ~rom normal human tissues :~2~ 6~

(i.e. endothelial or uterine cells), human tumors or cultured cells thereof have been reported (cf. Wilson et al, Cancer Res., 40, 933 - 938 (1980~ and Rijken and Collen J. Biol. Chem., 256, 7035 - 7041 (1981)).
It has been known that normal human tissue cells such as endothelial or uterine cell or tumorous cells such as human melanoma or mammary carcinoma cells would produce PA.
However normal tissue cells are inavailable for the industrial production of PA since they have finite life span.
On the other hand, human tumorous cells such as melanoma or mammary carcinoma cells have bean conventionally cultured in an anchorage dependent manner with the use of bottles or similar containers, which results in a poor productivity of PA and is also inavailable in an industrial scale.
It is generally believed that human PA may be ~ 20 classified into two types, i.e. the urokinase type having ; a poor affinity for fibrin and the tissue plasminogen activator (TPA) type having an excellent affinity for fibrin.
It has been well known that melanoma cells would produca PA of the TP~ type. Melanoma cells have s~

attracted a good deal of attention ~ince they would mainly produce PA of the I'PR type only. On the other hand, no other tumorous cells except some mammary carcinoma cells have been reported to produce PA of the TPA type only. Rhabdomyosarcoma cells have been hitherto thought to produce only PA of the urokinase type.
Summary of the Invention:
Under these circumstances, the present inventors tried to find out tumorous cells which would grow by suspended spinner culture and succeeded in isolating a variant which would grow in suspension from the KYM-1 cell cultures which had been established from human rhabdomyosarcoma cells (presented by Dr. Morimasa Sekiguchi of Institute of ~edicine, the University of Tokyo). This variant was named variant KYM-A. This variant KYM-A can be subcultured by suspended spinner culture. Though the deposition thereof was not accepted by the Fermentation Research Institute, Agency of Industrial Science and Technology in Ibaraki-ken, Japan, it has now been deposited with the Institute for Fermentation (foundation), in Osaka, Japan, under IFO
50030.

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Subsequent studies have revealed that the variant KYM-A might produce and accumulate a remarkable amount of PA in a medium. The PA thus obtained was named PA-K~fM.
No PA of the urokinase type was detected in the medium of the variant KYM-A.
Accordingly the PA-KYM of the present invention is a PA of a novel origin (i.e. human xhabdomyosarcoma cells).
It is further accepted as a novel PA of the TPA type because of its physicochemical properties.
Brief Description of the Drawings:
Fig. 1 is a graph of the activity curve of the PA-KYM at each pH value, Fig. 2 is a graph of the residual ; activity of the PA at each pH value, Fig. 3 is a graph of the activity curve at different temperatures, Fig. 4 is a graph of the residual activity of the PA after incubatlon at each temperature and Fig. 5 is an ultraviolet spectrum thereof.
Detailed Description of the Invention:
The properties of the variant K~M-A are as folIows.
1. Each cell is spherical in shape and highly refractile.

2. The cells, respect1vely, may be present as a single cell. Alternatively they may form a chain or ':

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spherical cluster together. A cluster contains approximately two to 100 cells.

3. When cultured in a plastic Petri dish or subjected to spinner culture in a tank, this variant may yrow mostly in the form of suspended cells.

4. This variant may be subcultured by suspended spinner culture.

5. This variant may be converted into anchorage dependent cells by, e.g., the use of a spent culture.
The converted cells are morphologically similar to epithelial cells.
A spent culture medium may ba a supernatant of cultures other than that of this variant which is prepared by culturing appropriate cells in an appropriate basal medium for one to 100 hours and removing the cells and cell debris from the medium. Examples of the available cells other than this variant are human hepatoma cells such as HuH6-Ce5 and HuH7 strains (cf.
Nakabayashi et al., Cancer Research, 42, 3858 - 3863 2U t1~82)).
Besides using the spent culture medium, this variant may be converted into anchorage-dependent cells by the use of a medium containing fibronectin. Alternatively substratum attached culture of this variant is also possible by treating the sur~ace of a Petri dish or 75~

similar containers with proteins such as collagen, gelatin, poly L-lysine or egg lysozyme.

6. Subcutaneous transplantation of 106 cells to a nude mouse or transplantation thereof to a cheek pouch of an ALS administered hamster would result in tumorigenesis.

7. Chromosome According to the conventional cytogenetic method, the most frequent chromosome number of this vaxiant are 46 and 47 with some distribution in the neighborhood thereof. This fact suggests that the chromosome number of the variant KYM-A is relatively close to that of the normal human diploid (i.e. 46) in spite oE its tumor cell origin.

8. This variant produces a remarkable amount of the PA-KYM.
In order to produce the PA-KYM of the present invention, the variant may be subjected either to substratum attached culture or suspension culture.
In order to produce the PA-KYM, the variant KYM-A
may be allowed to adhere to the surface of plastic or glass Petri dishes, roller bottles or microcarrier beads.
However suspension culture in spinner flasks is more suitable from the viewpoint of industrial productionO

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In order to produce the PA-KYM, it is preferable to culture the variant KYM-A in a medium containing serum and replace it with a medium containing no s~rum at the initiation of a stationary phase of the growth of the cells. Preferable serum media are those containing 10%
of bovine fetal serum to RPMIb, ~EM* or Fl2~ ~products of Flow Laboratories) or a mixture thereof.
Pre~erable non-serum media are those prepared by adding insulin, human transferrin, monoethanolamine and selenous acid to a basic medium such as RPMI, MEM, F12 or a mixture thereof. A medium containing the above-mentioned four components will be referred to as ITES-containing medium hereinafter.
In order to prod-~ca the PA-KYM by the variant KYM-A, the cell strain is subjected to suspension culture in a plastic Petri dish until an appropriate number of cells are obtained. Then it was subjected to culture in a spinner flask of 100 to 8000 m~ in volume. It is preferable to inoculate the cells at the density of 104 to ~0 105 cells/m~. A stationary state is reached at the density of 5 x 105 to 2 x 1o6 cells/me. The culture temperature may be from approximataly 30 to 40 C, pre~erably approximately 37C. The culture may be * Trade Mark z carried out in an atmosphere of 100% air, air containing 5 to 10% of carbon dioxide gas or air containing 5 to 100% of oxygen.
The culture may be carried out in a batchwise operation. Alternately, when the cells grow sufficiently, the media may be exchanged at an interval of one to four days to thereby obtain a medium containing the P~-KYM continuously for approximately one month after the initiation of the culture.
Since the PA-KYM of the variant KYM-A is an enzyme which activates plasminogen into plasmin and belongs to protein, it may be purified by any method generally used in purifying protein such as salting-out, affinity chromatography, ion-exchange chromatography, application ~ of molecular sieve, or combinations thereof. The purification may be carried out either continuously or in a batchwise operation depending on the efficiency and convenience.
The serum-free medium containing the PA-KYM is then loaded on a zinc-chelate agarose column which has been sufficiently e~uilibrated with a tris hydrochloride buffer solution containing sodium chloride~ This buffer solution also contains Tween 80 and sodium azide. These compounds will be added to all buffer solutions hereina~ter. After the completion of loading, the column ~;, ~L2~

is washed with the same buffer solution and subjected to gradient elution with the foregoing buffer solution and another buffer solution containing imidazole. The imidazole gradient fractions exhibit a PA KYM activity.
The active fractions are combined, concentrated with polyethylene glycol and dialyzed againlst a phosphate buffer solution at 4 C for 24 hours with several changes of the external solutionO
The dialysate exhibiting a PA-KYM activity is loaded on a concanavalin A Sepharose column which is sufficiently equilibrated with the same phosphate buffer solution as described above. After the completion of loading, the column is washed with the same buffer solution and subjected to gradient elution with a buffer solution containing potassium thiocyanate and ~-methyl-mannoside. The gradient fractions of potassium thiocyanate and ~-methylmannoside exhibit a PA-KYM
activity. The active fractions are co~bined, concentrated to an appropriate volume with polyethylene glycol and dialyzed against a physiological saline solution containing Tween 80~ and azide nitride at 4 C for 24 hours with several changes of the external solution.
After the completion of the dialysis, a white precipitate * Trade Mark ~s~

in the dialysate is recovered by centrifugation. ~ PA-KYM activity is observed mainly in the recovered precipitate. The precipitate is dissolved in a small amount of a phosphate buffer solution containing potassium thiocyanate and centrifuged, thus removing insoluble matters and giving a supernatant which exhibits a PA~KYM activity.
The supernatant thus obtained is loaded on a Sephadex~ (G-200) column suficiently washed with a phosphate buffer solution containing potassium thiocyanate and developed with the same buffer solution as described above to give a fraction which exhibits a P~-KYM activity.
The physicochemical properties of tbe PA-KYM thus purified are as follows.
a. The PA-KYM is a product of a variant ~YM-A
originating from human rhabdomyosarcoma cells.
b~ Molecular weight According to SDS-polyacrylamide gel electrophoresis, unreduced PA-KYM shows two closely migrating bands in a ; molecular weight range of approximately 56,000 to 62,000.
On the other hand, reduced PA-KYM shows two bands at approximately 32,000 and approximately 36,000 when measured in the same manner as described above.
c. Function and substrate speci~icity * Trade Mark ~r, ~t~'7 ~ ~ 2 The PA-XYM is an enzyme protein which causes fibrinolysis in the presence ctf plasminogen. Since plasminogen is essential in this reaction, this enzyme protein is one of typical plasminogen activators. The PA KYM is shown to have a ~uch higher affinity for fibrin than that of urokinase.
d. Optimum pH value The optimum pH value thereof is approximately 8 to 10. Fig. 1 shows an activity curve thereof.
e. Stable pH value The stable pH value thereof is approximately 5 to 11. FigO 2 shows the residual activity (%3 thereof.
f. Range of optimum temperature for function Fig. 3 shows the enzyme activity at various temperatures. The optimum temperature for function is between 30 and ~5 C.
g. Thermoresistance The PA-KYM lS hardly inactivated by heating to ~tO C
for 90 min. Its residual activity at 60 C or above is not higher than 60%. Fig. 4 shows the residual activity (%~ thereof.
h. Inhibition Table 1 shows the residual activities thereof when ~,~tjl - 12 - ~Z~5~6z exposed to each inhibitor in a concentration of 0.1 mM, 1 mM and 10 mM.
Table 1 0.1 ~q 1 mM 10 mM
__ __ CaC~2 94.9 104.4 85.
EDTA - 112.9 FeSOL~ - 103.3 KC~ - 99.8 MgC~2 g5-5 90.7 89.0 AgN03 87.2 79.6 13.6 CuS04 89.2 7Q.6 9.9 HgC~2 115.8 45.6 o MnC~2 95 5 26.6 5.1 ZnS04 34.3 15.6 0 An examination on the inhibition of the PA~-KYM is per~ormed by assigning a value of 100 % to the activity of the enzyme in the absence of any inhibltor.
i. Amino acid composition The PA-K~q, which is an enzyme protein, is hydroly-zed with 6 N hydrochloric acid and subJected to amino acid analysis. Table 2 show~ the result wherein each amino acid composition is shown in % based on the total number of amino acid residues.

- 13 _ ~7~2 Table 2 PA-KYM _ ASP 10.6 THR 5.3 GLU lO.9 PRO 6.3 GLY 9.5 ALA 7.2 VAL 5,o MET 1.0 ILE 3.5 LEU 8.1 TYR 4.5 I5 PHE 3.5 ARG 7,0 * Cysteine a~d tryptophan were not determined.

jO Ul-traviolet spectrum Fig. 5 shows an ultraviolet spectrum o~ an aqueous solution of the PA-KYM.
k. Solubility in solvents The PA-KYM is soluble in water or in salt solutions such as a phosphate buffer solution at a concentration up to approximately 50 ~g/m2. A solution of higher concentrations may be prepared in the presence o~ such chemicals as 1.6 M of potassium thiocyanate.
It is insoluble in organic solvents such as ethanol or ether.
1. Form The PA-KYM is in the form of a white powder when lyophilized.
m. Color reaction After suh~ected to SDS-polyacrylamide gel electrophoresis and to PAS reaction successively, the PA-KYM turns to pink characteristic of glycoproteins. Its ; affinity for a concanavalin A agarose resin further suggests that it is a glycoprotein.
n. Isoelectric point Chromatofocusing analysis of the PA-KYM in the presence of ~ M urea has revealed that the isoelectric pH
of its main component is 7.5 to 8.0 while that of the minor component is 7.0 to 7.5, suggesting that it is a mixture of weakly basic proteins.
Methods for determining the PA-KYM activity, which are similar to those conventionally employed for determining the PA activity, are as follows.
(i) Fibrin plate method (cf. Astlap et al~, Arch.

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Biochem~ Biophys., 40, 346 ~ 351 (1952)).
Thrombin is reacted in an agarose suspension containing fibrinogen and plasminogen at 37C to thereby prepare a fibrin plate containing plasminogen. A hole of 3 mm in diameter into which a sample is introduced is bored through the fibrin plate. Five to 10 ~B of the sample is introduced into the hole and the fibrin plate is placed in an incubator for a certain period.
Then it is taken out of the incubator and the diameter of -the distinct fibrinolysis zone is measured. Since the activity of the sample is correlated to the diameter of the fibrinolysis zone, the diameters of the zones are compared to determine the activity.
(ii) Method for determining the PA-KYM activity by a two stages reaction with the use of a synthetic substrate (S-2251).
Plasminogen dissolved in a 50 mM tris hydrochloride buffer solution (pH 7.4) containing 0.15 M of sodium chloride and a solution of the PA-KYM in a 50 mM tris hydrochloride buffer solution (pH 7.4) containing 0,15 M of sodium chloride are maintained at 37C for accura-tely 10 min (the first reaction). Then a 0.33 M
lysine solution in a 0.15 M tris hydrochloride buffer solution containing 0.15 M sodium chloride is immedia-tely added to stop the first reaction.

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Subsequently a solution of a synthetic substrate prepared by dissolving 3 mg/m~ of a syn~hetic substrate S-2251 in a 50 ~M tris hydrochloride buffer solution (pH 7.4) containing 0.15 M sodium chloride is main-tained at 37C for 30 min and the plasmin formed bythe first reaction is colorimetrically determined at a wavelength of 405 nm ~the second reaction). After maintaining for accura-tely 30 min, a 2.5 M acetic acid solution is immediately added to thereby stop the second reaction. The PA-KYM activity is determined based on the absorption at 405 nm.
(iii) Method for determining the PA-KYMN activity by a one stage reaction with the use of a synthetic substrate (S-2444).
To a PA-KYM solution in a 50 mM phosphate buffer solu-tion (pH 8.8) containing 0.15 M sodium chloride, a solution of a synthetic substrate prepared by dissolving 3 mg/m~ of a synthetic substrate S-2444 ln a 50 ~M
tris hydrochlorlde buffer solution (pH 8.8) containing 0.15 M sodium chloride is added and the mixture is maintained at 37C for accurately 90 min~ men a 2.5 M
solution of acetic acid is added to the mixture to thereby stop the reaction. The PA-KXM activity is .. l7 ~7~a~2 colorimetrically de-termined at a wavelength of 405 nm.
m e PA-KYM of ~he present invention is effective in lysing thrombi formed in blood.
The P~-KYM of the present invention is preferably formulated into pharmaceutical compositions suitable for intravenous administration. These compositions may contain VariOUS additives including stabilizers such as mannitol, albumin, gelatin or sodium bisulfite;
pH regulators such as sodium hydroxide or sodîum phos-phate; and isotonizing agents such as sodium chloride, mannitol or glucose.
The PA-KYM of the presen-t invention may be clinically administered by venoclysis, intravenous drip, instilla-tion or subconjuctival or retrobulbar injection in the 1~ form of a solution containing 100 to 100,000 IU thereof depending on the age, body weight and condition of a patient.
To further illustrate the present invention, the follo~ing examples will be given.
Example 1: Culture In order to culture the variant KYM-A, IFO 50030.
a medium RPMI-1640. MEM or F12 (products of Flow Labora tories, USA) containing 2.0 g of sOdium bicarbonate, 1~192 g o~ ~-2-hydroxyethylpiperazine-N'-2-ethanesulfonic 2~ acid an~ 60 mg of kanamycin sulfate per liter of the - 18 ~ ~ æ

medium and further containing decomplemented bovine fetal serum (a product of KC Biological, USA) to a final concen-tration of 10 %, or a mixture thereof a-t various ratios may be used.
A plastic Petri dish was inoculated with the KYM-A
cells. When approximately 100 m~ of a cell suspension was obtained, suspension culture was initiated with the use of a spinner flask. Suspension culture in a volume of up to 8 Q is able to be performed by gradually scaling up the culture. The initial inoculation ratio in each stage was 104 to 105 cells/m~. When it reached 5 x 105 to 2 x 106 cells/m~, the cells were inoculated into another flask.
Example 2: ~ollection When the cell concentration reached 5 x 105 to 2 x 106 cells/m~, the medium was replaced with a serum free medium comprising a medium RPMI-1640, MEM, F12 or a mixture thereof containing 2.0 g of sodium bicarbonate, 1.192 g of N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, ~275(~

60 mg of kanamycin sulate, 8.5 mg of insulin, 1 mg of human transferrin (a product of Sigma), 4.6 mg of ethanolamine and 13 ~g of selenous acid per liter and further containing 20 KIU/me of Aprotinin~ (a product of Sigma). The strain was subjected to suspension culture in this serum free medium for one to four days. ~fter repeating it four to lo times, the meclium was filtered and the obtained filtrate was subjected to purification.
Example 3: Purification The filtrates recovered in Example 2 were combined and Aprotinin, Tween 80 and sodium azide were added thereto to give a final concentration of 20 KIU/mQ, 0.01%
and 0.02%, respectively. These three substances were added to all buffer solutions as described hereinafter.
The sample thus obtained was purified in the following mannerO
(a) 30 e of the filtrate was loaded on a zinc chelate-agarose column (9 cm 0 x 21 cm) which had been adjusted according to the method reported by Porath et al~ ~cf~ Nature, 258, 958 - 959 (1975)) and sufficiently washed with a 20 mM tris hydrochloride buffer solution (pH 7.05) containing 1 M NaC~. After the completion of the loading, the column was washed with 3000 me of ths same buffer solution as described above. The filtrate * Trade Mark and the washing passed through the colu~n exhibited no PA-KYM activity. Then the oolumn was sub~ec-ted to ~radlent elution with 1500 m~ of the same buffer solu-tion as described above and another buffer solution (pH 7.3) prepared by addi.ng 150 mM imidazole to the abovementioned buffer solution to give fractions of 15-m~ portians. PA-KYM activity determ:ination on each I fraction revealed that fractions in ~he neighborhood ¦ of fraction No. 125 exhibited a remarkable PA-KYM
activity. These acti~e fractions were combined, intro-duced into a dialysis tube, dusted with dry polyethylene glycol 20,000 powder and concentrated at 4OC.
Subsequently it was dialyzed against a 0.01 M phosphate buffer solution ~pH 6.7) at 4C for 24 hours with several changes of the external solution. After the completlon of the dialysis, the solution in the tube was centri-: fuged at 10,000 rpm for 10 min to give a supernatan-t.
(b) 130 m~ of the solution containing the PA-KYM
thu~ obtained was loadrYi on a concanavalin A Sepharose*
column (1 x 20 cm; a product of Pharmacia Fine Chemicals) which had been sufficiently washed with a 0.01 M
phosphate ~uf~er solution (pH 6.7). After the comple-tion of the loading, the column was washed with the same buffer solution as de~cribed above and subjected ~o gradient elution with 150 m~ of the abovementioned * Trad~3 Mark ,i'.,.".~

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buffer solution ~nd 150 m~ of another bu~fer solution prepared by adding 0,6 M sodium thiocyanate and 3 M
a-methylmannoside to the abo~e buffer solution ~o give frac-tions of 3.5~m~ portions. Fractions i~ the neighborhood of fraction NoO 32 exhibit;ed a PA-KYM
activity.
The active fractions were combin~l and concen~rated to a volume of 5 m4 with the use of polyethylene glycol 207000. The concentrate was dialyzed against a physio-logical saline solution at 40C for 24 hours withseveral changes of the external solution. During the dialysis, the enzyme solution became turbid. The dialysate was centrifuged at 15,000 rpm for 30 min, and the precip-tate exhibited a P~-KYM activi-ty. At this stage, protein contaminants were removed which mainly comprising proteins of higher molecular weights than PA-~X~
by gel ~iltration. The precipitate obtained by the centrifuga~ion was dissolved in 2 m~ of 0~01 M
phosphate buffer olution (pH 6.7) containing 1.6 N
potassium ~hiocyanate~ Insoluble residue was removed by centrifugatlon to give a supernatant showing a PA-KYM ac~ vity. The solution thus obtained was loaded on a S~phadex G 200 *(a product of Pharmacia ~i~e Chemicals) column (1"6 x 85 cm) which had be~ suffi ciently washed with 0.01 M phosphate buffer solution * Trade Mark ~,.

22 _ ~2~

(pH 6~7) containing 1.6 N potassium thiocyanate and developed w,th the same buffer solution as described above to give fractions of 2.4-mB portions. The fractions in the neighborhood of fraction No, 35 exhibited a PA~K-YM activity.
As described above, a purified specimen of the PA-KYM was obtained by the process comprising four stages including fractional precipitation. Table 3 shows the degree of purification at each stage.

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Note to the Table:
~ A color reaction is carried out at 37C ~or 90 min with the use o~ an appropriate amount (a m~) of the er~me solution and a synthetic substrate S-Z4L~4.
When the degree of coloration at 405 nm (oD4 5) is b~
the enzyme concentration (C) can be determined in ~he following manner:
C = b x l.0/a.
~ Protein concentration is determined by Lowry's method and represented in ~g/m~.
The specific activity is given by ~
~ The degree o~ puri~ication is represented by an increase in ~he specific activity at each stage by assigning a value of 1.0 to the case o~ the medium.
5 E~ample 4: Molecular weight and purity e molecular weight and purity of the purified specimen as obtained in Example 3 were examined.
An unreduced specimen was subjected to electro-phoresis on SDS-polyacrylamide gels containing 10 ~ of acrylamide at room temperature with a current of 5 mA ~or 18 hours according to the method reported by Laemmli (c~. Nature, 227, ~80 (1970)). The gel thus obtained was washed with a 2.5 ~ aqueous solution of ..~ Triton X-100 for one hour to remove the SDS. Then it was overlaid on an agarose plate oontaining fibrin ~trade mc~k ~17S~

and plasminogen, which had been prepared according to the method reported by Granelli Piperno and Reich (J. Exp.
Med., 1~8, 223 - 23~ (1978)), and maintained at 37 C for one to five hours. Subsequent observation revealed that there was no fibrinolysis zone except two closely migrating zones in a molecular weight range of 56,000 to 62,000. On the other hand, no fibrinolysis zone was observed when the same procedure was followed exc~pt that no plasminogen was added to the agarose gel. These results suggested that the fibrinolysis would depend on plasminogen. When 20 ~g/me of an IgG fraction of antiurokinase antiserum was added to the fibrin plate, similar fibrinolysis zones to those obtained in the ; absence o~ IgG were observed. Therefore the two bands in the molecular weight range of 56,000 to 62,GOO should be assigned not to a plasminogen activator o~ the urokinase type but to that of the TPA type.
The unreduced purified specimen of the PA-KYM
(approximately 0.1 ~g1 obtained in Example 3 was separated, either as such or after reduced by adding 5% of 2-mercaptoethanol, by SDS-polyacrylamide gel electrophoresis in the same manner as described above.
Proteins contained in the gel thus obta~ned were stained according to the method reported by Oakly et al.

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(cf. Anal. Biochem., 105, 361 - 363 (1980)).
Consequently it was found that the unreduced puri~ied specimen exhibited two bands in a molecular weight range of 56,000 to 62,000 while the reduced specimen exhibited two bands at 32,000 and 36,000. No remarkable band other than those as ~escribed above was observed, which suggested that the specimen was purified almost completely.
The following standard proteins of known molecular weights were employed in the determination of the molecular weight; phosphorlyase b (94000), bovine serum albumin (67000), egg album.in (43000) and carbonic anhydrase (30,000).
Example 5: Examination on the affinity for fibrin 10 g of CNBr-activated Sepharose 4B (a product of Pharmacia Fine Chemicals) was swollen with two liters of 1 ~M Hce and washed. 1 g of fibrinogen (a product of Kabi) dissolved in 200 mQ o~ a coupling buffer solution of 0.5 m Nace/o . 1 M NaHC03 (pH 8.3) was then mixed with the resin slurry and allowed to stand at room temperature for 10 hours to be adsorbed by the swollen resin. The resulting resin wa~ introduced into 100 me of a 0.05 M
phosphate buf~er solution (pH 7.5) containing two units of thrombin (bovine thrombin; a product of Mochida ~1 ~L~7~062 Pharmaceutical Co., Ltd~) and maintained at 37 C for 1o min, thus giving a fibrin-Sepharose resin. The resin thus obtained was packed into a minicolumn (6 m~) and equilibrated with a 0.05 M phosphate buffer solution (pH
7.5) containing 0.01 % of Tween 80. 1 m2 of a PA-KYM
solution or a urokinase solution was loaded on the column and the column was washed with 15 me of a 0.05 M
phosphate buffer solution (pH 7.5) to collect fractions of 1-mQ portions. Then the column was developed with 25 me of the same buffer solution as described above containing 1.6 M potassium thiocyanate to collect fractions of l-m~ portions. The enzyme activities of the washing and the potassiu~ thiocyanate solution were determined with the use of a synthetic substrate S-2444 to determine the recovery. As shown in Table 4, the washing contained no PA-KYM and the PA-KYM require~
potassium thiocyanate for elution, which suggested a strong affinity thereof for fibrin. On the other hand, urokinase exhibited a poor affinity and the washing exhibited the activity.

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Table 4 PA-KYM Urokinase Loaded sample 1.218~ 0.970 Washing o 0.690 Potassium thiocyanate 1.072 0 eluate Recovery 88 % 71 %
_ _ _ _ * A color reaction was carried out with the use of an appropriate amount (i.e. 0.001 to 0.3 me~ of the enæy~e solution and a synthetic medium S-2444 at 37C for 90 min. The indicated value represents the degree o coloration at 405 nm calculated by assuming that the whole enzyme solution is used based on the absorbance measured at 405 nm obtained with aliquots of collected fractions.
For example, when 1 mQ of a sample was loaded in total and 0.1 me thereof is used in the color reaction at 37 C for 90 min. to give an absorbance (oD405) of 0.1218, the indicated value was calculated in the following manner:
~.128 x 1.0~0.1 = 1.218

Claims (2)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of plasminogen activator KYM which comprises culturing a human rhabdomyosarcoma cell variant KYM-A (IFO 50030) and collecting said plasmino-gen activator from the medium thus obtained, said plasmino-gen activator substance KYM having the following physico-chemical properties:
a molecular weight range of approximately 56,000 to 62,000 in the unreduced state, and a molecular weight range of approximately 32,000 to 36,000 in the reduced state as determined by SDS-polyacrylamide gel electro-phoresis;
said substance being an enzyme protein which causes fibrinolysis in the presence of plasminogen;
said substance having an approximate optimum pH value range of 8 to 10;
said substance having an approximate stable pH value range of 5 to 11, and corresponding residual activity in the range of 82% to 100%;
said substance having an optimum function temperature range of 30°C to 45°C;
said substance having thermo-resistance qualities such that by heating from 0°C to 50°C for 90 minutes said substance has a corresponding residual activity of approxi-mately 100% to 82% and that by heating to 60°C or above said substance has a residual activity of not more than 60%;
said substance when exposed to inhibitors in a concen-tration of 0.1 mM, 1.0 mM and 10 mM has the residual activities as follows:

wherein said substance has a residual activity value of 100% in the absence of any inhibitor;
said substance having an amino acid composition percent based on the total number of amino acid residues as follows when hydrolyzed with 6 N hydrochloric acid and subjected to amino acid analysis:
PA-KYM
ASP 10.6 THR 5.3 SER 9.7 GLU 10.9 PRO 6.3 GLY 9.5 ALA 7.2 VAL 5.0 MET 1.0 ILE 3.5 LEU 8.1 TYR 4.5 PHE 3.5 HIS 3.4 LYS 4.5 ARG 7.0 cysteine not determined tryptophan not determined said substance having an approximate spectrum as follows when in aqueous solution:
Wavelength Absorbance 230 0.183 235 0.122 240 0.064 245 0.045 250 0.038 255 0.035 260 0.036 265 0.039 270 0.041 275 0.043 280 0.043 285 0.040 290 0.032 300 0.026 310 0.011 320 0.008 350 0.006 400 0.004 said substance being soluble in water, salt or buffer solutions to about 50µg/ml, and being insoluble in organic solvents;
said substance when lyophilized having the form of a white powder;
said substance, when subjected to SDS-polyacrylamide gel electrophoresis and to PAS reaction successively, turns pink, characteristic of glycoproteins, and has an affinity for con-canavalin A-agarose resin also suggestive of a glycoprotein; and said substance having an isoelectric pH of its main component of 7.5 to 8.0, and an isoelectric pH of its minor component of 7.0 to 7.5, whereby it is concluded that said substance is a mixture of weakly basic proteins.

2. A process for the preparation of the plasminogen activator KYM as set forth in Claim 1, wherein said culture is suspension culture or substratum-attached culture.