Note: Descriptions are shown in the official language in which they were submitted.
~2~7~C36~
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~
-- 2
(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~
-- 3
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.
~275~
- 4
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
':
~2i~
-- 5 --
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
~2'751~
-- 7 --
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
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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~
-- 10 --
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
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~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
SER 9 7
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
HIS 3-4
LYS 4-5
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
62
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.
1~7S~P6Z
-- 15 --
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.
- 16 ~ ~ 7 ~ ~ ~
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
- 20 -
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'.,.".~
- 21 - ~2'~
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.
~2'7~
-- 23 --
O
'~1 $
o ~ O
a.,, ~ ~ ~ '
Q~ CH -t r~
h-,t ~ ~D
b~h r I
a~ ~
CH rl O ~ D O
O ~ O O
X,~ -~t t
r~l
~Q ~
,~
O
C~ O
S ~ O ~ ;i'
~ ~ U~
,9 h
C,~ p~ C)
O o
a) ~ u~ ~ ~o o
~,~ ~ ~ ~
N ~ Il~
s:~ a) (\
C~
O ~f
O
O OO O C)
S~ O,h ~) h
~X
o a)
~;a) +~ a) h ~d
5 ~
-24-~75~ %
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~
- 25 -
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.
~i
~L;Z/~5~
- 26 -
(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
- 27 -
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.
~275~
- 28 -
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
