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Characterization of the plasminogen activator isolated from human embryo kidney cells: Comparison with Urokinase
THROMBOSIS Printed
RESEARCH in the United
Vol.
1, pp. 201-208, 1972 Pergamon Press, Inc.
States
CHARACTERIZAnON OF THE PLASMINOGEN ACTIVATOR ISOLATED FROM HUMAN EMBRYO KIDNEY CELLS: COMPARISON WITH UROKINASE
Grant H. Barlow and LaVera Lazer Molecular Biology Department, Abbott Laboratories, North Chicago, Illinois 60064 U.S.A.
(Received 8.4. 1972. ABSTRACT.
Accepted by Editor L. Lorand)
Comparison is made between the plasminogen activator isolated from urine, Urokinase, and the one isolated from human embryo kidney cells grown in tissue culture. Physical, chemical, and immunological studies based primarily on activity measurements show all properties measured as being identical. It is therefore suggested that the two activators are the same. Introduction
Urokinase, the plasminogen activator from urine, has received much attention as a therapeutic agent in thrombo-embolic diseases (1).
Its use is ham-
pered by the lack of supply of the enzyme since at least 1500 liters of urine are required to produce one human dose and by the high cost for its collection, purification, and packaging.
These problems motivated researchers to search
for another source of this fibrinolytic agent.
In recent years, Bernik and Kwaan (2,3) have demonstrated (a) fibrinolytic activity in cultures of human kidneys, (b) that this fibrinolytic agent was produced to the greatest degree in cultures of human renal cells from 2632 week old feti.
These findings and the development by Weiss and Schleicher
(4,5) of cell culture equipment which allows for the culturing of cells on a large scale prompted us to produce this activator on a large scale. Since it is extremely important that this tissue activator be identical with the urinary Urokinase, the studies reported here were initiated in an
PLASMINOGEN
202
Vol.l,No.3
ACTIVATOR
attempt to establish this identity. Methods and Results Clinical urinary activator prepared by the method of White (6) was used to compare with tissue culture activator purified by essentially the same method to the same purity about 60,000 CTA units/A280
.
The first comparison was based on the ability of the two activators to hydrolyze acetyl-glycyl-lysyl-methyl ester (AGLMe). method of Walton (7) was used.
A modification of the
Ester hydrolysis was followed by a micro
titrimetric procedure in a pH stat maintained at 37'C.
The titrant was 0.01
M sodium hydroxide and the reaction was carried out in 0.15 M KCl.
Enzyme
solutions were prepared in a gelatin diluent to prevent absorption on glass surfaces (0.5% gelatin, 0.15 M KCl, 5 x 10m4 M Tris at pH 7.0). The effect of pH on the rate of activator catalyzed hydrolysis of AGLMe is shown in Figure 1.
The optimum pH in both the tissue and urinary activa-
tor was found at pH 7.8 and all further comparative data were obtained at this pH.
Fig. 1 pH optimum determination on hy rolysis of AGLMe. Concentration of AGLMe is 6 x lo-9 M. The varying heights of the curves are due to the different quantities of activator used in the assay mixture.
PLASMINOGEN
Vol.l,No.3
ACTIVATOR
203
Figure 2 shows the correspondence between esterase activity and the plasminogen activation activity as measured by the fibrin plate method of Brakman (8).
Y
d 50
, 100
150
200
250
CTA UNITS
Fig. 2 Comparison between the esterase activity (AGLMe hydrolysis) and the plasminogen activation activity by fibrin plate. Cont. of AGLMe is 6 x 10-3 M. A direct correlation was found between the value obtained by fibrin plate and AGLMe hydrolysis for the three activators tested since all the activities fall on the same straight line.
It was found that 100 CTA units of both tissue and
urinary activator will hydrolyze 1.7 micromoles of AGLMe per ten minutes when using a CTA vial of 4800 units as a standard. Figure 3 shows the pH-mobility curve obtained using the method of moving boundary "partition" electrophoresis developed by Barlow and Lazer (9). The electrophoretic mobility as a function of pH on urokinase and tissue activators showed that both materials follow the same relationship and had identical iso-electric points, namely 6.9.
A mixture of urokinase and tissue acti-
vator also gave the same elution pattern on iso-electric focusing using LKB Iso-electric focusing apparatus and carrier ampholyte at pH 3-10 range.
PLASMINOGEN
204
Vol.l,No.3
ACTIVATOR
Electrophoresis on a Brinkman Free-flow Electrophoretic Separator at pH 8.9 in tris-acetate buffer (ionic strength = 0.05) showed the same elution position for both activators.
PM
Fig. 3 pH electrophoretic mobility for activators by partition method. All buffers at ionic strength of 0.1. Above pH 8 = Tris buffer; pH 6 = Phosphate buffer; pH 5 = acetate buffer. The sedimentation coefficients were determined on tissue activator and urinary activator in a movable partion cell.
These measurements were carried
out in a phosphate buffer, (0.1 M phosphate, 0.1 M NaCl 0.001 M sodium vesanate) at pH 6.5 using a Spinco Model E Ultracentrifuge.
The biological acti-
vity was determined by esterolytic assay on the starting material and the material
in the top part of the partion cell after sedimentation.
The values
obtained by this method for tissue activator and urinary activator were identical, namely SEO,~ = 2.9. (10) Gel filtration chromatography of (a) urokinase, (b) tissue activator,
PLASMINOGEN
Vol.l,No.3
205
ACTIVATOR
and (c) a mixture of both activators was carried out on a column loaded with Sephadex G-200.
The samples were applied to a column 2.5 centimeters in dia-
meter and 50 centimeters in height.
The protein was eluted with 0.1 M phos-
phate buffer pH 6.5 containing 0.1 M sodium chloride and 0.001 M sodium versenate.
The enzymatic activity was measured in each fraction by the
plasminogen activator, fibrin plate method.
Figure 4 shows the similarity of
the two activators along with the mixture of the activators.
The bioactivity,
shown by the zone diameter - which is a measure of the fibrinolytic lysis, peaks at fraction 18 or 19 in the three samples shown.
5
10
15
20
25
FRACTION NO.
Fig. 4 Elution diagram from Sephadex G-200. Column = 2.5 x 50 cm. Eluted with 0.1 M phosphate at pH 6.5, containing 0.1 M NaCl and 0.001 M EDTA. The most convincing evidence for the identity of the two activators has been derived from immunological studies.
It has been known that the
fibrinolytic activity of the tissue culture material is quenched by the antibody to purified urinary urokinase. ated by gel diffusion. two activators.
This identity has been further substanti-
Figure 5 shows the gel diffusion pattern comparing the
This immunodiffusion pattern clearly shows lines of identity,
without spurring, between urinary and tissue culture activator.
PLASMINOGEN
206
ACTIVATOR
Vol.l,No.3
Fig. 5 Immuno-diffusion pattern on activator samples 1 and 4 contain tissue activator 2 and 5 contain urinary activator, clinical grade 3 and 6 contain urinary activator, purified 7 conta ins rabbit anti purified urinary activator Discussion All of.the parameters that have been studied show the two activators to be identical.
These parameters were selected so that location and interpre-
tation could be determined by measurement of either biological activity against a fibrin plate or by esterolytic activity against AGLMe.
The reason
for this is that neither enzyme is available in the pure state and therefore, normal detection methods such as refractive index or absorption could not be used.
It is tacitly assumed that the possible interaction with impurities is
identical.
Since no differences are shown, one must decide on how many
different parameters need be studied before identity is established.
The
only absolute proof would be to show identical amino acid sequences of the two enzymes, but unfortunately the likelihood of ever having enough pure material is very slim.
It is thus concluded by the data presented that these
two activators are identical.
PLASMINOGEN
Vol.l,No.3
ACTIVATOR
207
References 1.
Urokinase Pulmonary Embolism Trial, Phase I Results. 214 2163, 1970. -'
2. BERNIK, M. B. and KWAAN, H. C. Cultures of the Human Kidney.
J. An. Med. Ass.
Origin of Fibrinolytic Activity in J. Lab and Clin Med. 7&, 650, 1967.
3.
BERNIK, M. B. and KWAAN, H. C. Plasminogen Activator in Cultures From Human Tissues. An Immunological and Histochemical Study. J. Clin Invest. 48, 1740, 1969.
4.
WEISS, R. E. and SCHLEICHER, J. B. A Multisurface Tissue Propagator for the Mass-Scale Growth of Cell Monolayers. Biotech. and Bioeng. X, 601, 1968.
5.
SCHLEICHER, J. B., and WEISS, R. E. Application of a Multiple Surface Tissue Culture Propagator for the Production of Cell Monolayers, Virus, and Biochemicals. Biotech. and Bioeng. 5, 617, 1968.
6.
WHITE, W. F., BARLOW, G. H., and MOZEN, M. M. The Isolation and Characterization of Plasminogen Activators (Urokinase) from Human Urine. Biochem 2, 2160, 1966.
7. WALTON, P. L. by Urok nase.
The hydrolysis of cx-N-acetylglycyl-L-lysinemethyl ester Biochim et Biophys Acta 132, 104, 1967.
a.
BRAKMAN P. Fibrinolysis, A standardized fibrin plate method and a fibrin0 ytic assay of plasminogen. Amsterdam, Scheltema and Hokema, 1967.
9.
BARLOW, G. H. and LAZER, L. Anal. Biochem. 39, 1, 1971.
9
Moving boundary "partition" electrophoresis.
Ultracentrifugation, Diffusion, and Viscometry. In: Methods in Enzymology Vol. IV, New York - Academic Press, 1957, p. 59.
10. SCHACHMAN, H. K.
RESEARCH in the United
Vol.
1, pp. 201-208, 1972 Pergamon Press, Inc.
States
CHARACTERIZAnON OF THE PLASMINOGEN ACTIVATOR ISOLATED FROM HUMAN EMBRYO KIDNEY CELLS: COMPARISON WITH UROKINASE
Grant H. Barlow and LaVera Lazer Molecular Biology Department, Abbott Laboratories, North Chicago, Illinois 60064 U.S.A.
(Received 8.4. 1972. ABSTRACT.
Accepted by Editor L. Lorand)
Comparison is made between the plasminogen activator isolated from urine, Urokinase, and the one isolated from human embryo kidney cells grown in tissue culture. Physical, chemical, and immunological studies based primarily on activity measurements show all properties measured as being identical. It is therefore suggested that the two activators are the same. Introduction
Urokinase, the plasminogen activator from urine, has received much attention as a therapeutic agent in thrombo-embolic diseases (1).
Its use is ham-
pered by the lack of supply of the enzyme since at least 1500 liters of urine are required to produce one human dose and by the high cost for its collection, purification, and packaging.
These problems motivated researchers to search
for another source of this fibrinolytic agent.
In recent years, Bernik and Kwaan (2,3) have demonstrated (a) fibrinolytic activity in cultures of human kidneys, (b) that this fibrinolytic agent was produced to the greatest degree in cultures of human renal cells from 2632 week old feti.
These findings and the development by Weiss and Schleicher
(4,5) of cell culture equipment which allows for the culturing of cells on a large scale prompted us to produce this activator on a large scale. Since it is extremely important that this tissue activator be identical with the urinary Urokinase, the studies reported here were initiated in an
PLASMINOGEN
202
Vol.l,No.3
ACTIVATOR
attempt to establish this identity. Methods and Results Clinical urinary activator prepared by the method of White (6) was used to compare with tissue culture activator purified by essentially the same method to the same purity about 60,000 CTA units/A280
.
The first comparison was based on the ability of the two activators to hydrolyze acetyl-glycyl-lysyl-methyl ester (AGLMe). method of Walton (7) was used.
A modification of the
Ester hydrolysis was followed by a micro
titrimetric procedure in a pH stat maintained at 37'C.
The titrant was 0.01
M sodium hydroxide and the reaction was carried out in 0.15 M KCl.
Enzyme
solutions were prepared in a gelatin diluent to prevent absorption on glass surfaces (0.5% gelatin, 0.15 M KCl, 5 x 10m4 M Tris at pH 7.0). The effect of pH on the rate of activator catalyzed hydrolysis of AGLMe is shown in Figure 1.
The optimum pH in both the tissue and urinary activa-
tor was found at pH 7.8 and all further comparative data were obtained at this pH.
Fig. 1 pH optimum determination on hy rolysis of AGLMe. Concentration of AGLMe is 6 x lo-9 M. The varying heights of the curves are due to the different quantities of activator used in the assay mixture.
PLASMINOGEN
Vol.l,No.3
ACTIVATOR
203
Figure 2 shows the correspondence between esterase activity and the plasminogen activation activity as measured by the fibrin plate method of Brakman (8).
Y
d 50
, 100
150
200
250
CTA UNITS
Fig. 2 Comparison between the esterase activity (AGLMe hydrolysis) and the plasminogen activation activity by fibrin plate. Cont. of AGLMe is 6 x 10-3 M. A direct correlation was found between the value obtained by fibrin plate and AGLMe hydrolysis for the three activators tested since all the activities fall on the same straight line.
It was found that 100 CTA units of both tissue and
urinary activator will hydrolyze 1.7 micromoles of AGLMe per ten minutes when using a CTA vial of 4800 units as a standard. Figure 3 shows the pH-mobility curve obtained using the method of moving boundary "partition" electrophoresis developed by Barlow and Lazer (9). The electrophoretic mobility as a function of pH on urokinase and tissue activators showed that both materials follow the same relationship and had identical iso-electric points, namely 6.9.
A mixture of urokinase and tissue acti-
vator also gave the same elution pattern on iso-electric focusing using LKB Iso-electric focusing apparatus and carrier ampholyte at pH 3-10 range.
PLASMINOGEN
204
Vol.l,No.3
ACTIVATOR
Electrophoresis on a Brinkman Free-flow Electrophoretic Separator at pH 8.9 in tris-acetate buffer (ionic strength = 0.05) showed the same elution position for both activators.
PM
Fig. 3 pH electrophoretic mobility for activators by partition method. All buffers at ionic strength of 0.1. Above pH 8 = Tris buffer; pH 6 = Phosphate buffer; pH 5 = acetate buffer. The sedimentation coefficients were determined on tissue activator and urinary activator in a movable partion cell.
These measurements were carried
out in a phosphate buffer, (0.1 M phosphate, 0.1 M NaCl 0.001 M sodium vesanate) at pH 6.5 using a Spinco Model E Ultracentrifuge.
The biological acti-
vity was determined by esterolytic assay on the starting material and the material
in the top part of the partion cell after sedimentation.
The values
obtained by this method for tissue activator and urinary activator were identical, namely SEO,~ = 2.9. (10) Gel filtration chromatography of (a) urokinase, (b) tissue activator,
PLASMINOGEN
Vol.l,No.3
205
ACTIVATOR
and (c) a mixture of both activators was carried out on a column loaded with Sephadex G-200.
The samples were applied to a column 2.5 centimeters in dia-
meter and 50 centimeters in height.
The protein was eluted with 0.1 M phos-
phate buffer pH 6.5 containing 0.1 M sodium chloride and 0.001 M sodium versenate.
The enzymatic activity was measured in each fraction by the
plasminogen activator, fibrin plate method.
Figure 4 shows the similarity of
the two activators along with the mixture of the activators.
The bioactivity,
shown by the zone diameter - which is a measure of the fibrinolytic lysis, peaks at fraction 18 or 19 in the three samples shown.
5
10
15
20
25
FRACTION NO.
Fig. 4 Elution diagram from Sephadex G-200. Column = 2.5 x 50 cm. Eluted with 0.1 M phosphate at pH 6.5, containing 0.1 M NaCl and 0.001 M EDTA. The most convincing evidence for the identity of the two activators has been derived from immunological studies.
It has been known that the
fibrinolytic activity of the tissue culture material is quenched by the antibody to purified urinary urokinase. ated by gel diffusion. two activators.
This identity has been further substanti-
Figure 5 shows the gel diffusion pattern comparing the
This immunodiffusion pattern clearly shows lines of identity,
without spurring, between urinary and tissue culture activator.
PLASMINOGEN
206
ACTIVATOR
Vol.l,No.3
Fig. 5 Immuno-diffusion pattern on activator samples 1 and 4 contain tissue activator 2 and 5 contain urinary activator, clinical grade 3 and 6 contain urinary activator, purified 7 conta ins rabbit anti purified urinary activator Discussion All of.the parameters that have been studied show the two activators to be identical.
These parameters were selected so that location and interpre-
tation could be determined by measurement of either biological activity against a fibrin plate or by esterolytic activity against AGLMe.
The reason
for this is that neither enzyme is available in the pure state and therefore, normal detection methods such as refractive index or absorption could not be used.
It is tacitly assumed that the possible interaction with impurities is
identical.
Since no differences are shown, one must decide on how many
different parameters need be studied before identity is established.
The
only absolute proof would be to show identical amino acid sequences of the two enzymes, but unfortunately the likelihood of ever having enough pure material is very slim.
It is thus concluded by the data presented that these
two activators are identical.
PLASMINOGEN
Vol.l,No.3
ACTIVATOR
207
References 1.
Urokinase Pulmonary Embolism Trial, Phase I Results. 214 2163, 1970. -'
2. BERNIK, M. B. and KWAAN, H. C. Cultures of the Human Kidney.
J. An. Med. Ass.
Origin of Fibrinolytic Activity in J. Lab and Clin Med. 7&, 650, 1967.
3.
BERNIK, M. B. and KWAAN, H. C. Plasminogen Activator in Cultures From Human Tissues. An Immunological and Histochemical Study. J. Clin Invest. 48, 1740, 1969.
4.
WEISS, R. E. and SCHLEICHER, J. B. A Multisurface Tissue Propagator for the Mass-Scale Growth of Cell Monolayers. Biotech. and Bioeng. X, 601, 1968.
5.
SCHLEICHER, J. B., and WEISS, R. E. Application of a Multiple Surface Tissue Culture Propagator for the Production of Cell Monolayers, Virus, and Biochemicals. Biotech. and Bioeng. 5, 617, 1968.
6.
WHITE, W. F., BARLOW, G. H., and MOZEN, M. M. The Isolation and Characterization of Plasminogen Activators (Urokinase) from Human Urine. Biochem 2, 2160, 1966.
7. WALTON, P. L. by Urok nase.
The hydrolysis of cx-N-acetylglycyl-L-lysinemethyl ester Biochim et Biophys Acta 132, 104, 1967.
a.
BRAKMAN P. Fibrinolysis, A standardized fibrin plate method and a fibrin0 ytic assay of plasminogen. Amsterdam, Scheltema and Hokema, 1967.
9.
BARLOW, G. H. and LAZER, L. Anal. Biochem. 39, 1, 1971.
9
Moving boundary "partition" electrophoresis.
Ultracentrifugation, Diffusion, and Viscometry. In: Methods in Enzymology Vol. IV, New York - Academic Press, 1957, p. 59.
10. SCHACHMAN, H. K.