Purification of High Molecular Weight Urokinase from Human Urine and Comparative Study of Two Active Forms of Urokinase

Takeji Shibatani; Toshio Kakimoto; Ichiro Chibata

doi:10.1055/s-0038-1657329

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1983; 49(02): 091-095
DOI: 10.1055/s-0038-1657329

Original Article

Schattauer GmbH Stuttgart

Purification of High Molecular Weight Urokinase from Human Urine and Comparative Study of Two Active Forms of Urokinase

Takeji Shibatani

The Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co. Ltd., Osaka, Japan

,

Toshio Kakimoto

The Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co. Ltd., Osaka, Japan

,

Ichiro Chibata

The Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co. Ltd., Osaka, Japan

› Author Affiliations

Abstract

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Summary

An improved method for the purification of high molecular weight urokinase to homogeneity from human urine was established. A yield of 32% with a 3,100-fold purification was obtained by Hyflo Super-Cel treatment, heat treatment at 60° C for 10 hr, serial column chromatography on DEAE-Sepharose CL-6B and 0-[3-(p-sulfophenylamino)-2-hydroxypropyl]-cellulose (SFOP-cellulose), and gel filtration on Ultrogel AcA 54. The low molecular weight form of urokinase was also purified to homogeneity by chromatography on hydroxyl apatite and gel filtration on Sephadex G-75 after the SFOP-cellulose column step. The high molecular weight urokinase had only one isoelectric form with a pi of 9.7, whereas the low molecular weight form had six isoelectric subforms with pi values between 9.4 and 6.4. The absorption coefficients at 280 nm of both urokinase forms were 13.61 and 13.50, respectively. Fibrinolytic and esterolytic activities of the two urokinase forms were compared in various assay methods.

Keywords

Purification - High and low molecular weight urokinase - Fibrinolysis - Fibrin-tube method - Fibrin-plate method - Two - stage lysis time method - terolytic activity

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References

References
1 Williams JR B. The fibrinolytic activity of urine. Br J Exp Pathol 1951; 32: 530-537
2 Astrup T, Stemdorff I. An activator of plasminogen on normal urine. Proc Soc Exp Med 1952; 81: 675-678
3 Sobel GW, Mohler SR, Jones NW, Dowdy ABC, Guest MM. An activator of profibrinolysin extracted from urine. Am J Physiol 1952; 171: 768-769
4 Painter RH, Charles AF. Characterization of a soluble plasminogen activator from kidney cell cultures. Am J Physiol 1962; 202: 1125-1130
5 Barlow GH, Lazer LV. Characterization of plasminogen activator isolated from human embryo kidney cells. Comparison with urokinase. Thromb Res 1972; 1: 201-207
6 Lewis LJ. Plasminogen activator (urokinase) from cultured cells. Thromb Haemostas 1979; 42: 895-900
7 Lesuk A, Terminiello L, Traver JH. Crystalline human urokinase: Some properties. Science 1965; 147: 880-882
8 White WF, Barlow GH, Mozen MM. The isolation and characterization of plasminogen activators (urokinase) from human urine. Biochemistry 1966; 5: 2160-2169
9 Ogawa N, Yamamoto H, Katamine T, Tajima H. Purification and some properties of urokinase. Thrombos Diathes Haemorrh 1975; 34: 194-209
10 Holmberg L, Bladh B, Astedt B. Purification of urokinase by affinity chromatography. Biochim Biophys Acta 1976; 445: 215-222
11 Soberano M, Ong EB, Johnson AJ, Levy M, Schoellmann G. Purification and characterization of two forms of urokinase. Biochim Biophys Acta 1976; 445: 763-773
12 Nobuhara M, Sakamaki M, Ohnishi H, Suzuki Y. A comparative study of the high molecular weight urokinase and low molecular weight urokinase. J Biochem 1981; 90: 225-232
13 Miwa N, Takayanagi H, Suzuki A. Comparative studies on two active enzyme forms of human urinary urokinase. I. Purification by serial column chromatography and homogeneity analyses of molecular weight and isoelectric point. Chem Pharm Bull 1981; 29: 463-471
14 Huber K, Kirchheimer J, Binder BR. Rapid isolation of high molecular weight urokinase from native human urine. Thromb Haemostas 1982; 47: 197-202
15 Suyama T, Nishida M, Iga Y, Naito R. Difference in thrombolytic effect between higher and lower molecular weight forms of urokinase. Thromb Haemostas 1977; 38: 48 (Abstr.)
16 Samama M, Cazenave B, Otero AM. Urokinase I and II activity. Thromb Haemostas 1978; 40: 578-580
17 Ploug J, Kjeldgaard NO. Urokinase. An activator of plasminogen from human urine. I. Isolation and properties. Biochim Biophys Acta 1957; 24: 278-282
18 Nishizaki S, Kawamura J. Studies on the determination of the potency of urokinase preparations. Iyakuhin Kenkyu 1974; 5: 295-308
19 Morita T, Kato H, Iwanaga S, Takada K, Kimura T, Sakakibara S. New fluorogenic substrates for α-thrombin, factor Xa, kallikreins, and urokinase. J Biochem 1977; 82: 1495-1498
20 Claeson G, Friberger P, Knos M, Eriksson E. Methods for determination of prekallikrein in plasma, glandular kallikrein and urokinase. Haemostasis 1978; 7: 76-78
21 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275
22 Weber K, Osborn M. The reliability of molecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 1969; 244: 4406-4412
23 Vesterberg O, Svensson H. Isoelectric fractionation, analysis, and characterization of ampholytes in natural pH gradients. IV. Further studies on the resolving power in connection with separation of myoglobins. Acta Chem Scand 1966; 20: 820-834
24 Porath J, Fomstedt N. Group fractionation of plasma proteins on dipolar ion exchangers. J Chromatogr 1970; 51: 479-489
25 Bergstrom K. A preparative method for the partial purification of human urokinase. Arkiv Kemi 1963; 21: 535-546
26 Higashi N, Arimura H, Ishikawa H. Inactivation of viruses intentionally added to urokinase samples by heat-treatment. Chem Pharm Bull 1977; 25: 3366-3369