structural relationship between human high and low molecular mass urokinase

Hoppe-Seyler's Z. Physiol. Chem.Bd. 363, S. 133-141, Februar 1982

Structural Relationship Between Human High and Low Molecular MassUrokinase

Wolfgang A. GüNZLER3 , Gerd J. STEFFENS3, Fritz OrriNG3, Gerhard BusEb andLeopold FLOHE3

3 Grünenthal GmbH, Forschungszentrum Aachen-Eilendorf, Abteilung Proteinchemieb Rheinisch-Westfälische Technische Hochschule Aachen, Abteilung Physiologische Chemie

(Received 15 October 1981)

Summary: Human low molecular mass urokinasewas demonstrated to consist of two polypeptides.The peptide chains of about 30000 and of2427 Da, respectively, were isolated by gel filtra-tion after reductive cleavage of a single interchaindisulfide bridge. The complete amino acid se-quence of the 2427-Da chain consisting of 21amino acids was determined. Stoichiometricamounts of hexosamines were not found in the2427-Da chain.The isolated 30000-Da chains of both, humanlow and high molecular mass urokinases were

found to be identical in terms of amino acid com-position, of hexosamine content, of TV- and ofC-terminal amino acid sequences.The amino acid sequence of the 2427-Da chainof the low molecular enzyme was found to bedifferent from the TV-terminal sequence of the20000-Da chain of the high molecular enzyme.The transformation of high to the low molecularform is considered a limited proteolytic degrada-tion of the 20000-Da chain of high molecularmass urokinase, exclusively.

Strukturelle Verwandtschaft zwischen menschlicher hoch- und niedermolekularer Urokinase

Zusammenfassung: Es wurde gezeigt, daß mensch-liche niedermolekulare Urokinase aus zwei Peptid-ketten besteht. Die beiden Peptidketten von etwa30000 Da und von 2427 Da Molekularmassewurden nach reduktiver Spaltung einer einzigenkettenverbindenden Disulfidbrücke isoliert. Dievollständige Aminosäuresequenz der aus 21 Ami-nosäuren bestehenden 2427-Da-Kette wurde be-stimmt. Hexosamine waren in dieser Kette nichtin stöchiometrischen Mengen nachzuweisen.Die isolierten 30000-Da-Ketten von nieder- undhochmolekularer Urokinase erwiesen sich als

identisch nach Maßgabe von Aminosäurezusam-mensetzung, Hexosamingehalt,7V- und C-termina-ler Aminosäuresequenz.Die Aminosäuresequenz der 2427-Da-Kette un-terscheidet sich von der TV-terminalen Sequenzder 20000-Da-Kette des hochmolekularen En-zyms. Es wird geschlossen, daß die limitierte Pro-teolyse bei der Umwandlung von der hochmole-kularen in die niedermolekulare Urokinase aus-schließlich die 20000-Da-Kette des hochmoleku-laren Enzyms betrifft.

Enzymes:Carboxypeptidase A (EC 3.4.17.1); Urokinase (EC 3.4.21.31).A bbreviations:DITC-glass, diisothiocyanate-activated glass; -Nan, p-nitroamlide.

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134 W. A. G nzler, G. J. Steffens, F. tting, G. Buse and L. Flohe Bd. 363(1982)

Key words: Human urokinases, subunit characterization, arrangement of peptide chains, partial amino acidsequence.

Human urokinase is reported to exist in activeforms of different molecular size, high and lowmolecular mass urokinases with values of about50000 and 30000 Da, respectively I1'2!. Uponreduction with thiols, the high molecular form iscleaved into two subunits with values of about30000 and 20000 Da, respectively, while nochange in molecular mass has been observed withlow molecular urokinase under reducing conditionsas judged by dodecyl-sulfate gel electrophoresisl2!.On the other hand, the high molecular enzymecan be transformed into the low molecular formby mild proteolytic digestion!3'4!. Accordingly,both procedures, the reduction and limitedproteolysis of high molecular urokinase, yieldproducts of the apparently identical size of30000 Da which each contain an active siteserine residue^2' but cannot be entirely identical.Since low molecular urokinase is derived from ahigh molecular form containing two peptidechains^4!, the possibility that it also consists oftwo peptide chains is worthwile considering. De-pending on the site of proteolytic attack in thehigh molecular enzyme, three alternatives forthe subunit composition of a 30000-Da lowmolecular form can be proposed in principal(Scheme 1).a) A small peptide bearing an interchain disul-fide-linkage is cleaved from a terminal region ofthe 30000-Da chain: the low molecular formwould consist of a single chain differing from the30000-Da chain by its C- or TV-terminus.

c j 30000-Da Chain a, c3

Ι Φ

ί ς20 000-Da Chain

1

SI

tb,c2

Scheme 1. Working hypothesis on transformation ofhigh into low molecular mass urokinase.The arrows indicate hypothetical proteolytic degrada-tion sites.

b) Only the 20000-Da chain is degraded, result-ing in a small, easily overlooked peptide, whichremains disulfide-linked to the original 30000-Dachain: the low molecular form would consist oftwo peptide chains, one of them identical to the30000-Da chain.c) Both chains of the high molecular urokinaseare proteolytically attacked: the low molecularform would consist of two or three disulfide-linked peptide chains, the main chain being dif-ferent from the 30000-Da chain by its C- and/orTV-terminus.The present report confirms the existence of twopeptide chains in the low molecular form and itsderivation from high molecular urokinase, asproposed in alternative b).

Material and Methods

Urinary urokinases obtained from Ares (Geneva, Switzer-land) were further purified by affinity chromatographyon benzamidine Sepharosel5!. The purity of the uro-kinase preparations was checked by dodecyl-sulfatepolyacrylamide gel electrophoresis, essentially accordingto Laemmlif6!. The enzymatic activity of urokinase wasassayed with ^Glu-Gly-Arg-Nan (S-2444, Kabi Diagnosti-ca, M nchen) as a substrate, according to Claeson etalJ7 '. Purified samples of both enzyme forms were re-duced and iS-carboxymcthylated, essentially accordingto Crestfield et al.f8l The sample was dissolved in 1.5MTris/HCl buffer pH 9 containing 8M deionized urea and5mM EDTA. The solution was flushed with nitrogenand incubated with 2-mercaptoethanol (about 50-foldmolar excess over cystine residues) for 3 h at roomtemperature. 0.2 μιτιοί (10 μΟί) iodo-[2-14C]acetic acid(Amersham Buchler, Braunschweig) was added. The mix-ture was incubated for 30 min in the dark, treated with100 μΐ 2-mercaptoethanol, and after further 90 min ofincubation, mixed with 260 mg unlabelled iodoaceticacid in 2M NaOH. After 15 min in the dark, the mixturewas applied to an appropriate column for gel filtration.In the case of the low molecular mass urokinase the reac-tion mixture was separated on a 2.8 χ 50 cm column ofBiogel P-10 (200-400 mesh, Biorad, M nchen) in 70%acetic acid. The chains of the S-carboxymethylated highmolecular enzyme were separated on a 5 χ 20 cm columnof Biogel P-100 (- 400 mesh, Biorad, M nchen) in 3%sodium dodecyl sulfate.



CorrectionVolume 363, No. 2Structural Relationship between Human High and Low Molecular Mass Urokinase, by W. A. Giinzler,G. J. Steffens, F. Ötting, G. Buse and L. FlohePage 135, Fig. 1: The photograph was inverted and should be replaced by the corrected figure below.

Lane 1 2

155000—

68000—

39000—

21500 —

17000—

14400-

8200—

6200-

Lane 1 2Pep tide Protein

Standards

3 4Reduced Native

Low molecular form

5 6Reduced Native

High molecular form

7 8Protein Pep tide

Standards

Fig. 1. Disc dodecyl-sulfate polyacrylamide gel electrophoresis of urokinase forms.Resolving gel: 12.5% acrylamide. Lanes 1 and 8: Peptide standards 17000,14400, 8200 and 6200 Da (Fluka, Neu-Ulm); lanes 2 and 7: Protein standards 155000, 68000, 39000 and 21500 Da (Boehringer, Mannheim); lanes 3-6:reduced and native urokinases as indicated.



Bd. 363 (1982) Human Urokinases 135

The amino acid composition was determined after acidhydrolysis (6M HC1, 0.1% thioglycolic acid, 20 h at110 °C) with a LC 6001 amino acid analyzer (Biotronik,Frankfurt) using a 0.6 χ 21 cm column of DC 6 A resineand a separation program capable of separating hexo-saminesl9]. Tryptophan was determined after acid hy-drolysis in the presence of 4% thioglycolic acid. Gluco-samine and galactosamine were quantitated after 15 hhydrolysis in 3M HC1 at 110 °C. A reference sample ofJV-acetylglucosaminitol was kindly supplied by Dr.E. Ohst,RWTH Aachen.

Automated sequence analyses were performed using aBeckman sequencer model 890 C or an LKB solid-phasesequencer model 4020. The N-terminal sequence runsof the unreduced enzyme preparations were performed,utilizing the fast Quadrol protein program'10!. Forautomated sequencing of the small peptide of the lowmolecular mass urokinase, we used the peptide programof Crewther and Inglis'111 which was adapted to the890 C sequencer! 121. Furthermore, this peptide wascovalently bound to DITC-glass, according to Machleidtand Wachteri 131 and degraded according to Laursen' 141.

8

155000—

68000-

39000—

21500 —

17000-

14400-

8200—

6200 —

Lane 1 2Peptide Protein

Standards

3 4Reduced Native

Low molecular form

5 6Reduced Native

High molecular form

7 8Protein Peptide

Standards

Fig. 1. Disc dodecyl-sulfate polyacrylamide gel electrophoresis of urokinase forms.Resolving gel: 12.5% acrylamide. Lanes 1 and 8: Peptide standards 17000,14400, 8200 and 6200 Da (Fluka, Neu-Ulm); lanes 2 and 7: Protein standards 155000, 68000, 39000 and 21500 Da (Boehringer, Mannheim); lanes 3-6:reduced and native urokinases as indicated.



136 W. A. Günzler, G. J. Steffens, F. Ötting, G. Buse and L. Flohe Bd. 363 (1982)

The 2-anilino-l,3-thiazolin-5-one amino acids were con-verted at 55 °C in 20% trifluoroacetic acid for 30 min.The phenylthiohydantoin derivatives of amino acidswere identified and quantified by high performanceliquid chromatography on a Spectra Physics model SP8000, according to Zimmerman et alJ l51. For rapididentification of these amino acid derivatives, thin-layerchromatography on Merck HPTLC plates (Kieselgel 60 FC 254) was used as described elsewhere(16LFor C-terminal sequence analysis samples of isolated30000-Da chains of both enzyme forms or of performic

t-[min] ->·

Fig. 2. Gradient high performance liquid chromatogramsof the phenylthiohydantoin derivatives of the first twosteps from a liquid phase Edman-degradation of un-reduced low molecular mass urokinase.Column: Zorbax ODS (25 cm 4.6 mm). Solvent A:O.OlM sodium acetate (pH 4.5), Solvent B: acetoni-trile. Gradient from 22% to 48% acetonitrile in 6 min.Flow rate: 1 m//min, temperature: 50 °C.Sample size of the references: proline derivative (P)1.4 nmol; lysine derivative (K) 1.1 nmol;isoleucinederivative (I) 0.95 nmol.

acid-oxidized low molecular mass urokinase were incu^bated with carboxypeptidase A (Sigma, München) at37 °C in buffer of pH 8.0 containing 0.1 % sodium dode-cyl sulfate and glucosamine as an internal standard. Ali-quot portions were withdrawn at the times indicated,acidified and analyzed for free amino acids.

Results

In agreement with published data(2J dodecyl-sul-fate polyacrylamide gel electrophoresis revealedno substantial differences between low molecularmass urokinase and the 30000-Da chain of re-duced high molecular form or between reducedand unreduced low molecular enzyme (Fig. 1).However, with the method applied here the re-duced low molecular enzyme and the 30000-Dachain of reduced high molecular form were con-sistently found to be moving slightly faster thanthe unreduced low molecular form, indicating asomewhat lower molecular weight for the latterafter reduction. As judged from the resolvingpower of the electrophoretic system employed,this difference was estimated to be less than 3000.A peptide of this size, however, would not showup in our electrophoretic system.

TV-terminal sequence analysis of the unreducedlow molecular enzyme provided evidence for theexistence of a second peptide chain as isoleucineand lysine were detected in almost equal amounts(Fig. 2). This double sequence (Table 1) was ob-served over 12 degradation steps. Thereafter, onlyone sequence could be detected.

Separation of two peptide chains of the lowmolecular mass urokinase after reduction with

Table 1. W-terminal double sequences of unreducedurokinases.

Step

1234567

Unreduced lowmolecular form

Ile/LysHe/ProGly/SerGly/SerGlu/ProPhe/ProThr/Glu

Unreducedhigh molecularform

Ile/SerIle/AsnGly/GluGly/LeuGlu/HisPhe/GlnThr/Val




Fig. 3. Separation of lowmolecular mass urokinase pep-tide chains after reduction andS-carboxymethylation with[14C]iodoacetate on BiogelP-10.

l·lο"ο /

0.10

25 30 35Fr act. no.

dpm-

40 45

2-mercaptoethanol and S-carboxymethylationwith [14C]iodoacetic acid was achieved by gelchromatography on Biogel P-10. Apart from the30000-Da chain, a distinct radioactive peak with-out significant absorbance at 280 nm was eluted(Fig. 3). Amino acid analysis revealed that thissmall peptide consists of 21 amino acids includingone labelled S-carboxymethylcysteine (Table 2).Since the radioactivity of this single residue ob-viously exceeds the amount predictable from theratio of cysteine residues in the peptide chains ofthe low molecular enzyme, a preferred reductionof the interchain disulfide can be assumed underthe conditions employed.The complete amino acid sequence of this smallchain of 2427 Da was determined by both solidand liquid phase techniques, utilizing a specialpeptide program for liquid phase sequencing! 101.Using the solid phase technique, the peptide wascoupled at its lysine residues to DITC-glass. Gapsresulting from fixed amino acids were observed inpositions 1,10, and 16. After the 16th step theremaining peptide consisting of the last five resi-dues was washed out and saved. This pentapeptidewas subsequently sequenced by liquid phase tech-nique in order to establish the C-terminal se-quence (Table 3). In retrospect, the position ofthe disulfide bridge in the 2427-Da peptide ex-

Table 2. Amino acid analysis and amino acid composi-tion of the second peptide chain of low molecular massurokinase (2427-Da chain).Values are given as mol of residue per mol of peptide(nearest integer in parentheses); - means not significant.

Amino acid

AsxThrSerGlxProGlyAlaCysValMetHeLeuTyrPheGlcNGalNHisTrpLysArg

Total

_1.112.053.803.971.26-0.90a

---1.91-1.12----3.041.85

(0)(1)(2)(4)(4)(1)(0)(1)(0)(0)(0)(2)(0)(1)(0)(0)(0)(0)(3)(2)

21a Determined as 5-carboxymethylcysteine.



138 W. A. Günzler, G. J. Steffens, F. Ötting, G. Buse and L. Flohe Bd. 363 (1982)

Table 3. Complete amino acid sequence of the second peptide chain of low molecular mass urokinase (2427-Dachain).->· Sequenced by liquid phase (peptide program); —· sequenced by solid phase; -^sequenced by liquid phase (pep-tide program) after solid phase sequencing from position 1 to 16.

1 10 20 21H-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-Leu-Lys-Phe-Gln-Cys-Gly-Gln-Lys-Thr-Leu-Arg-Pro-Arg-OH

plains the limitation of the double sequence inunreduced low molecular enzyme to 12 degrada-tion steps: subsequently, the remaining octapep-tide was washed out due to the conditions em-ployed in the protein program^10! of the liquidphase sequencing technique.

The amino acid sequences 3-5 and 17-20 ofthe 2427-Da chain fit into a coding sequence forO-glycosylation, Ser/Thr-X-X-Pro, which was pro-posed by Jolles et alJ17K However, no glucosa-mine or galactosamine could be detected in hy-drolysates of this peptide, only a small unidenti-fied peak was eluted with a retention time cor-responding to glucosaminitol.

As anticipated, the sequence of the 2427-Dapeptide accounts for one half of the doublesequence obtained with the unreduced low mo-lecular enzyme. This assignment was confirmedby the sequence of the 21 TV-terminal aminoacids of the isolated 30000-Da chain of this en-zyme, which matched the other half of thedouble sequence (Table 4).

As the 2427-Da chain is blocked by a C-terminalarginine residue, treatment with carboxypeptidaseA of both the low molecular enzyme and its30000-Da chain, yielded qualitatively identicalresults. Leucine and alanine were released suc-

cessively, indicating Ala-Leu-OH as C-terminusof the 30000-Da chain (Fig. 4a). Since consider-ably more than 1 mol leucine per mol of peptidecan be obtained, it is suggested that a second leu-cine residue is linked to the amino end of alanine.As to the question of whether the 30000-Da chainof high molecular mass urokinase might be de-graded during proteolytic conversion into the lowmolecular form, the sequences of the TV- and C-terminus, respectively, of this peptide chain weredetermined and compared to those of the 30000-Da chain of the low molecular form. TV-terminalsequence analysis of unreduced high molecularenzyme resulted in two main sequences, one ofwhich turned out to be identical to that of the30000-Da chain of the low molecular form (Ta-ble 1). This assignment was confirmed by TV-ter-minal sequence analysis of the isolated 30000-Da chain and the main 20000-Da chain of thehigh molecular enzyme (Table 4). C-terminalanalysis of the isolated 30000-Da chain of thehigh molecular form with carboxypeptidase A,again yielded leucine and alanine (Fig. 4b).Additional evidence for the identity of the30000-Da chains in both enzyme forms wasprovided by the amino acid composition and bythe hexosamine content of the isolated chains(Table 5).

Table 4. JV-terminal sequences of the separated reduced and 5-carboxymethylated 30000-Da chains from low orhigh molecular mass urokinase and of the 20000-Da chain of the high molecular form.

30000-Da chains1 10 20 21

H-Ile-Ile-Gly-Gly-Glu-Phe-Thr-Thr-Ile-Glu-Asn-Gln-Pro-Trp-Phe-Ala-Ala-Ile-Tyr-Arg-Arg . . .

20000-Da chain

H-Ser-Asn-Glu-Leu-His-Gln-Val. . .Brought to you by | provisional account

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1.0.ίll 0.5(0o'i

2 Wt [min]

Fig. 4. Release by carboxypeptidase A ofC-terminal amino acids from 5-carboxy-methylated, isolated 30000-Da chains oflow (a) and high (b) molecular mass uro-kinase.

2.0

2 W 120t [min]

Discussion

In early experiments, a single TV-terminus (iso-leucine) was found in high molecular mass uro-kinasel3'. The conclusion that it consists of asingle amino acid chain is not consistent withavailable evidence^2'18!. Lesuk et aU3l reportedlysine as the single TV-terminal amino acid in thelow molecular enzyme derived from high molecu-lar form by proteolysis. In contrast, Studer et alJ19

and Henschen et aU 181 presented a single TV-termi-nal amino acid sequence of low molecular formstarting with isoleucine. Very recently, however,the former group reported two TV-terminal aminoacid sequences in unreduced low molecular uro-kinase starting with isoleucine and Iysinef20lOur results definitely prove that the low molecu-lar enzyme consists of two chains linked by adisulflde bridge. The present view of its structure



140 W. A. G nzler, G. J. Steffens, F. tting, G. Buse and L. Flohe Bd. 363 (1982)

Table 5. Amino acid analysis [mol/100 mol] of the30000-Da chains of low and high molecular mass uro-kinase.

Amino acid

AsxThrSerGlxProGlyAlaCysValMetlieLeuTyrPheGlcNGalNHisTrpLysArg

Low molecularform30000-Da chain

7.797.287.71

10.135.539.314.753.31a

4.401.715.417.644.642.770.780.433.661.91b

5.455.38

High molecularform30000-Da chain

7.937.427.859.456.488.954.882.97a

4.881.765.727.624.692.850.820.353.631.485.705.00

3 Determined as S-carboxymethy Icy steine.b Determined after acid hydrolysis in the presence of4% thioglycolic acid.

is depicted in Scheme 2. The 30000-Da chainstarts with a TV-terminal isoleucine as describedpreviously for the high molecular form by Lesuket aU3l and corresponds to the sequence reportedby Studer et aU19! and Henschen et aU18J forthe TV-terminus of the low molecular form. This30000-Da subunit is identical in both enzyme spe-cies. The sequence of the 2427-Da chain corres-

ponds to the TV-terminus ascribed to the lowmolecular form by Lesuk et alJ3l and to the TV-terminal sequence of a low molecular mass com-ponent found by Gillessen et alJ2°l.As to the problem of how the low molecular en-zyme is derived from the high molecular form,our results are only consistent with one of thetheoretical possibilities outlined in Scheme 1.Therefore, it seems safe to assume that1) the transformation of the high into the low

molecular form represents a limited proteoly-tic attack exclusively at the 20000-Da chainof the former,

2) the 20000-Da chain is degraded from the TV-terminus, and

3) a 2427-Da "core" peptide of 21 amino acidsis generated, which remains linked to the30000-Da chain by a single disulfide bridge.

Gradual proteolysis of the 20000-Da chain mostprobably explains by-products consistently ob-served in high molecular mass urokinase usingelectrophoresis, gel Chromatographie separationand TV-terminal analysis. As a final degradationproduct, the 2427-Da chain still contains two po-tential sites for tryptic cleavage at positions 10and 16, respectively. Probably, these sites areburried and are not available for further proteoly-tic attack.The enzymatically active protease forms, high andlow molecular mass urokinase, share the propertyof containing two different peptide chains withother serine proteases e.g. plasmin. While the20000-Da chain of the high molecular enzymemay be compared to the heavy (A) chain of hu-man plasmin in terms of variable size, the 30000-Da subunit of urokinase and the light (B) chainof human plasmin obviously correspond in termsof size, amino acids of the active site, invariability

30000-Da Chain Carbohydrate

H -[ I I G G E F T T I E N Q P W F A A I Υ R R ) ( approx. 230 amino acids ((A L [-QH

Η-[""κ P S S P P E E L K F Q C G Q K T L R P R~]-OH

2427-Da ChainScheme 2. Structure of low molecular mass urokinase according to the results presented.



Bd. 363(1982) Human Urokinases 141

in different enzyme forms, and TV-terminal aminoacid sequence 1211. Striking homologies existbetween the TV-terminal amino acid sequences ofthe 30000-Da subunit of urokinase and of por-cine pancreatic or human urinary kallikrein[22,23,18]. 9 ollt Of the flrst 21 amino acid posi-tions are found to be identical. The relationshipof urokinase with other serine proteases will,most probably, facilitate further work on itsamino acid sequence.

This work was supported by grant PTB 8239 from theBundesministerium für Forschung und Technologie.

Literature

1 White, W.F., Barlow, G.H. & Mozen, M.M. (1966)Biochemistry 5, 2160-2169.

2 Soberano, M.E., Ong, E. B., Johnson, A.J., Levy,M. & Schoellmann,G. (I9l6)ßiochim. Biophys.Acta 445, 763-773.

3 Lesuk, A., Terminiello, L., Traver, J.H. & Groff, J.L.(1967) Fed. Proc. 26,647.

4 Soberano, M.E., Ong, E.B. & Johnson, A.J. (1976)Thromb. Res. 9, 675-681.

5 Holmberg, L., Bladh, B. & Astedt, B. (1976) Bio-chim. Biophys. Acta 445, 215-222.

6 Laemmli, U.K. (1970) Nature (London) 227,680-685.

7 Claeson, G-, Friberger, P., Knös, M. & Eriksson, E.(1978) Haemostasis 7, 76-78.

8 Crestfield, A.M., Moore, S. & Stein, W.H. (1963) /.Biol. Chem. 238, 622-627.

9 Biotronik (1981) Applikationsreport ASA 981/2,München.

10 Edman, P. (1975) in Molecular Biology, Biochemi-stry Biophysics, vol. 8: Protein Sequence Determina-tion (Needleman, S.B., ed.) pp. 214-226, SpringerVerlag, Berlin.

11 Crewther, W.G. & Inglis, A.S. (1975) Anal. Bio-chem. 68,572-585.

12 Steffens, G. J. (1980) Habilitationsschrift, RWTH-Aachen, pp. 37-39.

13 Machleidt, W. & Wächter, E. (1977) Methods En-zymol. 47, 263-277.

14 Laursen, R.A. (1970) Eur. J. Biochem. 20, 89-102.15 Zimmerman, C.L., Appella, E. & Pisano, J.J. (1980)

Anal. Biochem. 77, 569-573.16 Steffens, G.J. & Buse, G. (1976) Hoppe-Seyler's Z.

Physiol. Chem. 357, 1125-1137.17 Jolles, J., Schentgen, F., Alais, C., Fiat, A.M. &

Jolles, P. (1972) Helv. Chim. Acta 55, 2872-2883.18 Henschen, A., Reich, E., Sauser, D., & Lottspeich, F.

(1981) in Verhandlungsber. d. 25. Tag. d. Dtsch.Arbeitsgem. f. Blutgerinnungsforsch, in München,Febr. 1981 (Blümel, G. & Haas, S., eds.) pp.367-370, Schattauer-Verlag, Stuttgart.

19 Studer, R.O., Roncari, G. & Lergier, W. (1977) inThrombosis and Urokinase (Paoletti, R. & Sherry,S., eds.) pp. 89-90, Academic Press, New York.

20 GUlessen, D., Lergier, W., Studer, R.O., Schaller, J.,Nick, H. & Rickli, E.E. (1981) Serono Sympos.Genf, Abstr. p. 38.

21 Wiman, B. (1977) Eur. J. Biochem. 76,129-137.22 Fiedler, F., Ehret, W., Godec, G., Hirschauer, C.,

Kutzbach, C., Schmidt-Kastner, G. & Tschesche, H.(1977) in Kininogenases-Kallikrein 4 (Haberland, G.,Rohen, J.W. & Suzuki, T., eds.) pp. 7-14, Schat-tauer-Verlag, Stuttgart.

23 Lottspeich, F., Geiger, R., Henschen, A. & Kutzbach,C. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360,1947-1950.

Dr. W.A. Günzler, PD Dr. G.J. Steffens, Dr. F. Ötting und Prof. Dr. L. Flohe, Grünenthal GmbH, Forschungs-zentrum,Zieglerstraße 6, D-5100 Aachen-Eilendorf.Prof. Dr. G. Buse, RWTH Aachen, Abteilung Physiologische Chemie,Melatener Straße 211, D-5100 Aachen.



structural relationship between human high and low molecular mass urokinase

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