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Biological Chemistry Hoppe-Seyler

Volume 371 Supplement Issue

Proteinase Inhibitors and Biological Control

Selected Contributions presented at

Brdo, Jugoslavia June 25-28,1989

Edited by Hans Fritz, I . Schmidt, and Vito Turk

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DE

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1990 Walter de Gruyter · Berlin · New York

Biological Chemistry Hoppe-Seyler THE OFFICIAL ORGAN OF THE GESELLSCHAFT FÜR BIOLOGISCHE CHEMIE

This Journal was founded in 1877 as Zeitschrift für Physiologische Chemie by F. Hoppe-Seyler and was continued after his death under the editorship of A . Kossei, F. Knoop , K.Thomas, F. Lynen, A . Butenandt and G.Weitzel as

Hoppe-Seyler s Zeitschrift für Physiologische Chemie [Volume 21 (1895) -Vo lume 365 (1984)].

Volume 371 May 1990 Supplement

CONTENTS

Serine Proteinases and Their Inhibitors Serpins: Structure and Mechanism of Action ./. Travis, A. Guzdek, J. Potempa and W. Watorek 3

Structure of Inter-a-Inhibitor (Inter-α-Trypsin Inhibitor) and Pre-«-Inhibitor: Current State and Proposition of a New Terminology W. Gebhard, Κ. Hochstraßer, Η. Fritz, J. J. Enghild, S. V. Pizzo and G. Salvesen I 3

The Squash Inhibitor Family of Serine Proteinases 7. Odewski 2 3

Human Leukocyte Elastase Inhibitors: Designed Variants of Human Pancreatic Secretory Trypsin Inhibitor (hPSTI ) 7. Collins, M. Szardenings, F. Maywald, H. Blöcker, R. Frank, H.-J. Hecht, Β. Vasel, D. Schomburg, E. Fink and H. Fritz 2^

Aprotinin and Aprotinin Analogues Expressed in Yeast K. Nortis, F Norris, S.E. Bj0rn, I. Diets and L.C. Petersen 3^

Design of an Aprotinin Variant with Inhibitory Activity against Chymotrypsin and Cathepsin G by Recombinant D N A Technology T. Brinkmann and H. Tschesche ^3

Refolding of Proteinase Inhibitor Variants on Trypsin-Sepharose as a Matrix with Complementary Structure Ρ Flecker 5 3

Irregular Elastase Complexes: Crystallographic and Molecular Dynamics Studies of Small Inhibitors and Water E. Meyer and M. Geller 59

A New, Highly Sensitive Enzymic Assay for Human Tryptase and its Use for Identification of Tryptase Inhibitors S.C. Dietze, E.-A. Auerswald and Η. Fritz ^

Inhibition of Histamine Release from Human Mast Cells E x Vivo by Natural and Synthetic Chymase Inhibitors S. C. Dietze, C. Ρ Sommerhoff and Η. Fritz ^

Regulation of Mouse Τ Cell Associated Serine Proteinase-1 (MTSP-1) by Proteinase Inhibitors and Sulfated Polysaccharides M.M. Simon, T. Tran, U. Fruth, D. Gurwitz and M.D. Kramer 81

Inhibitors of the Cell Surface Protease Guanidinobenzoatase FS. Steven, M.M. Griffin, H. Maierandl.C. Talbot 89

Response to I L - 6 Stimulation of Human Hepatoblastoma Cells: Production of Pancreatic Secretory Trypsin Inhibitor T. Yasuda, M. Ogawa, A. Murata, Y. Oka, K.-i. UdaandT. Mori 95

Polyethylene Glycol Modification of Serpins Improves Therapeutic Potential A. E. Mast, G. Salvesen, FH. Brucato, H.-P Schnebli and S. V. Pizzo 1 0 1

Cysteine Proteinases and Their Inhibitors Mechanism of Interaction of Cysteine Proteinases and Their Protein Inhibitors as Compared to the Serine Proteinase-Inhibitor Interaction W. Bode, R. Engh, D. Musil, B. Laber, M. Stubbs, R. Η über and V. Turk

Equilibrium and Kinetic Studies of the Interaction of Chicken Cystatin with Four Cysteine Proteinases /. Βjörk, Κ. Ylinenjärvi and Ρ Lindahl

Copyright © by Walter de Gruyter & Co · Berlin · New York

May 1990 Contents Vol. 371 Suppl. (1990)

N-Terminal Variants of Recombinant Stefin B: Effect on Affinity for Papain and Cathepsin Β U. Thiele, I. Assfalg-Machleidt, W. Machleidt and E.-A. Auerswald 125

Synthesis of Cysteine Proteinase Inhibitors Structurally Based on the Proteinase Interacting N-Terminal Region of Human Cystatin C A. Grubby M. Abrahamson, I. Olafsson, J. Trojnar, R. Kasprzykowska, F. Kasprzykowskiand Z. Grzonka 137

Efficient Preparation of Human Recombinant Cystatin A by Escherichia coli Η. Kaji, Τ Samejima, Ι. Kumagai, Τ Hihino, K.-i. Μ iura and A. Takeda 145

Refolding of Chicken Cystatin and Human Stefin A Studied by Fast Protein Liquid Chromatography ( F P L C ) E. Zerovnik, J. Kos, Β. Lenarcic and V. Turk 151

Mutations in the Q V V A G Region of the Cysteine Proteinase Inhibitor Stefin Β R. Jerala, M. Trstenjak-Prebanda, L. Kroon-Zitko, B. Lenarcic and V. Turk 157

Rat Cystatin C : The Complete Amino Acid Sequence Reveals a Site for N-Glycosylation F. Esnard, A. Esnard, D. Faucher, J . - R Capony, J. Derancourt, M. Brillard and F. Gauthier 161

Low Molecular Mass Protein Inhibitor of Cysteine Proteinases from Soybean J. Brzin, A. Ritonja, Τ Popovic and V Turk 167

A Novel Member of the Calcium-dependent Cysteine Protease Family H. Sorimachi, S. Ohmi, Y. Emori, H. Kawasaki, T.C Saido, S. Ohno, Y Minami and K. Suzuki 171

Effects of Autolysis on the Catalytic Properties of the Calpains V Ε Thompson, D. E. Göll and W. C. Kleese 177

Tissue and Biological Fluid Distribution of Cysteine Proteinases Inhibitor: Rat Cystatin C C. Tavern, D. Prevot, J. Ρ Girolami, J. Leung-Tack and A. Colic 187

Role for Cathepsin Β and Cystatins in Tumor Growth and Progression Β. Ε Sloane, J. Rozhin, D. Robinson and Κ. V. Ho/in 193

Cysteine Proteinase Inhibitors in Human Cancerous Tissues and Fluids Τ Τ Lah, Μ. Kokalj-Kunovar and V Turk 199

Activities of Calcium-Activated Neutral Proteases and its Endogenous Inhibitor in Skeletal Muscle of Dystrophic Hamster S. Kawashima, M. Nakamura and M. Hayashi 205

Cathepsin Β - Indicator for the Release of Lysosomal Cysteine Proteinases in Severe Trauma and Inflammation /. Assfalg-Machleidt, M. Jochum, D. Nast-Kolb, M. Siebeck, A. Billing, Τ Joka, G. Rothe, G. Valet, R. Zauner //. - P. Scheu her and W. Machleidt 211

Mode of Activation of the Precursor to Cathepsin L : Implication for Matrix Degradation in Arthritis R.A. Macicwicz, R.J. Wardale, S.E Wotton, V.C. Duance and D.J. Etherington 223

Cystatin C Mutation Causing Amyloid Angiopathy and Brain Hemorrhage 0. Jensson, A. Pälsdottir, L. Thorsfeinsson, A. Arnason, M. Abrahamson, /. Olafsson and A. Grubb 229

Evidence of Protease Activity Producing NH 2-Terminal Truncated Form of Rat Cystatin A in Newborn Rat Epidermis A. Takeda, Y Nakamura, hi. Kaji and T. Samcjima 233

„In Vitro 4 k Digestion of Intakt Bovine Lens Capsules by Four Human Lysosomal Cysteine-Proteinases N. Guinec, M. Pagano, V. Dalei-Fumeron and R. Engter 239

Rat Cystatin C : Inhibitor of Granulocyte Phagocytic Functions J. Leung-Tack, C. Tavern, A. Er-Raki, M.C. Gensac and A. Colic 255

High Affinity Binding of Rat Brain Cathepsin Β to Purified Rat/Bovine Cerebral Mannose 6-Phosphate Receptors L.-M. Chi, M.-X. Zhu and N. Marks 259

Use of Experimental Models for the Study of Collagen Degradation by Macrophages I. N. Bird, LA. Silver, R.A. Maciewicz and D.J. Etherington 265

Aspartic Proteinases and Their Inhibitors Expression in Escherichia coli of the Aids Virus Aspartic Protease through a Protein Fusion B. D. Korant and C.J. Rizzo 271

Characterization and Inhibition of the Retroviral HIV-Proteinase K. von der Helm, S. Seelmeir and U. Junker 277

Vol. 371 Suppl. (1990) Contents May 1990

Structural and Functional, Analogies between Fibronectin and Retroviral Proteinases V. Keil-Dlouha 2 8 3

A Kinetic Analysis of the Activation of Pig Pepsinogen by Chemical and Physical Techniques D.M. Click / 2 8 9

Metalloproteinases Partial Amino Acid Sequence of Human PMN Leukocyte Procollagenase V. Knüuper, S. Kramer, iL Reinke and Ii. Tschesche 2 ^ 5

The Role of Matrix Metalloproteinase 3 in the Stepwise Activation of Human Rheumatoid Synovial Procollagenase Κ. Suzuki, Η. Nagase, Α. I to, J.J. Enghild and G. Salvescn 305

A New Look at Pz-Peptidase A.J. Barrett 311

Human Liver Aminopeptidase Μ and its Regulation by Bile Acids M. Sasaki, M. Nakanishi and A. Moriyama 321

Protease Mechanism Common Feature of the Four Types of Protease Mechanism L . Pol gar 327

Biol . Chem. Hoppe-Seyler Vol. 371. Suppl., pp. 211-222. May 1990

Cathepsin Β - Indicator for the Release of Lysosomal Cysteine Proteinases in Severe Trauma and Inflammation

IRMGARD ASSFALG-MACHLEIDT", MARIANNE JOCHUM1", DIETER NAST-KOLB C , MATTHIAS STEBECKC, A R E N D B I L L I N G 0 , THEO JOKA C , GREGOR ROTHE 1, GÜNTER VALET 1 , RITA ZAUNER 3 , HEINZ-PETER SCHEUBER1" AND WERNER MACHLEIDT 3

a Inst i tut für Physiologische Chemie, Physikalische Biochemie und Zel lbiologie der Universität München, Goethestr. 33, D-8000 München 2, FRG h Ab te i lung für Klinische Chemie und Klinische Biochemie in der Chirurgischen K l i n i k Innenstadt der Universität München, Nußbaumstr. 20, D-8000 München 2 c Chirurgische K l in ik Innenstadt der Universität München, Nußbaumstr. 20. D-8000 München 2 d Chirurgische K l in ik und Pol ikl inik der Universität München. K l i n i k u m Großhadern. Marchioninistr . 15. D-8000 München 70 0 Chirurgische Universitätskliniken Essen, D-4300 Essen 1 Arbeitsgruppe Zel lbiochemie, Max-Planck-Institut für Biochemie. A m Klopferspitz 18a. D-8033 Mart insr ied

Int roduct ion

Lysosoma l e l a s tase f rom p o l y m o r p h o n u c l e a r (PMN) g r a n u l o c y t e s is b e l i e v e d t o be an i m p o r t a n t nonspec i f i c m e d i a t o r o f i n f l a m m a t i o n c o n t r i b u t i n g t o t h e d e v e l o p m e n t o f s e p s i s - and t r a u m a - r e l a t e d o r gan f a i l u r e [1 ] . We h a v e f o u n d p r e v i o u s l y t h a t a lso t h e l y sosoma l c y s t e i n e p r o t e i n a s e (CP) c a t h e p s i n Β or a c a t h e p s i n B - l i k e enzyme is r e l eased i n t o t h e b lood p l a s m a o f p a t i e n t s d u r i n g s ep t i c shock [21. I n c o n t r a s t t o PMN e l a s t a s e , r e l e a s e d c a t h e p s i n Β can be d e t e c t e d by i t s enzymat i c a c t i v i t y because i t i s o n l y l o o s e l y b o u n d t o t h e p lasma i n h i b i t o r s and d i s soc i a t e s r e a d i l y u p o n d i l u t i o n .

Here we w a n t t o r e p o r t f u r t h e r e v i d e n c e f o r t h e r e l ease o f c y s t e i n e p r o t e i n ases i n t o t h e c i r c u l a t i o n of p o l y t r a u m a t i z e d p a t i e n t s a n d , l o c a l l y , i n t o t h e a l v e o l a r e p i t h e l i a l l i n i n g f l u i d a n d p u r u l e n t p e r i t o n e a l e x u d a t e s . I n c r e a s e d c y s t e i n e p r o t e i n a s e a c t i v i t y was a lso o b s e r v e d i n t h e b l o od p l asma o f p i gs sub j e c t ed t o e x p e r i m e n t a l s ep t i c shock . I n a l l cases t h e re l eased c y s t e i n e p r o t e i n a s e a c t i v i t y was i d e n t i f i e d as t h a t o f c a t h e p s i n Β o r a c a t h e p s i n B -l i k e enzyme. I t s i n t e r a c t i o n w i t h t h e p l a sma c y s t e i n e p r o t e i n a s e i n h i b i t o r s was i n v e s t i g a t e d i n v i t r o u s i n g samples f r om i n d i v i d u a l p a t i e n t s . I n d i r e c t e v i d ence w i l l be p r o v i d e d t h a t t h e m a i n p o r t i o n o f t h e r e l eased l y s o s o m a l c y s t e i n e p r o t e inase ( s ) s h o u l d o r i g i n a t e f r o m ce l l s o t h e r s t h a n PMN g r a n u l o cy t es , most p r o b a b l y f rom macrophages , a n d may p l a y a ro l e as n o n s p e c i f i c m e d i a t o r ( s ) o f i n f l a m m a t i o n .

Enzymes: C a t h e p s i n Β (EC 3.4 .22.1 ) ; C a t h e p s i n Η (3 .4 .22 .16 ) ; C a t h e p s i n L ( 3 .4 .22 .15 ) ; L e u k o c y t e e las tase (EC 3.4 .21.37) . Abbreviations: 012M, a 2 - m a c r o g l o b u l i n ; (XiPI, α ι -p ro t e inase i n h i b i t o r ; ARDS, a d u l t r e s p i r a t o r y d i s t r e s s syndrome; BALF, b r o n c h o a l v e o l a r l a v a g e f l u i d ; CP, c y s t e i n e p r o t e i n a s e ( s ) ; ELISA, e n z y m e - l i n k e d i m m u n o s o r b e n t assay ; E - 6 4 , l-(trans-e p o x y s u c c i n y l - L - l e u c y l a m i n o ) - 4 - g u a n i d i n o b u t a n e ; FPLC, f a s t p r o t e i n l i q u i d c h r o m a t o g r a p h y ; NMec, ( 4 - m e t h y l - 7 - c o u m a r y l ) a m i d e ; PMN, p o l y m o r p h o n u c l e a r ; SEM, s t a n d a r d e r r o r o f t h e mean; Z, b e n z y l o x y c a r b o n y l .

Copyright © by Walter de Gruyter & Co · Berlin · New York

212 I . Assfalg-Machleidt et al . Vol.371 Suppl. (1990)

Mater ia l s and Methods

Patients and samples

D e t a i l s a b o u t t h e o r g a n i z a t i o n o f t h e c l i n i c a l s tud i e s and samp l i ng p r o t o c o l s are p r o v i d e d i n r e f . [3] f o r p o l y t r a u m a t i z e d p a t i e n t s , i n re f . [4] f o r BALF and i n r e f . [5 ,6 ] f o r p e r i t o n i t i s e x u d a t e s . E x p e r i m e n t a l s ep t i c shock i n anes t e s i z ed p igs was p e r f o r m e d as d e s c r i b e d [7J. A l l samples were s t o r e d f r o z en and t h a w e d o n l y once i m m e d i a t e l y be fore d e t e r m i n a t i o n .

Separation and lysis of blood cells

H e p a r i n i z e d h u m a n b l o o d , d i l u t e d w i t h t i s s u e c u l t u r e med ium RPMI 1640 (Gibco) was l a y e r e d o v e r l y m p h o c y t e s e p a r a t i o n medium MSL ( d = 1.077, E u r o b i o ) and c e n t r i f u g e d . T h e m o n o n u c l e a r c e l l s were r e c e n t r i f u g e d i n RPMI and l y s e d i n 50 mM s o d i u m a c e t a t e b u f f e r pH 5.5 c o n t a i n i n g 0.5 % T r i t o n X - 1 0 0 , 0.3 mM EDTA a n d 0 .01 % B r i j 35 . For p r e p a r a t i o n o f PMN g r a n u l o c y t e s M o n o - P o l y R e s o l v i n g Med ium ( F l ow ) was used .

Assay for cysteine proteinase activity

CP a c t i v i t y was m e a s u r e d i n c o n t i n u o u s assays w i t h t h e f l uo r ogen i c s u b s t r a t e s Z - P h e - A r g - N M e c , Z - A r g - A r g - N M e c a n d A r g - N M e c u s i n g the spec i f i c i n h i b i t o r E - 6 4 as d e s c r i b e d [ 2 ] .

Stopped assay for papain inhibiting capacity

I n c r e a s i n g v o l u m e s o f sample d i l u t i o n s were i n c u b a t e d w i t h p a p a i n (7 nM) i n a c e t a t e b u f f e r pH 5.5, 1 mM d i t h i o t h r e i t o l , 2 mM EDTA, 0.015 % Br i j 35, f o r 25 m i n a t 30 °C. A f t e r d i l u t i o n t h e s u b s t r a t e Z - P h e - A r g - N M e c (10 μΜ) was added a n d t h e r e a c t i o n was s t opped w i t h monoch l o r oac e t a t e a f t e r 1 5 - 2 5 m i n .

ELISA for human cathepsin Β

I m m u n o s e l e c t e d p o l y c l o n a l a n t i b o d i e s a g a i n s t h u m a n c a t h e p s i n Β were p r epa r ed f r om sheep a n t i s e r u m w i t h c a t h e p s i n Β immob i l i z ed on C N B r - a c t i v a t e d Sepharose . C a t h e p s i n Β b o u n d t o m i c r o t i t e r p l a t e s coated w i t h t h e a n t i b o d i e s was d e t e r m i n e d i n a s a n d w i c h ELISA u s i n g b i o t i n y l a t e d a n t i b o d i e s and b i o t i n -a v i d i n - p e r o x i d a s e f o r d e t e c t i o n .

ELISA for human elastase

G r a n u l o c y t i c e l a s t a s e was e s t i m a t e d i n complex w i t h a i - p r o t e i n a s e i n h i b i t o r a c c o r d i n g t o r e f . [5 ] u s i n g a spec i f i c t w o - s i t e ELISA.

Gel chromatography

S e p a r a t i o n o f p a t i e n t p l a s m a on a Superose 12 FPLC co lumn was per fo rmed as d e s c r i b e d [ 2 ] . I n h i b i t o r y a c t i v i t y o f t h e i n d i v i d u a l f r a c t i o n s was d e t e r m i n e d i n s t o p p e d a s says as a b o v e u s i n g 1/40 o f each f r a c t i o n (0.4 ml ) w i t h 200 pM p a p a i n .

Vol. 371 Suppl. (1990) Cathepsin Β Activity inTrauma and Inflammation 213

R e s u l t s

CP a c t i v i t y in blood plasma of poly t raumat i zed p a t i e n t s

CP a c t i v i t y o f b lood p lasma was f o l l o w e d i n 69 i n t e n s i v e ca r e p a t i e n t s due to s e v e r e t r a u m a w i t h a mean i n j u r y s e v e r i t y score (1SS) a c c o r d i n g t o B a k e r e t a l . [8] o f 36 (see [3] f o r d e t a i l s ) . W i t h i n 6 h a f t e r t h e i n j u r y , i n c r e a s e d l e v e l s o f CP a c t i v i t y ( mean: 5 - 6 - f o l d o f n o r m a l ) were f o u n d i n t h e b l o o d p l a sma o f p a t i e n t s who deve l oped l e t h a l ( F i g . 1 A) or r e v e r s i b l e ( F i g . 1 B) m u l t i p l e o r g a n f a i l u r e s e ve ra l days l a t e r . I n i t i a l CP a c t i v i t y was s i g n i f i c a n t l y l owe r (mean : 3 - f o l d o f no rma l ) i n p o l y t r a u m a t i z e d p a t i e n t s w i t h o u t s u b s e q u e n t o r gan f a i l u r e (F i g . 1 C). In a l l t h r e e g roups CP a c t i v i t y r e a c h e d i t s m a x i m u m 6 h be fo re c u P I - b o u n d PMN e las tase , and r e t u r n e d t o v a l u e s a b o u t 2 - f o l d o f t h e n o r m a l w i t h i n 3 days .

0 1 2 3 4 5 6 7 8 9 10 11 12 13 Η 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Η

Time [d] Time [d]

15 -| 1 1 1 1 1 1 1 I 1 1 I I L

0 Η 1 1 1 1 1 1 1 1 1 1 1 1 1

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Time [dj

Fig . 1. Catheps in Β a c t i v i t y and αιΡΙ-bound e l a s t a s e l e v e l s in blood p lasma of polytraumatized pat ients expressed as the mul t ip le of normal v a l u e s (50 mU/1 f o r c a t h e p s i n B, 90 ng/ml f o r e l a s t a s e ) . A, p a t i e n t s w i t h l e t h a l o r g a n f a i l u r e ( n = l l ) ; B, p a t i e n t s w i t h r e v e r s i b l e o r gan f a i l u r e ( n = 2 9 ) ; C, p a t i e n t s w i t h o u t o rgan comp l i c a t i ons ( n = 2 9 ) .

214 I . Assfalg-Machleidt et al. Vol. 371 Suppl. (1990)

As r e p o r t e d p r e v i o u s l y [2 ] , d i l u t i o n e x p e r i m e n t s r e v e a l e d t h a t t h e C P - a c t i v i t y o f b lood p lasma is i n e q u i l i b r i u m w i t h r e v e r s i b l e c y s t e i n e p r o t e i n a s e i n h i b i t o r s . E v a l u a t i o n o f d i l u t i o n e x p e r i m e n t s p e r f o r m e d w i t h p l a s m a o f i n d i v i d u a l p o l y t r a u m a t i z e d p a t i e n t s (F i g . 2) p r o v i d e d e s t i m a t e s o f Km = 98 μΜ and It/Κι = 1 5 - 2 0 (Km, M i chae l i s c o n s t a n t ; I t , t o t a l i n h i b i t o r c o n c e n t r a t i o n ; K i , i n h i b i t i o n c o n s t a n t ) . The r o u t i n e assay used i n t h e c l i n i c a l s t u d i e s w i t h p o l y t r a u m a t i z e d p a t i e n t s (80 μ] P lasma i n 1.5 ml t o t a l v o l u m e , 150 μΜ s u b s t r a t e ) d e t e c t s app rox . 60 % o f t o t a l C P - a c t i v i t y as f r ee , a c t i v e enzyme .

CP a c t i v i t y i n bronchoa lveo lar l avage f lu id

One o f t h e f a t a l c o m p l i c a t i o n s o f p o l y t r a u m a t i z e d p a t i e n t s i s t h e a d u l t r e s p i r a t o r y d i s t r e s s syndrome (ARDS). We f o u n d t h a t b r o n c h o a l v e o l a r l a vage f l u i d (BALF) o f a p a t i e n t w i t h ARDS c o n t a i n e d h i g h l e v e l s o f CP a c t i v i t y compared t o t h a t o f a p o l y t r a u m a t i z e d p a t i e n t w i t h o u t t h i s c o m p l i c a t i o n (F ig . 3 ) . For c ompar i s on w i t h PMN e l as tase , t h e CP a c t i v i t y was c o n v e r t e d i n t o ng/ml c a t h e p s i n Β assuming a spec i f i c a c t i v i t y o f 80 ü/mg f o r p u r i f i e d c a t h e p s i n Β [21. The m a x i m a l l e v e l s o f α ιΡ Ι -bound e l a s t a s e were a p p r o x . 2 0 -f o l d h i g h e r t h a n those o f c a t h e p s i n Β a n d d i d n o t a l w a y s c o i n c i d e w i t h t h e l a t t e r . I n some samples an e l a s t a s e / c a t h e p s i n Β r a t i o c lose t o 1.0 was obse r v ed .

C a t h e p s i n Β c o n c e n t r a t i o n s measured w i t h an ELISA u s i n g spe c i f i c a n t i b o d i e s a g a i n s t h u m a n l y sosoma l c a t h e p s i n Β c o r r e l a t e d w e l l w i t h t h e c o n c e n t r a t i o n s c a l c u l a t e d f r o m a c i t i v i t y (Tab le 1). These r e s u l t s i n d i c a t e t h a t , i n t h i s p a t i e n t , v i r t u a l l y a l l measured c y s t e i n e p r o t e i n a s e a c t i v i t y was due to c a t h e p s i n Β (or a c r o s s - r e a c t i v e p roenzyme ) a n d most o f t h e i m m u n o l o g i c a l l y d e t e c t a b l e enzyme was e n z y m a t i c a l l y a c t i v e . T i t r a t i o n o f p a p a i n w i t h BALF r e v e a l e d t h e absence o f c y s t e i n e p r o t e i n a s e i n h i b i t o r s ( l ess t h a n 10 nM compared to 5 μΜ i n n o r m a l p lasma) and no d i s s o c i a t i o n was o b s e r v e d i n d i l u t i o n e x p e r i m e n t s .

0 10 20 30 40 50 60 70 80 90 100 -100 -80 -60 -40 -20 0 20 40 60 80 100

Plasma [μΙ] Plasma [μΙ]

Fig . 2. D i lut ion a n a l y s i s of CP a c t i v i t y in blood p lasma of a po lyt raumat ized pat ient . F rom a r e p l o t a c c o r d i n g t o D i x o n [9} ( l owe r pane l ) Km was e s t i m a t e d as 98 μΜ and a I t / K i o f 16 was c a l c u l a t e d f o r c o m p e t i t i v e i n h i b i t i o n (Km, Michae l i s c o n s t a n t ; I t , t o t a l i n h i b i t o r c o n c e n t r a t i o n ; K i , i n h i b i t i o n c o n s t a n t ) .


Ο

140 τ

120

1 0 0 -

8 0 -

6 0 -

4 0

2 0 -

0

γ 2 5 0 0

- 2 0 0 0

1 - 1500

- 1 0 0 0 2 CO «3

ÜJ - 500 Ο

0 1 2 4 5 6 7 8 9 10 11 12 13 14 21

Time Cd]

F i g . 3. L e v e l s of c a t h e p s i n Β ( f i l led c i r c l e s ) and αιΡΙ-bound e l a s t a s e (open c i r c l e s ) i n b ronchoa lveo la r l avage f luid of po lyt raumat ized pa t i en t s wi th ARDS (A) and wi thout ARDS (B). T h e c a t h e p s i n Β l e v e l was c a l c u l a t e d f r om e n z y m a t i c a c t i v i t y a s s u m i n g a spec i f i c a c t i v i t y o f 80 U/mg.

T a b l e 1. C o r r e l a t i o n between CP a c t i v i t y and immuno-react ive c a t h e p s i n Β (ELISA ) i n BALF of a p a t i e n t w i th ARDS

Sample 3 CP a c t i v i t y

tmU/1]

E n z y m a t i c a l l y a c t i v e

c a t h e p s i n B b

[ng/ml]

Immune-reactive

c a t h e p s i n Β [ng/ml]

431/1 3 267 40 .8 39.7 431/5 618 7.7 8.0 431/7 9 194 114.9 121.7 431/8 4 429 55.4 46.6 431/10 2 232 27.9 23.6 431/11 618 7.7 10.8 431/12 1 622 20.3 26.2

a see F i g . 3 f o r c o m p a r i s o n C a l c u l a t e d a s s u m i n g a spec i f i c a c t i v i t y o f 80 U/mg


1000

800 -

-5 600 ü CO

CD JZ CO Q .

φ

400

£ 200

α

Time [h]

F i g . 4. C a t h e p s i n Β a c t i v i t y i n b l ood p l a s m a o f p i g s s u b j e c t e d t o e x p e r i m e n t a l e n d o t o x i n shock . 19 a n e s t e s i z e d m i n i a t u r e p igs r e c e i v e d an i . v . i n f u s i o n o f b a c t e r i a l l i p o p o l y s a c c h a r i d e f r o m 5. abortus equi f o r 6 h o u r s a n d d e v e l o p e d a s e v e r e sep t i c shock w i t h low a r t e r i a l mean p r e s s u r e ( s o l i d t r i a n g l e s ) . C a t h e p s i n Β a c t i v i t y i n c r e a s e d ( s o l i d c i r c l e s ) as compared t o c o n t r o l a n i m a l s ( n = 7 ) r e c e i v i n g an NaCl i n f u s i o n (open c i r c l e s ) . D a t a are mean v a l u e s + / -SEM.

CP a c t i v i t y i n e x p e r i m e n t a l e n d o t o x i n shock o f p i g s

A r e p r o d u c i b l e i n c r e a s e o f CP a c t i v i t y o f b l ood p l a sma was o b s e r v e d i n p igs s u b j e c t e d t o s e v e r e s e p t i c shock by i n f u s i o n o f b a c t e r i a l e n d o t o x i n . C a t h e p s i n Β a c t i v i t y c o r r e l a t e d w i t h p h y s i o l o g i c a l shock p a r a m e t e r s l i k e decrease o f a r t e r i a l mean p r e s s u r e ( F i g . 4 ) .

1 I c

CD

CO Q . <D

SZ +-» CO

Ο ο in -

ο clear exudates • acute peritonitis • Etappentavage A pancreatitis

Ik 30000 60000 90000

Elastase [ng/ml]

1 r

120000 150000

F i g . 5. C a t h e p s i n Β v e r s u s c u P I - b o u n d e l a s t a s e l e v e l s i n p e r i t o n e a l e x u d a t e s . C a t h e p s i n Β l e v e l s we re c a l c u l a t e d f r om e n z y m a t i c a c t i v i t y a s s u m i n g a spec i f i c a c t i v i t y o f 80 U/mg; c u P I - b o u n d e l as tase was d e t e r m i n e d by ELISA [5 ] . Open c i r c l e s , c l ea r e x u d a t e s ( n = 9 ) ; s o l i d c i r c l e s , a c u t e p e r i t o n i t i s ( n = 9 ) ; s o l i d s q u a r e s , p e r s i s t e n t p e r i t o n i t i s t r e a t e d by E t a p p e n l a v a g e ( n = 9 ) ; s o l i d t r i a n g l e s , p a n c r e a t i t i s ( n = 2 ) .


CP a c t i v i t y in pe r i tonea l e xuda te s

P e r i t o n i t i s e x u d a t e s c o n t a i n h i g h l e v e l s o f o t i P I - b o u n d a n d e v e n f ree e l a s t a s e w h i c h i s b e l i e v e d t o c o n t r i b u t e t o n o n s p e c i f i c p r o t e o l y t i c b r e a k d o w n o f o p s o n i n s l i k e C3 and IgG r e s u l t i n g i n d i m i n i s h e d o p s o n i z i n g a c t i v i t y [5) . We f o u n d a l so h i g h l e v e l s o f CP a c t i v i t y i n most o f t h e s e e x u d a t e s (F i g . 5, Tab l e 2 ) . CP a c t i v i t y d i d n o t a l w a y s p a r a l l e l o u P I - b o u n d e l a s t a s e . H i g h CP a c t i v i t y a n d m o d e r a t e e l a s t a s e l e v e l s were f o u n d i n cases o f a c u t e p e r i t o n i t i s whereas b o t h CP a c t i v i t y a n d e l a s t a s e c o n c e n t r a t i o n t e n d e d t o be e x t r e m e l y e l e v a t e d i n p a t i e n t s w i t h p e r s i s t e n t p u r u l e n t p e r i t o n i t i s t r e a t e d b y E t a p p e n l a v a g e [6 ] . CP a c t i v i t y a n d e l a s t a s e were low i n c l e a r e x u d a t e s . L i k e i n p l a sma , CP a c t i v i t y i n p e r i t o n e a l e x u d a t e s d i s s o c i a t e s r e v e r s i b l y f r om c o m p l e x e s u p o n d i l u t i o n . T y p i c a l Km a n d It/Κι were d e t e r m i n e d as 150 μΜ and 5 0 , r e s p e c t i v e l y .

T a b l e 2 . E l a s t a s e and c a t h e p s i n Β i n p e r i t o n e a l exudates

Samples E l a s t a s e C a t h e p s i n Β E l a s t a s e / mean mean c a t h e p s i n Β ± SEM ± SEM

[ng/ml] [ng/ml a ]

C l e a r exudates 1 502 ± 848 30 ± 14 49 (n = 9) Acute p e r i t o n i t i s 13 809 ± 5 584 146 ± 58 95 (n = 9) Etappen lavage 74 900 ± 13 219 198 + 62 378 (η = 9)

C a l c u l a t e d f r om e n z y m a t i c a c t i v i t y a s s u m i n g a s p e c i f i c a c t i v i t y o f 80 U/mg

CP a c t i v i t y of blood monocytes and g ranu locytes

Lysed i s o l a t e d g r a n u l o c y t e s (98 % o f t o t a l c e l l s ) f r o m n o r m a l p e r i p h e r a l b l o od were f o u n d t o h a v e a low b u t s i g n i f i c a n t CP a c t i v i t y o f 115 rnU/10 6 c e l l s c o r r e s p o n d i n g t o 1.4 ng/10 6 c e l l s o f c a t h e p s i n B. 1 0 - 2 0 - f o l d h i g h e r CP a c t i v i t y was f o u n d i n l y s a t e s o f a f r a c t i o n c o n t a i n i n g 14.5 % monocy t e s , 83.9 % l y m p h o c y t e s and o n l y 0.16 % g r a n u l o c y t e s .

Subs t r a t e spec i f i c i t y of CP a c t i v i t y

The s p e c i f i c i t y o f CP a c t i v i t y i n b l o od p l asma , BALF and p e r i t o n e a l e x u d a t e s f o r s m a l l s y n t h e t i c p e p t i d e s u b s t r a t e s c l o s e l y r e semb l es t h a t o f i s o l a t e d h u m a n c a t h e p s i n Β ( Tab l e 3 ) . As j u d g e d f r o m a c t i v i t y w i t h t h e a m i n o p e p t i d a s e s u b s t r a t e A r g - N M e c , less t h a n 1 % o f t h e e n d o p e p t i d a s e a c t i v i t y w i t h Z - P h e -A r g - N M e c can be a t t r i b u t e d t o c a t h e p s i n H . Because o f t h e low spec i f i c a c t i v i t y o f c a t h e p s i n Η (1.5 U/mg as c a l c u l a t e d f r o m k c a t a n d Km i n re f . [ 11 ] ,

218 l . Assfalg-Machleidt et al. Vol.371 Suppl. (1990)

i t s p resence i n a p p r e c i a b l e a m o u n t s c a n n o t be e x c l u d e d . A s i g n i f i c a n t c o n t r i b u t i o n o f c a t h e p s i n L ( spec i f i c a c t i v i t y 36 U/mg w i t h Z - P h e - A r g - N M e c a c c o r d i n g t o re f . [ 1 2 ] , h o w e v e r , is r u l e d o u t b y t h e e s t i m a t e d Km v a l u e s (see Tab l e 3) a n d t h e o b s e r v e d It/Κι (see D i s c u s s i o n ) .

Table 3 . S u b s t r a t e s p e c i f i c i t y of CP a c t i v i t y

Sample R a t i o of a c t i v i t i e s Km

wi th (μΜ) 3

Z--Arg-Arg-NMec/ R-NMec/ Z--Phe-Arg-NMec Z--Phe-Arg-NMec

[145 μΜ] [56 μΜ]

I s o l a t e d l y so soma l c a t h e p s i n Η 17 c a t h e p s i n L < 0 .0002 2 . 4 b

c a t h e p s i n Β 0 .22 < 0 .00005 160 c

Human monocytes' 1 0.20 n . d . n . d . Human g r anu l ocy t e s ' 1 0.19 n . d . n . d . P e r i t o n i t i s exudate 0 .19 0 .014 150 BALF ( p a t i e n t w i t h ARDS) 0 .12 0 .006 225 Plasma (polytrauma) 0 .07 0 .04 95 Plasma ( s e p s i s ) 0 .22 0 .08 170 c

a w i t h Z - P h e - A r g - N M e c , d e t e r m i n e d f r om d i l u t i o n e x p e r i m e n t s un l ess o t h e r w i s e i n d i c a t e d b f r o m re f . [12] c f r o m r e f . [ 2 ] d f r o m l y s a t e s o f i s o l a t e d ce l l s o f n o r m a l p e r i p h e r a l b l ood

Endogenous inh ib i to r s of c y s t e i n e prote inases

The i n h i b i t o r y c a p a c i t y f o r CP was d e t e r m i n e d by t i t r a t i o n o f a c o n s t a n t a m o u n t o f p a p a i n w i t h i n c r e a s i n g a m o u n t s o f samples (p lasma, e x u d a t e , BALF ) f r om i n d i v i d u a l p a t i e n t s . E q u i v a l e n c e was assumed w h e n a c o n s t a n t e n z y m a t i c a c t i v i t y was r e a ched . T h i s c o n s t a n t a c t i v i t y i s due t o p a p a i n e n t r a p p e d i n CX2M w h e r e i t r e m a i n s s t i l l a c t i v e a g a i n s t t h e s m a l l s y n t h e t i c s u b s t r a t e [ 13 ] . The r e m a i n i n g a c t i v i t y ( 1 0 - 4 0 % ) was s u b t r a c t e d f r o m t h e i n i t i a l a c t i v i t y o f p a p a i n . The p a p a i n i n h i b i t i n g c a p a c i t y o f n o r m a l b l o od p l asma is a r o u n d 5 μΜ. Reduced i n h i b i t o r y c a p a c i t y was f o u n d i n p l asma samples o f some p o l y t r a u m a t i z e d p a t i e n t s . B A L F c o n t a i n e d less t h a n 10 nM f ree i n h i b i t o r s (Tab le 4 ) . C h r o m a t o g r a p h y o f p l a s m a f r o m a p o l y t r a u m a t i z e d p a t i e n t on a Superose 12 FPLC c o l u m n s e p a r a t e d t w o m a i n peaks o f i n h i b i t o r y a c t i v i t y c o r r e s p o n d i n g t o m o l e c u l a r masses o f 70 k D a a n d 15 kDa , r e s p e c t i v e l y ( d a t a n o t s h o w n ) . Weak i n h i b i t i o n o f p a p a i n a c t i v i t y was a lso f o u n d i n t h e h i g h m o l e c u l a r mass r e g i o n where c t 2 M is e l u t e d .


T a b l e 4 . I n h i b i t o r y c a p a c i t y for papa in

Sample I n h i b i t o r y c a p a c i t y N o n - t i t r a b l e f o r p a p a i n a c t i v i t y

[ u m o l / l ] [% o f t o t a l ]

No rma l p lasma (n=2) 4 . 2 - 5 .6 23 - 29 P o l y t r a u m a p lasma (n=15) 1.4 - 5 .6 14 - 26 P e r i t o n e a l e x u d a t e (n=6) 0 .6 - 2 .5 9 - 14 BALF (n=3) < 0 . 0 1 n . d .

D i s c u s s i o n

Nature and c e l l u l a r or ig in of r e l eased CP a c t i v i t y

T h e p r e s e n t e d r e s u l t s o f a c l i n i c a l s t u d y w i t h p o l y t r a u m a t i z e d p a t i e n t s c o n f i r m e d and e x t e n d e d p r e v i o u s o b s e r v a t i o n s t h a t , b es ides t h e l y s o s o m a l s e r i n e p r o t e i n a s e s PMN e l a s t a s e a n d c a t h e p s i n G, a l so c y s t e i n e p r o t e i n a s e s a re r e l e a s e d i n t o t h e c i r c u l a t i o n u n d e r c o n d i t i o n s o f s e v e r e t r a u m a and i n f l a m m a t i o n . H i g h l e v e l s o f t h i s e n z y m a t i c a c t i v i t y we re d e t e c t e d l o c a l l y i n b r o n c h o a l v e o l a r l a v a g e f l u i d and p e r i t o n e a l e x u d a t e s . As i n p r e v i o u s wo rk , t h e s p e c i f i c i t y o f o u r assay f o r t h i s c lass o f p r o t e i n a s e s was e n s u r e d by t h e use o f t h e spec i f i c a c t i v e - s i t e i n a c t i v a t o r E - 6 4 [ 14 ] . A c t i v i t y measuremen ts u s i n g d i f f e r e n t p e p t i d e s u b s t r a t e s a n d Km d e t e r m i n a t i o n s f r o m D i x o n p l o t s (see T a b l e 3) i n d i c a t e d t h a t t h e e n z y m a t i c a c t i v i t y o f a l l s t u d i e d samples s h o u l d be m a i n l y due t o c a t h e p s i n Β or a c a t h e p s i n B - l i k e enzyme. I n t h e case o f BALF t h i s was d i r e c t l y c o n f i r m e d w i t h a s p e c i f i c ELISA fo r h u m a n c a t h e p s i n B.

T h o u g h Z - A r g - A r g - N M e c w o u l d be more spec i f i c f o r c a t h e p s i n Β (cf. Tab l e 3 ) , Z - P h e - A r g - N M e c has been se l e c t ed f o r r o u t i n e assay , because t h i s s u b s t r a t e i s 4 t i m e s more s e n s i t i v e f o r c a t h e p s i n B - l i k e a c t i v i t y a n d l ess s e n s i t i v e to E - 6 4 - r e s i s t a n t n o n - c y s t e i n e p r o t e i n a s e a c t i v i t y f o u n d i n some samples [2|.

I n b l ood p l a sma as w e l l as i n l o c a l s e c r e t i o n s t h e o b s e r v e d c a t h e p s i n B - l i k e a c t i v i t y c o r r e l a t e s w i t h t h e seve reness o f t h e c l i n i c a l m a n i f e s t a t i o n o f o r gan d y s f u n c t i o n s . H i gh c a t h e p s i n Β a c t i v i t y o f b l ood p l a s m a d e t e c t a b l e soon a f t e r t h e t r a u m a t i c e v e n t seems t o be a s e n s i t i v e and s p e c i f i c p a r a m e t e r f o r t h e p r e d i c t i o n o f t r a u m a - r e l a t e d o r gan f a i l u r e (see r e f . [3] f o r d e t a i l s ) .

As we h a v e c o n f i r m e d i n t h i s w o r k , PMN g r a n u l o c y t e s o f n o r m a l b l ood c o n t a i n o n l y m i n o r a m o u n t s o f c a t h e p s i n Β (1.5 ng/10 6 c e l l s ) , c o m p a r e d t o 5 - 9 u g / l o 6

c e l l s o f e l a s t a s e [ 15 ] . The r a t i o o f c e l l u l a r e l a s t a s e / c a t h e p s i n Β c o n t e n t ( 3 0 0 0 - 6 0 0 0 ) i s r o u g h l y 1 0 - 3 0 - f o l d h i g h e r t h a n t h e r a t i o o f r e l eased e l a s t a s e / c a t h e p s i n Β i n b l ood p lasma o f p o l y t r a u m a p a t i e n t s ( 2 0 0 - 3 0 0 ) and i n p e r i t o n i t i s e x u d a t e s ( 1 0 0 - 4 0 0 ) . T h e r e f o r e s i m u l t a n e o u s r e l ease o f b o t h enzymes f r o m PMN g r a n u l o c y t e s can p r o b a b l y n o t a c c o u n t f o r t h e obse r ved


c a t h e p s i n Β l e v e l s . M e a s u r e m e n t o f i n t r a c e l l u l a r c a t h e p s i n Β a c t i v i t y b y f l ow c y t o m e t r y i n d i c a t e d , h o w e v e r , t h a t PMN g r a n u l o c y t e s may inc r ease t h e i r c a t h e p s i n Β c o n t e n t u n d e r i n f l a m m a t o r y c o n d i t i o n s (G. Rothe and G. V a l e t , u n p u b l i s h e d r e s u l t s ) . Moreove r , i n p e r i t o n e a l e x u d a t e s , a c o n t r i b u t i o n o f b a c t e r i a l c y s t e i n e p r o t e i n a s e s has t o be c o n s i d e r e d .

A n e l a s t a s e / c a t h e p s i n Β r a t i o c lose t o 1.0 was f o u n d i n BALF w h i c h is k n o w n t o c o n t a i n l a r g e n u m b e r s o f a l v e o l a r macrophages [ 16 ] . A c c o r d i n g t o t h e c e l l u l a r c o n t e n t s [ 1 7 , 1 8 ] , macrophages m u s t be c o n s i d e r e d as a m a i n source f o r r e l e a s e d c a t h e p s i n B. I n c e l l l y s a t e s , m o n o c y t e s o f n o r m a l p e r i p h e r a l b l ood c o n t a i n 1 0 - 2 0 t i m e s more c a t h e p s i n Β a c t i v i t y t h a n g r a n u l o c y t e s . Recen t l y , a s e n s i t i v e f l o w c y t o m e t r i c a ssay f o r i n t r a c e l l u l a r c a t h e p s i n Β a c t i v i t y has been d e v e l o p e d (G. Ro the e t a l . , u n p u b l i s h e d ) . Us ing t h i s assay , a 5 5 - f o l d h i g h e r c a t h e p s i n Β a c t i v i t y was d e t e c t e d i n l i v i n g h u m a n monocy t e s as compared t o n e u t r o p h i l s . T h e 5 - f o l d h i g h e r c y t o m e t r i c r esponse o f t h i o g l y c o l a t e - e l i c i t e d r a t p e r i t o n e a l m a c r o p h a g e s i n c ompar i s on t o r e s i d e n t p e r i t o n e a l macrophages sugges t s t h a t c a t h e p s i n Β a c t i v i t y i s i n d e e d a c e l l u l a r m a r k e r f o r a c t i v a t i o n a n d d i f f e r e n t i a t i o n o f mac rophages . I n b l o o d p l a sma a n d s e c r e t i ons , i n c r eased c a t h e p s i n Β a c t i v i t y a c compan i ed b y an decreased e l a s t a s e / c a t h e p s i n Β r a t i o may t u r n o u t t o be a n e x t r a c e l l u l a r p l a s m a t i c m a r k e r f o r macrophage a c t i v a t i o n .

I n t e r a c t i o n of CP a c t i v i t y w i t h endogenous prote in inh ib i to r s

P a p a i n , a p l a n t c y s t e i n e p r o t e i n a s e , was used t o d e t e r m i n e t h e i n h i b i t o r y c a p a c i t y f o r c y s t e i n e p r o t e i n a s e s , because t h i s enzyme is v e r y s i m i l a r t o l y s o s o m a l c a t h e p s i n L. B o t h p r o t e i n a s e s b i n d t i g h t l y t o a l l k n o w n c y s t e i n e p r o t e i n a s e i n h i b i t o r s o f b l o o d a n d t i s s u e s [19 ] . A c c o r d i n g t o t h e r e s u l t s o f t h e p a p a i n assay , b l o o d p l a s m a a n d p e r i t o n e a l e x u d a t e c o n t a i n a h i g h i n h i b i t o r y c a p a c i t y f o r c y s t e i n e p r o t e i n a s e s . The i n h i b i t o r y c a p a c i t y o f b lood p lasma was f o u n d t o be r e d u c e d t o a b o u t 30 % i n some p o l y t r a u m a p a t i e n t s . I t r e t u r n s t o n o r m a l o r s l i g h t l y r e d u c e d v a l u e s w i t h i n t w o weeks .

D i x o n p l o t a n a l y s i s o f p l a s m a samples f r o m i n d i v i d u a l p a t i e n t s (see F i g . 2) p r o v i d e d e s t i m a t e s o f t h e r a t i o It/Κι. A r a t i o o f 20 f o u n d i n p lasma o f p o l y t r a u m a t i z e d p a t i e n t s p o i n t s t o t h e k i n i n o g e n s ( e xpec t ed It/Κι = 8 - 1 6 0 ) a n d c y s t a t i n C ( e x p e c t e d It/Κι = 5 0 - 1 2 0 ) as t h e most i m p o r t a n t i n h i b i t o r s i n v o l v e d i n t h e e q u i l i b r i u m w i t h c a t h e p s i n Β [2 ] . T h i s i s c o n s i s t e n t w i t h t h e m o l e c u l a r masses o f t h e obse r v ed i n h i b i t o r y f r a c t i o n s i n b lood p lasma. C a t h e p s i n L i s b o u n d t o k i n i n o g e n a n d c y s t a t i n C m u c h more t i g h t l y t h a n c a t h e p s i n Β a n d w o u l d n o t be d e t e c t a b l e as a c t i v e enzyme (It/Κι o f 3 χ 10 5

w i t h k i n i n o g e n a n d 2 χ 1 0 4 w i t h c y s t a t i n C). S u r p r i s i n g l y , o n l y t r aces o f c a t h e p s i n Β a c t i v i t y h a v e been f o u n d a s s o c i a t e d w i t h 0L2M whereas p a p a i n is e n t r a p p e d r e a d i l y [ 2 ] .

Pathob iochemica l r e l e v a n c e of c a theps in Β

I t i s t e m p t i n g t o s p e c u l a t e t h a t i n c r e a s e d c a t h e p s i n B - l i k e a c t i v i t y i s d i r e c t l y i n v o l v e d i n p a t h o m e c h a n i s m s l e a d i n g t o t r a u m a - r e l a t e d o r gan f a i l u r e , such as ARDS, o r p e r s i s t e n t p e r i t o n i t i s . As f a r as d a t a are a v a i l a b l e , i s o l a t e d c a t h e p s i n Β i s n o t v e r y a c t i v e on p r o t e i n s u b s t r a t e s [ 20 ] , b u t t h i s may be


d i f f e r e n t f o r c a t h e p s i n Β p r e c u r s o r s w h i c h h a v e been f o u n d i n s p u t u m [21} a n d i n t u m o r s [22 ] . The p r o t e o l y t i c p o t e n t i a l o f t h e r e l e a s e d c a t h e p s i n B - l i k e a c t i v i t y f o r t h e c l eavage o f p h y s i o l o g i c a l l y i m p o r t a n t p r o t e i n s needs t o be i n v e s t i g a t e d i n more d e t a i l . Bes ides i t s own p r o t e o l y t i c a c t i v i t y , c a t h e p s i n Β may be e n v i s a g e d as an i n d i c a t o r f o r t h e s i m u l t a n e o u s r e l e a s e o f o t h e r c y s t e i n e p r o t e i n a s e s . U n l i k e c a t h e p s i n B, c a t h e p s i n L has been s h o w n t o c l eave e l a s t i n [23] a n d t o i n a c t i v a t e αιΡΙ [ 2 4 ] . The d e c r eas ed i n h i b i t o r y c a p a c i t y obse r v ed i n b l o o d p l a sma o f p a t i e n t s a n d i n B A L F m a y e n h a n c e t h e h a r m f u l e f f e c t s o f r e l eased l y s o s o m a l c y s t e i n e p r o t e i n a s e s .

Acknowledgement

The a u t h o r s w i s h to t h a n k Prof . Dr. Hans F r i t z , A b t e i l u n g für K l i n i s c h e Chemie u n d K l i n i s c h e B iochemie i n der C h i r u r g i s c h e n K l i n i k I n n e n s t a d t der Univers i tät München, f o r s t i m u l a t i n g a n d h e l p f u l d i s c u s s i o n s a n d Pro f . Dr . V i t o T u r k , D e p a r t m e n t o f B i o c h e m i s t r y , Jose f S t e f a n I n s t i t u t e , L j u b l j a n a , f o r p r o v i d i n g p u r i f i e d h u m a n c a t h e p s i n Β as a n t i g e n f o r t h e p r o d u c t i o n o f sp e c i f i c a n t i b o d i e s . The engaged t e c h n i c a l a s s i s t a n c e o f Mrs . C l a u d i a M a n t h e y i s g r e a t l y a p p r e c i a t e d . The w o r k was f i n a n c i a l l y s u p p o r t e d b y t h e S o n d e r f o r s c h u n g s b e r e i c h 207 o f t h e U n i v e r s i t y o f München ( g r a n t s G - l t o W.M., G - 5 t o M.J. a n d G - 6 t o G.V.) .

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