Bayer-Symposium V Proteinase Inhibitors Proceedings of the 2nd International Research Conference Edited by H. Fritz, H. Tschesche, L. J. Greene, and E. Truscheit Springer-Verlag New York· Heidelberg· Berlin 1974 Dr. HANS FRITZ, Institut flir Klinische Chemie und Klinische Biochemie der Universitat Miinchen, 8000 Munich 2, Fed. Rep. Germany Dr. HARALD TSCHESCHE, Organisch-Chemisches Laboratorium der Technischen Universitat Miinchen, 8000 Munich 2, Fed. Rep. Germany Dr. LEWIS 1. GREENE, Brookhaven National Laboratory, Department of Biology, Upton, NY 11973, USA Dr. ERNST TRUSCHEIT (Editor-in-Chief), Biochemisches Laboratorium der Bayer AG, 5600 Wuppertall, Fed. Rep. Germany The editors are indebted to Miss M. L. CHAMBERLAIN and Dr. W. WINGENDER for their valuable assistance and in particular to Mrs. B. NAGEL for careful proof-reading and assistance in the preparation of the subject index Bayer-Symposium V (2nd International Research Conference on Proteinase Inhibitors) held at Grosse Ledder near Cologne, Fed. Rep. Germany October 16th-20th, 1973 With 410 Figures. A stereo viewer for the stereo pair figures on pages 476,488,489,493,502,504, 505,507,509,515, and 519 is attached inside the back cover ISBN-13: 978-3-642-87968-5 e-ISBN-13: 978-3-642-87966-1 DOl: 10.1007/978-3-642-87966-1 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under § 54 of the German Copyright Law, where copies are made for other than private use, a fee is payable to the publisher, the amount of the fee to be determined by agreement with the publisher. © by Springer-Verlag Berlin· Heidelberg 1974. Library of Congress Catalog Card Number 74-6880. Softcover reprint of the hardcover 1st edition 1974 The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Typesetting, printing, and binding: BrUhlsche Universitatsdruckerei, GieBen. Preface These proceedings contain the papers presented at the Bayer-Symposium V which was held in Grosse Ledder on October 16-21, 1973 and which was simultaneously the Second International Research Conference on Proteinase Inhibitors. The cordial atmosphere, the frank discussions and the profitable exchange of ideas during the First International Research Conference on Proteinase Inhibitors which was held on November 4--6, 1970 in Munich were a stimulant for us to consider holding a Second Conference especially as the steadily increasing research activities in this and related fields called for a new assessment of recent efforts in proteinase inhibitor research. In the three years since the First Conference was held, this field has developed particularly in the areas of molecular aspects of protein-protein interactions and in the extension to inhibitors of nonserine proteinases and peptidases. In planning this meeting the Organizing Committee was faced with the problem of selecting a limited number of participants and we decided that it was appropriate and necessary to focus in detail on the chemistry of proteinase inhibitors, their interac tions with a variety of enzymes and also to invite more general papers to survey the physiological and clinical aspects of the subject which we felt would be of importance in the future. The organizers have been aware of the fact that not all of the more recent aspects could be considered. The number of participants and papers presented has more than doubled since the First Conference, despite the careful selection of the contributions, thus the size of the Conference Proceedings has considerably increased from about 300 to 800 pages. This explains why it was unfortunately impossible in this volume to include the extended, lively and extremely stimulating discussions which were enjoyed by the participants during the Conference. The contributions of Dr. G. FEINSTEIN, University of Tel-Aviv, who was unfortunately prevented from attending have, with the permission of all partici pants, been included in this volume without having been presented at the Conference. We are particularly grateful to Professor M. LASKOWSKI,Jr. for his excellent resume of the symposium. The editors are greatly indebted to all contributors and to all those who gave assistance without which the production of this volume would not have been accomplished. The preface permits the editors to acknowledge the conscientious work of the publisher. It is with great respect and affection that the organizers of the symposium, on behalf of all participants and the Bayer AG, dedicate this volume to Professor MICHAEL LASKOWSKI, Sr. for his significant, extensive research in the area of proteinase inhibitors and for his example to us both as a man and as a scientist. HANS FRITZ . HARALD TSCHESCHE . LEWIS J. GREENE . ERNST TRUSCHEIT * Contents A Turbulent Life: M. LASKOWSKI, Sr. 3 Welcome: E. TRUSCHEIT 11 Section I Plasma Proteinase Inhibitors Biochemistry of Proteinase Inhibitors from Human Plasma: A Review of Recent Development: N. HEIMBURGER. With 2 Figures .. . . . .. 14 Purification, Properties, and Composition of oc r Trypsin Inhibitor from Human Plasma: L. F. KRESS and M. Laskowski, Sr. With 7 Figures 23 Properties of Human ocl-Antitrypsin:J. Travis, D. JOHNSON, and R. PANNELL. With 8 Figures ........................ 31 Human oci-Antitrypsin Phenotypes: F. KOPPERS. With 3 Figures 40 Isolation and Fragmentation of ocrAntitrypsin: J.-O. Jeppsson and c.-B. LAURELL. With 3 Figures .................... 47 The Interaction of Human Cationic Trypsin and Chymotrypsin II with Human Serum Inhibitors: J. Bieth, M. AUBRY, and J. TRAVIS. With 7 Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Studies on the Quaternary Structure of Human Serum OCr Macroglobulin : R. C. Roberts, W. A. RIESEN, and P. K. HALL. With 5 Figures . . . . . 63 The Unique Nature of the Interaction of oc2-Macroglobulin with Proteinases: A.J.Barrett, P. M. STARKEY, and E. A. MUNN. With 2 Figures . . . . 72 Biology and Pathology of Plasma Proteinase Inhibitors: M.Steinbuch and R. AUDRAN. With 4 Figures . . . . . . . . . . . . . . . . . . . 78 Interaction between Endogenous Proteases and Plasma Protease Inhibitors in vitro and in vivo: K. OHLSSON. With 11 Figures . . . . . . . . . 96 Plasma Clearance of Human Antiproteinase/Proteinase Complexes: C. BLA- TRIX, J. ISRAEL, R. AUDRAl". J. DROUET, P. AMOUCH, and M. Steinbuch 106 Protease Inhibitors in Human Synovial Fluids of Patients with J oint Diseases: G. Feinstein!, G. SHTACHER, and R. MAAYAN. With 1 Figure 109 Proteinase Inhibitors of the Respiratory Tract: Studies on the Structural Relationship between Acid-Stable Inhibitors Present in the Respiratory Tract. Plasma and Urine: K. Hochstrasser, H.FEUTH, and K. HocH- GESAND 111 * In the case of several authors bold-face type indicates the name of the author who presented the paper at the conference. 1 Prevented from attending. paper not presented at the conference. VIII Contents Section II Proteinases and Proteinase Inhibitors in Fertilization Biochemistry and Clinical Significance of the Trypsin-Like Proteinase Acrosin from Boar and Human Spermatozoa: H.Fritz, W.D.SCHLELJ NING, and W.-B. SCHILL. With 10 Figures . . . . . . . . . . . . . 118 Studies on the Purification and Characterization of Boar Acrosin: K. L. Polakoski and W. L. WILLIAMS. With 9 Figures ........ 128 Proteinase Inhibitors and Proteinases of Human Semen: L. J. D. Zaneveld, G. F. B. SCHUMACHER, P. F. TAUBER, and D. PROPPING. With 6 Figures 136 Characterization of Two Proteinase Inhibitors from Human Seminal Plasma and Spermatozoa: H.Schiessler, M.ARNHOLD, and H.FRITZ. With 7 Figures ........................... 147 Isolation of Proteinase Inhibitors from Boar Sperm Acrosomes and Boar Seminal Plasma and Effect on Fertilization: K. L. Polakoski and W. L. WILLIAMS. With 8 Figures 156 Purification, Characterization, and Structural Studies of Proteinase Inhibitors from Boar Seminal Plasma and Boar Spermatozoa: H. Tschesche, S. KUPFER, O. LENGEL, R. KLAUSER, M. MEIER, and H. FRITZ. With 12 Figures ........................... 164 Proteinase Inhibitors in Human Cervical Mucus and Their in vitro Inter actions with Human Acrosin: G. F. B. Schumacher and L. J. D. ZAN EVELD. With 3 Figures . . . . . . . . . . . . . . . . . . . . . . . . 178 Section II I Pancreatic Proteinase Inhibitors Human Pancreatic Secretory Trypsin Inhibitor: L. J. Greene, D. E.RoARK, and D.C.BARTELT. With 8 Figures . . . . . . . . . . . . . . . . 188 Isolation of Human Pancreatic Inhibitor and the Study of Its Interaction with Mammalian and Human Proteases: G. Feinstein 2, R. HOFFSTEIN, and M. SOKOLOVSKY. With 11 Figures ................ 199 Studies on Inhibition of the Two Human Trypsins: C. Figarella, G. A. NEGRI, and O.GUY. With 13 Figures . . . . . . . . . . . . . . . . . . 213 Sequence of Cleavages in Temporary Inhibitors: Porcine Inhibitor Form II and Bovine Isoinhibitors A and C: S. L. SCHNEIDER, L. ST ASIUK, and M. Laskowski, Sr. With 9 Figures ................ 223 Temporary Inhibition - A Result of Consecutive Peptide Bond Hydrolysis: H. Tschesche, G.REIDEL, and M.SCHNEIDER. With 3 Figures ..... 235 Measurement of the Bovine Pancreatic Trypsin Inhibitors by Radioim munoassay: E. Fink and L. J. GREENE. With 6 Figures . . . . . . . . 243 Properties and Inhibition Spectrum of a New Human Pancreatic Protease: P. A. MALLORY and J. Travis . . . . . . . . . . . . . . . . . . . 250 2 Prevented from attending, paper not presented at the conference. Contents IX Section IV Characterization and Structure of New Inhibitors Amino Acid Sequence of Snail Inhibitor K and Correlation of Structure and Specificity: T. Dietl and H. TSCHESCHE. With 8 Figures ... . . . . 254 Novel Proteinase Inhibitors in Snake Venoms: Distribution, Isolation, and Amino Acid Sequence: H. TAKAHASHI, S.IwANAGA, T.KITAGAWA, Y.HOKAMA, and T.Suzuki. With 8 Figures ............ 265 Broad Specificity Inhibitors from Sea Anemones: G. Wunderer, K.KuMMER, H. FRITZ, L. BERESS, and W. MACHLEIDT. With 4 Figures . . . . . . . 277 Discussion Remark: A Polyvalent Proteinase Inhibitor in the Earthworm (Lumbricus terrestris): K. ILLCHMANN and E. WERLE: Presented by H. FRITZ .......................... 282 Discussion Remark: Proteinase Isoinhibitors from Cuttle Fish (Loligo vul garis): H. Tschesche and A. VON RUCKER. With 1 Figure . . . . . . . 284 The Partial Linear Sequence of Garden Bean Inhibitor II and Location of the Protease Reactive Sites: K. A. Wilson and M. LASKOWSKI, Sr. With 2 Figures ........................... 286 Elastase Inhibitors as Impurities in Commercial Preparations of Soybean Trypsin Inhibitor (Kunitz): J.Bieth and J.-C.FRECHIN. With 11 Figures 291 A New Alkaline Proteinase Inhibitor from Streptomyces albogriseolus: T.IKENAKA. With 1 Figure . . . . . . . . . . . . . . . . . . . 305 Section V Structure-Function Relationships Replacements, Insertions, and Modifications of Amino Acid Residues in the Reactive Site of Soybean Trypsin Inhibitor (Kunitz): D. Kowalski, T.R.LEARY, R.E.McKEE, R.W.SEALOCK, D.WANG, and M.LASKOW- SKI, Jr. With 8 Figures. . . . . . . . . . . . . . . . . . . . . . 311 Chemical Structure and Inhibitory Activities of Soybean Proteinase Inhibitors: T.Ikenaka, S.ODANI, and T. KOIDE. With 23 Figures 325 Structure-Function Relationships in Lima Bean Protease Inhibitor: F. C. Stevens, S. WUERZ, and J. KRAHN. With 8 Figures . . . . . . . 344 Structure-Activity Relationships of Several Trypsin and Chymotrypsin Inhibitors from Legume Seeds: Y.BIRK. With 1 Figure ....... 355 Reactive Site Cleavage, Thermodynamic Control Resynthesis, and Properties of Chemically Derivatized Trypsin-Kallikrein Inhibitors: H. TscHEscHE, H.Jering, G.SCHORP, and T.DIETL. With 15 Figures ........ 362 The Interaction of Catalytically Inactive Proteolytic Enzymes with Protein Inhibitors. Methyl-Chymotrypsin: D. S. RYAN and R. E. Feeney. With 3 Figures ........ ........ . . . . . 378 Discussion Remark: Inhibition of Anhydro-Trypsin and Anhydro- Chymotrypsin: C. A. RYAN . . . . . . . . . . . . . 384 x Contents Section V I Physical-Chemical Studies of Enzyme-Inhibitor Interactions Kinetics and Thermodynamics of the Interaction of Proteinases with Protein Inhibitors: W. R. Finkenstadt, M. A. HAMID, J. A. MATTIS, J. SCHRODE, R. W. SEALOCK, D. WANG, and M. LASKowsKI,Jr. With 16 Figures 389 Kinetic Studies of the Binding of Bovine Basic Pancreatic Trypsin Inhibitor to a-Chymotrypsin: J. Engel, U. QUAST, H. HEUMANN, G. KRAUSE, and E.STEFFEN. With 5 Figures ................... 412 The Mechanism of Association of Trypsin (or Chymotrypsin) with the Pancreatic Trypsin Inhibitors (Kunitz and Kazal), Kinetics and Thermo dynamics of the Interaction: M.Lazdunski, J.-P. VINCENT, H. SCHWEITZ, M.PERoN-RENNER, and J.PUDLES. With 7 Figures ......... 420 Kinetic Studies on the Interactions between Native, Acetylated and Suc cinylated Trypsin and Natural Proteinase Inhibitors: N. Levilliers, M.PERoN-RENNER, and J.PUDLES. With 5 Figures ......... 432 The Use of Fluorescence Techniques in the Study of the Interaction of the Basic Trypsin Inhibitor of Bovine Pancreas, Selectively Labelled at Lysine 15, with Chymotrypsin and Trypsin: Y.ELKANA (presented by M.RIGBI). With 5 Figures ........................ 445 Raman Studies of Native and Partially (Cys 14-Cys 38) Reduced Basic Pancreatic Trypsin Inhibitor: H. BRuNNER and M. HOLZ (presented by H.JERING). With 2 Figures ... . . . . . . . . . . . . . . . . 454 A Study on the Interaction of Trasylol with Trypsin and Kallikrein by Circular Dichroism: H.RoSENKRANZ. With 4 Figures . . . . . . . . 458 Some Kinetic Consequences of the Tight Binding of Protein-Proteinase Inhibitors to Proteolytic Enzymes and Their Application to the Deter mination of Dissociation Constants: J. BIETH. With 2 Figures . . . . 463 Section VII Molecular Aspects of Enzyme-Inhibitor Interactions Stereochemistry of Substrate Binding and Hydrolysis in the Trypsin Family of Enzymes: D.M.BLOw. With 9 Figures . . . . . . 473 The Model of the Basic Pancreatic Trypsin Inhibitor Refined at 1.5 A Resolution: J.Deisenhofer and W.STEIGEMANN. With 11 Figures 484 Structure of the Complex Formed by Bovine Trypsin and Bovine Pancreatic Trypsin Inhibitor, Refinement of the Crystal Structure Analysis: R.Hu ber, D.KuKLA, W.STEIGEMANN, J.DEISENHOFER, and A. JONES. With 9 Figures ........................... 497 The Mode of Action of Soybean Trypsin Inhibitor as Revealed by Crystal Structure Analysis of the Complex with Porcine Trypsin: J.JANIN, R.M.Sweet, and D.M.BLOw. With 6 Figures ........... 513 Contents XI Section VIII Organic Chemical Models and Low-Molecular-Weight Inhibitors Organic Chemical Models for Proteinase Inhibitors: E. T. KAISER 523 Progress in Designing Small Inhibitors Which Discriminate among Trypsin- Like Enzymes: E.SHAW. With 8 Figures ..... . . . . . . . . 531 Homoarginine Oligomers as Trypsin and Chymotrypsin Substrates and In hibitors: M.Rigbi, Y.ELKANA, N.SEGAL, D.KuGER, and L.SCHWARTZ. With 6 Figures ........................ 541 Proteinase Inhibitors of Microbial Origin. A Review: W. WINGENDER. With 5 Figures .......................... 548 Discussion Remark: Low Molecular Weight Carboxydipeptidase Inhibitors from Bothropsjararaca Venom: L.J.GREENE ............ 560 Section IX Inhibition of Non-Serine Prot eases Carboxypeptidase Inhibitor from Potatoes. Properties and Amino Acid Sequence: C.A.Ryan, G.M.HASS, R.W.KuHN, and H.NEURATH. With 7 Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . 565 Inhibitors of Lysosomal Proteinases: A. J. BARRETT. With 3 Figures . . . . 574 Inhibition of Insulin Specific Protease by Proteinase Inhibitors from the Snail (Helix pomatia): J.S.BRUSH and H.Tschesche. With 3 Figures 581 An Insulin Degrading Proteinase from Human Erythrocytes and Its In hibition by Proteinase Inhibitors: H. Tschesche, T. DIETL, H. J. KOLB, and E.STANDL. With 6 Figures ................. 586 Section X Evolution of Specificity of Proteinase Inhibitors Evolution of Specificity of Protein Proteinase Inhibitors: M.Laskowski,Jr., I. KATO, T. R. LEARY, J. SCHRODE, and R. W. SEALOCK. With 10 Figures 597 Section XI Proteinases and Inhibitors in Biological Systems Proteases and Protease Inhibitors in Neoplasia: H. P. SCHNEBLI . . . . . 615 Secretion of Enzymes by Neoplastic Cells and Macrophages: E. REICH. With 4 Figures ........................ 621 Plasminogen Activators of Normal and Transformed Cells: A.R.Goldberg and S.G.LAZAROWITZ. With 5 Figures .............. 631 XII Contents Proteinase Inhibitors from Ascaris lumbricoides: Properties and Their Physiological Role: R.J.Peanasky, G.M.ABU-ERREISH, C.R.GAusH, G.A.HOMANDBERG, D.O'HEERoN, R.K.LINKENHEIL, U.KUCICH, and D.R.BABIN. With 18 Figures .................. 649 W ound-Induced Accumulation of Proteinase Inhibitor I in Tomato Leaves: C.A.RYAN. With 2 Figures .............. . . . . . 667 Section XII General Discussion Remark, Resume, Concluding Remarks General Discussion Remark: Energetics of Inhibitor Binding. D. M. BLOW 677 Resume: M.LASKOWSKI,Jr. ............... 679 Concluding Remarks: H. TscHEscHE, L. J. GREENE, and H. FRITZ 685 Author Index 687 Subject Index 735