Use of Protease Inhibitors as Probes for Biological Functions: Conditions, Controls, and Caveats

  • Dorothy Hudig
  • James C. Powers


A new subfamily of serine proteases which are unique to lymphocytes has recently been discovered (Jenne et al., 1989). These proteases are induced when cytotoxic function is induced (Gershenfeld and Weissman, 1986; Lobe et al., 1986) and are stored in intracellular granules at low pH (Masson et al., 1990). When the granules are released during cytolysis, the proteases control cytolysis (Chapter 27, this volume) and may cleave perforin to activate pore formation. The proteases are also likely to produce localized mediators and systemic physiological signals (see review by Simon et al., 1989a). It has been suggested that lymphocyte proteases can cleave the HIV gp120/160 surface protein to promote viral entry into lymphocytes (Maraganore J, Biogen, personal communication), cleave secreted pro-interleukin-1 β (Sleath et al., 1990) to an active form, and arrest tumor cell growth (Sayers et al., 1992). To date, we have identified five different substrate specificities in rodents (Chapter 27, this volume), indicating the potential to cleave, activate, and inactivate numerous substrates. The human lymphocyte serine proteases are not as well characterized, though Asp-ase and tryptase activities have been purified (Poe et al., 1991; Fruth et al., 1987).


Serine Protease Inhibitor Sulfonyl Fluoride Serine Protease Activity Chloromethyl Ketone Porcine Pancreatic Elastase 
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  1. Bajusz S, Barabas E, Tolnay P, Szell E, Bagdy D (1978): Inhibition of thrombin and trypsin by tripeptide aldehydes. J Pep Pro Res 12: 217–221CrossRefGoogle Scholar
  2. Barrett AJ (1981): Alpha-2 Macroglobulin. Methods Enzymol 80: 737–754CrossRefGoogle Scholar
  3. Barrett AJ (1986): An introduction to the proteinases. In: Proteinase Inhibitors, Barrett AJ, Salvesen G, eds. Amsterdam: ElsevierGoogle Scholar
  4. Barrett AJ, Salvesen G, eds. (1986): Proteinase Inhibitors. Amsterdam: ElsevierGoogle Scholar
  5. Baugh RJ, Travis J (1976): Human leukocyte granule elastase: Rapid isolation and characterization. Biochemistry 15: 836–841CrossRefGoogle Scholar
  6. Beatty K, Bieth J, Travis J (1980): Kinetics of association of serine proteinases with native and oxidized a, -proteinase inhibitor and a, -antichymotrypsin. J Biol Chem 255: 3931–3934Google Scholar
  7. Beynon RJ, Bond JS, eds. (1989): Proteolytic Enzymes: A Practical Approach. Oxford: IRL PressGoogle Scholar
  8. Beynon RJ, Salvesen G (1989): Appendix III. Commercially available protease inhibitors. In: Proteolytic Enzymes: A Practical Approach, Beynon RJ, Bond JS, eds. Oxford: IRL PressGoogle Scholar
  9. Castillo MJ, Nakajima K, Zimmerman M, Powers JC (1979): Sensitive substrates for human leukocyte and porcine pancreatic elastase: A study of the merits of various chromophoric and fluorgenic leaving groups in assays for serine proteases. Anal Biochem 99: 53–64CrossRefGoogle Scholar
  10. Cho K, Tanaka T, Cook RR, Kisiel W, Fujikawa K, Kurachi K, Powers JC (1984): Active-site mapping of bovine and human blood coagulation serine proteases using synthetic peptide 4-nitroanilide and thin ester substrates. Biochemistry 23: 644–650CrossRefGoogle Scholar
  11. Cohen JA, Oosterbaan RA, Berends F (1967): Organophosphorus compounds. Methods Enzymol 11: 686–702CrossRefGoogle Scholar
  12. Farney DE, Gold A (1963): Sulfonyl fluorides as inhibitors of esterases: 1. Rates of reaction with acetylcholinesterase, alpha-chymotrypsin and trypsin. J Am Chem Soc 85: 977–1000Google Scholar
  13. Finkenstadt WR, Hamid MA, Mattis JA, Schrode J, Sealock RW, Wand D, Laskowski W, Jr (1974): In: Bayer Symposium V “Proteinase Inhibitors”, Fritz H, Tschesche H, Greene LJ, eds. New York: Springer-VerlagGoogle Scholar
  14. Fruth U, Sinigaglia F, Schlesier M, Kilgus J, Kramer MD, Simon MM (1987): A novel serine proteinase (HuTSP) isolated from a cloned human CD8+ cytolytic T cell line is expressed and secreted by activated CD4+ and CD8+ lymphocytes. Eur J Immunol 17: 1625–1633CrossRefGoogle Scholar
  15. Gershenfeld HK, Weissman IL (1986): Cloning of a cDNA for a T cell-specific serine protease from a cytotoxic T lymphocyte. Science 232: 854–857CrossRefGoogle Scholar
  16. Harper JW, Powers JC (1985): Reaction of serine pro-teases with substituted 3-alkoxy-4-chloroisocoumarins and 3-alkoxy-7-amino-4-chloroisocoumarins: New reactive mechanism-based inhibitors. Biochemistry 24: 7200–7213CrossRefGoogle Scholar
  17. Harper JW, Cook RR, Roberts CJ, McLaughlin BJ, Powers JC (1984): Active site mapping of the serine proteases human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II, bovine chymotrypsin A alpha, and Staphylococcus aureus protease V-8 using tripeptide thiobenzyl ester substrates. Biochemistry 23: 2995–3002CrossRefGoogle Scholar
  18. Harper JW, Hemmi K, Powers JC (1985): Reaction of serine proteases with substituted isocoumarins: Discovery of 3,4-dichloroisocoumarin, a new general mechanism-based serine protease inhibitor. Biochemistry 24: 1831–1841CrossRefGoogle Scholar
  19. Hein GE, Niemannn C (1961): An interpretation of the kinetic behavior of model substrates of alpha-chymotrypsin. Proc Natl Acad Sci USA 47: 1341–1344CrossRefGoogle Scholar
  20. Henkart PA, Berrebi GA, Takahama H, Mungar WE, Sitkovsky M (1987): Biochemical and functional properties of serine esterases in acidic cytoplasmic granules of cytotoxic T lymphocytes. J Immunol 139: 2398–2405Google Scholar
  21. Hori H, Yatsutake A, Minematsu Y, Powers JC (1985): Inhibition of human leukocyte elastase, porcine pancreatic elastase and cathepsin G by peptide ketones. In: Peptides: Structure and Function, Deber CM, Hruby VJ, Kopple KD, eds. Rockford, IL: Pierce Chemical CoGoogle Scholar
  22. Hudig D, Allison NJ, Pickett TM, Kam C-M, Powers JC (1991): The function of lymphocyte proteases: Inhibition and restoration of granule-mediated lysis with isocoumarin serine protease inhibitors. J Immunol 47: 1360–1368Google Scholar
  23. Hudig D, Callewaert DM, Redelman D, Gregg NJ, Krump M, Tardieu B (1988): Lysis by RNK-16 cytotoxic-lymphocyte granules: Rate assays and conditions to study control of cytolysis. J Immunol Methods 115: 169–177CrossRefGoogle Scholar
  24. Hudig D, Gregg NJ, Kam C-M, Powers JC (1987): Lymphocyte granule-mediated cytolysis requires serine protease activity. Biochem Biophys Res Commun 149: 882–888CrossRefGoogle Scholar
  25. Hudig D, Haverty T, Fulcher C, Redelman D, Mendelsohn J (1981): Inhibition of human natural cytotoxicity by macromolecular antiproteinases. J Immunol 126: 1569–1574Google Scholar
  26. Hudig D, Powers JC, Allison NJ, Kam C-M (1989): Selective isocoumarin protease inhibitors block RNK-16 lymphocyte granule-mediated cytolysis. Mol Immunol 26: 793–798CrossRefGoogle Scholar
  27. James GT (1978): Inactivation of the proteinase inhibitor phenylmethylsulfonyl fluoride in buffers. Anal Biochem 86: 574–579CrossRefGoogle Scholar
  28. Jenne DE, Masson D, Zimmer M, Haefliger JA, Li WH, Tschopp J (1989): Isolation and complete structure of the lymphocyte serine protease granzyme G, a novel member of the granzyme multigene family in murine cytolytic T lymphocytes. Evolutionary origin of lymphocyte proteases. Biochemistry 28: 7953–7961CrossRefGoogle Scholar
  29. Kam CM, Fujikawa K, Powers JC (1988): Mechanism-based isocoumarin inhibitors for trypsin and blood coagulation serine proteases: New anticoagulants. Biochemistry 27: 2547–2557CrossRefGoogle Scholar
  30. Kam CM, McRae BJ, Harper JW, Niemann MA, Volanakis JE, Powers JC (1987): Human complement proteins D, C2 and B. Active site mapping with peptide thioester substrates. J Biol Chem 262: 3444–3451Google Scholar
  31. Keesey J (1987): Biochemical Information: A Revised Biochemical Reference Source. Indianapolis: Boehringer MannheimGoogle Scholar
  32. Kettner C, Shaw E (1981): Inactivation of trypsin-like enzymes with peptides of arginine chloromethyl ketone. Methods Enzymol 80: 826–842CrossRefGoogle Scholar
  33. Krahn J, Stevens FC (1972): Lima bean protease inhibitor: Comparative study of the trypsin and chymotrypsin inhibitor activity of the four chromatographic variants. FEBS Lett 28: 313–316CrossRefGoogle Scholar
  34. Laura R, Robinson DJ, Bing DH (1980): (p-Amidinophenyl)methanesulfonyl fluoride, an irreversible inhibitor of serine proteases. Biochemistry 19: 4859–4864Google Scholar
  35. Lijnen HR, Collen D (1986): Alpha-2-Antiplasmin. In: Proteinase Inhibitors, Barrett AJ, Salvesen G, eds. Amsterdam: ElsevierGoogle Scholar
  36. Lively MO, Powers JC (1978): Specificity and reactivity of human granulocyte elastase and cathepsin G, porcine pancreatic elastase, bovine chymotrypsin and trypsin toward inhibition with sulfonyl fluorides. Biochem Biophys Acta 525: 171–179Google Scholar
  37. Lobe CG, Finlay BB, Paranchych W, Paetkau VH, Bleackley RC (1986): Novel serine proteases encoded by two cytotoxic T lymphocyte-specific genes. Science 232: 858–861CrossRefGoogle Scholar
  38. Lorand L, ed. (1976): Proteolytic enzymes, Part B. Methods Enzymol 45Google Scholar
  39. Lorand L, ed. (1981): Protoelytic enzymes, Part C. Methods Enzymol 80Google Scholar
  40. Luthy JA, Praissman M, Finkenstadt WR, Laskowski M, Jr (1973): Detailed mechanism of interaction of bovine ß-trypsin with soybean trypsin inhibitor (Kunitz) J Biol Chen 248: 1706–1766Google Scholar
  41. Masson D, Nabholtz M, Estrade C, Tschopp J (1986): Granules of cytotoxic lymphocytes contain two serine esterases. EMBO J 5: 1595–1600Google Scholar
  42. Masson D, Peters PJ, Geuze HJ, Borst J, Tschopp J (1990): Interaction of chondroitin sulfate with perforin and granzymes of cytolytic T-cells is dependent upon pH. Biochemistry 29: 11229–11235CrossRefGoogle Scholar
  43. Matheson NR, van Halbeek H, Travis J (1991): Evidence for a tetrahedral intermediate complex during serpinproteinase interactions. J Biol Chem 266: 13489–13491Google Scholar
  44. Naughton MA, Sanger F (1961): Purification and specificity of pancreatic elastase. Biochem J 78: 156–163Google Scholar
  45. Odake S, Kam C-M, Narasimhan L, Poe M, Blake JT, Krahenbuhl O, Tschopp J, Powers JC (1991): Human and murine cytotoxic T lymphocyte serine proteases: Subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins. Biochemistry 30: 2217–2227CrossRefGoogle Scholar
  46. Odani S, Ikenaka T (1973): Studies on soybean trypsin inhibitors VIII. Disulfide bridges in soybean Bowman-Birk trypsin inhibitor. J Biochem 74: 857–860Google Scholar
  47. Oleksyszyn J, Powers JC (1989): Irreversible inhibition of serine proteases by peptidyl derivatives of alphaaminoalkylphosphonate diphenyl esters. Biochem Biophys Res Commun 161: 143–149CrossRefGoogle Scholar
  48. Oleksyszyn J, Powers JC (1991): Irreversible inhibition of serine proteases by peptide derivatives of (alphaaminoalkyl)phosphonate diphenyl esters. Biochemistry 30: 485–493CrossRefGoogle Scholar
  49. Poe M, Blake JT, Boulton DA, Gammon M, Sigal NH, Wu JK, Zweerink HJ (1991): Human cytotoxic lymphocyte granzyme B: Its purification from granules and the characterization of substrate and inhibitor specificity: J Biol Chem 266: 98–103Google Scholar
  50. Powers JC, Harper JW (1986): Inhibitors of serine proteinases. In: Proteinase Inhibitors, Barrett AJ, Salvesen G, eds. Amsterdam: ElsevierGoogle Scholar
  51. Powers JC, Gupton BF, Harley AD, Nishino N, Whitley RJ (1977): Specificity of porcine pancreatic elastase, human leukocyte elastase and cathepsin G. Inhibition with peptide chloromethyl ketones. Biochim Biophys Acta 485: 156–166Google Scholar
  52. Powers JC, Kam CM, Narasimhan L, Oleksyszyn J, Hernandez MA, Ueda T (1989): Mechanism-based isocoumarin inhibitors for serine proteases: Use of active site structure and substrate specificity in inhibitor design. J Cell Biochem 39: 33–46CrossRefGoogle Scholar
  53. Ristow SS, Starkey JR, Hass GM (1983): In vitro effects of protease inhibitors on murine natural killer cell activity. Immunology 48: 1–8Google Scholar
  54. Rusbridge NM, Beynon RJ (1990): 3,4-Dichlorisocoumarin, a serine protease inhibitor, inactivates glycogen phosphorylase b. FEBS Lett 268: 133–136CrossRefGoogle Scholar
  55. Salvesen GS, Sayers CA, Barrett AK (1981): Further characterization of the covalent linking reaction of a, macroglobulin. Biochem J 195: 453–461Google Scholar
  56. Sayers TJ, Wiltrout TA, Sowder R, Munger WL, Smyth MJ, Henderson LE (1992): Purification of a factor from the granules of a rat natural killer cell line (RNK) that affects tumor cell growth and morphology: Molecular identity with a granule serine protease (RNK-PI). J Immunol (in press)Google Scholar
  57. Schechter I, Berger A (1967): On the size of the active site in proteases. I. Papain. Biochem Biophys Res Conunun 27: 157–162CrossRefGoogle Scholar
  58. Schechter NM, Sprows JL, Schoenberger OL, Lazarus GS, Cooperman BS, Rubin H (1989): Reaction of human skin chymotrypsin-like proteinase chymase with plasma proteinase inhibitors. J Biol Chem 264: 21308–21315Google Scholar
  59. Simon MM, Fruth U, Simon HG, Gay S, Kramer MD (1989): Evidence for multiple functions of T-lymphocytes associated serine proteinases. Adv Exp Med Biol 247A: 609–613Google Scholar
  60. Umezawa H, Aoyagi T (1977): Activities of proteinase inhibitors of microbial origin. In: Proteinases in Mammalian Cells and Tissues, Barrett AJ ed. Amsterdam: ElsevierGoogle Scholar
  61. Yoshimura T, Barker LN, Powers JC (1982): Specificity and reactivity of human leukocyte elastase, porcine pancreatic elastase, human granulocyte cathepsin G, and bovine pancreatic chymotrypsin with arylsulfonyl fluorides. Discovery of a new series of potent and specific irreversible elastase inhibitors. J Biol Chem 257: 5077–5084Google Scholar
  62. Zunino SJ, Allison NJ, Kam C-M, Powers JC, Hudig D (1988): Localization, implications for function, and gene expression of chymotrypsin-like proteinases of cytotoxic RNK-16 lymphocytes. Biochim Biophys Acta 967: 331–340CrossRefGoogle Scholar

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© Birkhäuser Boston 1993

Authors and Affiliations

  • Dorothy Hudig
  • James C. Powers

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