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. 1980 Jan 1;84(1):102–119. doi: 10.1083/jcb.84.1.102

Elastase-like enzymes in human neutrophils localized by ultrastructural cytochemistry

PMCID: PMC2110538  PMID: 6765949

Abstract

The thiol ester N-t-Boc-L-alanine-p-nitrothiophenyl ester (Boc-Ala-SNp) was synthesized and applied as an ultrastructural cytochemical substrate for intracellular elastase-like enzymes. Mature human neutrophils incubated with Boc-Ala-SNp and gold ions generate an electron-dense reaction product, gold p-nitrothiophenolate, which is found in the nuclear membrane, Golgi complex, endoplasmic reticulum, mitochondria, and granules of these cells. Enzyme activity against Boc- Ala-SNp is also observed in developing monkey bone marrow neutrophils and in other blood cells. The intracellular neutrophil enzyme activity is elastase-like because it is characterized by a slightly alkaline pH optimum and is inactivated by exposure of the cells to general and specific active site inhibitors of neutrophil elastase. This substrate appears to have important potential for use in ultrastructural studies of intracellular elastase-like enzymes.

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Selected References

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  1. Anderson D. R. Ultrastructure of normal and leukemic leukocytes in human peripheral blood. J Ultrastruct Res. 1966 Sep;9:1–42. doi: 10.1016/s0022-5320(66)90002-5. [DOI] [PubMed] [Google Scholar]
  2. Aoki Y. Crystallization and characterization of a new protease in mitochondria of bone marrow cells. J Biol Chem. 1978 Mar 25;253(6):2026–2032. [PubMed] [Google Scholar]
  3. Aoki Y., Urata G., Takaku F., Katunuma N. A new protease inactivating delta-aminolevulinic acid synthetase in mitochondria of human bone marrow cells. Biochem Biophys Res Commun. 1975 Jul 22;65(2):567–574. doi: 10.1016/s0006-291x(75)80184-7. [DOI] [PubMed] [Google Scholar]
  4. Baggiolini M., Bretz U., Dewald B., Feigenson M. E. The polymorphonuclear leukocyte. Agents Actions. 1978 Jan;8(1-2):3–10. doi: 10.1007/BF01972395. [DOI] [PubMed] [Google Scholar]
  5. Bainton D. F. Sequential degranulation of the two types of polymorphonuclear leukocyte granules during phagocytosis of microorganisms. J Cell Biol. 1973 Aug;58(2):249–264. doi: 10.1083/jcb.58.2.249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Baugh R. J., Travis J. Human leukocyte granule elastase: rapid isolation and characterization. Biochemistry. 1976 Feb 24;15(4):836–841. doi: 10.1021/bi00649a017. [DOI] [PubMed] [Google Scholar]
  7. Breton-Gorius J., Guichard J. Etude au microscope électronique de la localisation des peroxydases dans les cellules DE LA MOELLE OSSEUSE HUMAINE. Nouv Rev Fr Hematol. 1969 Sep-Oct;9(5):678–687. [PubMed] [Google Scholar]
  8. Breton-Gorius J., Reyes F. Ultrastructure of human bone marrow cell maturation. Int Rev Cytol. 1976;46:251–321. doi: 10.1016/s0074-7696(08)60993-6. [DOI] [PubMed] [Google Scholar]
  9. Bretz U., Baggiolini M. Biochemical and morphological characterization of azurophil and specific granules of human neutrophilic polymorphonuclear leukocytes. J Cell Biol. 1974 Oct;63(1):251–269. doi: 10.1083/jcb.63.1.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Briggs R. T., Drath D. B., Karnovsky M. L., Karnovsky M. J. Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method. J Cell Biol. 1975 Dec;67(3):566–586. doi: 10.1083/jcb.67.3.566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Davies M., Barrett A. J., Travis J., Sanders E., Coles G. A. The degradation of human glomerular basement membrane with purified lysosomal proteinases: evidence for the pathogenic role of the polymorphonuclear leucocyte in glomerulonephritis. Clin Sci Mol Med. 1978 Mar;54(3):233–240. doi: 10.1042/cs0540233. [DOI] [PubMed] [Google Scholar]
  12. Dewald B., Rindler-Ludwig R., Bretz U., Baggiolini M. Subcellular localization and heterogeneity of neutral proteases in neutrophilic polymorphonuclear leukocytes. J Exp Med. 1975 Apr 1;141(4):709–723. doi: 10.1084/jem.141.4.709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dunn W. B., Hardin J. H., Spicer S. S. Ultrastructural localization of myeloperoxidase in human neutrophil and rabbit heterophil and eosinophil leukocytes. Blood. 1968 Dec;32(6):935–944. [PubMed] [Google Scholar]
  14. Galdston M., Janoff A., Davis A. L. Familial variation of leukocyte lysosomal protease and serum 1 -antitrypsin as determinants in chronic obstructive pulmonary disease. Am Rev Respir Dis. 1973 May;107(5):718–727. doi: 10.1164/arrd.1973.107.5.718. [DOI] [PubMed] [Google Scholar]
  15. Gerber A. C., Carson J. H., Hadorn B. Partial purification and characterization of a chymotrypsin-like enzyme from human neutrophil leucocytes. Biochim Biophys Acta. 1974 Sep 11;364(1):103–112. doi: 10.1016/0005-2744(74)90137-5. [DOI] [PubMed] [Google Scholar]
  16. Graham R. C., Jr, Karnovsky M. J. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem. 1966 Apr;14(4):291–302. doi: 10.1177/14.4.291. [DOI] [PubMed] [Google Scholar]
  17. Granelli-Piperno A., Vassalli J. D., Reich E. RNA and protein synthesis in human peripheral blood polymorphonuclear leukocytes. J Exp Med. 1979 Jan 1;149(1):284–289. doi: 10.1084/jem.149.1.284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Granelli-Piperno A., Vassalli J. D., Reich E. Secretion of plasminogen activator by human polymorphonuclear leukocytes. Modulation by glucocorticoids and other effectors. J Exp Med. 1977 Dec 1;146(6):1693–1706. doi: 10.1084/jem.146.6.1693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hirsch J. G., Fedorko M. E. Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and "postfixation" in uranyl acetate. J Cell Biol. 1968 Sep;38(3):615–627. doi: 10.1083/jcb.38.3.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jackson R. C., Blobel G. Post-translational cleavage of presecretory proteins with an extract of rough microsomes from dog pancreas containing signal peptidase activity. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5598–5602. doi: 10.1073/pnas.74.12.5598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Janoff A. Alanine p-nitrophenyl esterase activity of human leucocyte granules. Biochem J. 1969 Aug;114(1):157–159. doi: 10.1042/bj1140157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Janoff A., Scherer J. Mediators of inflammation in leukocyte lysosomes. IX. Elastinolytic activity in granules of human polymorphonuclear leukocytes. J Exp Med. 1968 Nov 1;128(5):1137–1155. doi: 10.1084/jem.128.5.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Janoff A., Sloan B., Weinbaum G., Damiano V., Sandhaus R. A., Elias J., Kimbel P. Experimental emphysema induced with purified human neutrophil elastase: tissue localization of the instilled protease. Am Rev Respir Dis. 1977 Mar;115(3):461–478. doi: 10.1164/arrd.1977.115.3.461. [DOI] [PubMed] [Google Scholar]
  24. Loeven W. A. Elastolytic enzymes in the vessel wall. J Atheroscler Res. 1969 Jan-Feb;9(1):35–45. doi: 10.1016/s0368-1319(69)80064-5. [DOI] [PubMed] [Google Scholar]
  25. Malemud C. J., Janoff A. Human polymorphonuclear leukocyte elastase and cathepsin G mediate the degradation of lapine articular cartilage proteoglycan. Ann N Y Acad Sci. 1975 Jun 13;256:254–262. doi: 10.1111/j.1749-6632.1975.tb36051.x. [DOI] [PubMed] [Google Scholar]
  26. Odeberg H., Olsson I., Venge P. Cationic proteins of human granulocytes. IV. Esterase activity. Lab Invest. 1975 Jan;32(1):86–90. [PubMed] [Google Scholar]
  27. Powers J. C., Gupton B. F., Harley A. D., Nishino N., Whitley R. J. Specificity of porcine pancreatic elastase, human leukocyte elastase and cathepsin G. Inhibition with peptide chloromethyl ketones. Biochim Biophys Acta. 1977 Nov 23;485(1):156–166. doi: 10.1016/0005-2744(77)90203-0. [DOI] [PubMed] [Google Scholar]
  28. Rindler-Ludwig R., Schmalzl F., Braunsteiner H. Esterases in human neutrophil granulocytes: evidence for their protease nature. Br J Haematol. 1974 May;27(1):57–64. doi: 10.1111/j.1365-2141.1974.tb06774.x. [DOI] [PubMed] [Google Scholar]
  29. Shaw E. Selective chemical modification of proteins. Physiol Rev. 1970 Apr;50(2):244–296. doi: 10.1152/physrev.1970.50.2.244. [DOI] [PubMed] [Google Scholar]
  30. Smith R. E., van Frank R. M. The use of amino acid derivatives of 4-methoxy-beta-naphthylamine for the assay and subcellular localization of tissue proteinases. Front Biol. 1975;43(4):193–249. [PubMed] [Google Scholar]
  31. Spitznagel J. K., Dalldorf F. G., Leffell M. S., Folds J. D., Welsh I. R., Cooney M. H., Martin L. E. Character of azurophil and specific granules purified from human polymorphonuclear leukocytes. Lab Invest. 1974 Jun;30(6):774–785. [PubMed] [Google Scholar]
  32. Vatter A. E., Reiss O. K., Newman J. K., Lindquist K., Groeneboer E. Enzymes of the lung. I. Detection of esterase with a new cytochemical method. J Cell Biol. 1968 Jul;38(1):80–98. doi: 10.1083/jcb.38.1.80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Visser L., Blout E. R. The use of p-nitrophenyl N-tert-butyloxycarbonyl-L-alaninate as substrate for elastase. Biochim Biophys Acta. 1972 Apr 7;268(1):257–260. doi: 10.1016/0005-2744(72)90223-9. [DOI] [PubMed] [Google Scholar]
  34. West B. C., Rosenthal A. S., Gelb N. A., Kimball H. R. Separation and characterization of human neutrophil granules. Am J Pathol. 1974 Oct;77(1):41–66. [PMC free article] [PubMed] [Google Scholar]
  35. Whitaker J. R., Perez-Villase ñor J. Chemical modification of papain. I. Reaction with the chloromethyl ketones of phenylalanine and lysine and with phenylmethyl-sulfonyl fluoride. Arch Biochem Biophys. 1968 Mar 20;124(1):70–78. doi: 10.1016/0003-9861(68)90304-4. [DOI] [PubMed] [Google Scholar]
  36. Zimmerman M., Ashe B. M. Sbustrate specificity of the elastase and the chymotrypsin-like enzyme of the human granulocyte. Biochim Biophys Acta. 1977 Jan 11;480(1):241–245. doi: 10.1016/0005-2744(77)90337-0. [DOI] [PubMed] [Google Scholar]

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