Purification and Characterisation of an Extracellular Serine Proteinase From Aspergillus Fumigatus and Its Detection in Tissue

J Med Microbiol. 1990 Dec;33(4):243-51. doi: 10.1099/00222615-33-4-243.

Abstract

A serine proteinase (Alp) from the culture supernate of a clinical isolate of Aspergillus fumigatus was purified to virtual homogeneity at a yield of 41%. The procedure involved affinity chromatography on agarose-epsilon-amino-caproyl-D-tryptophan methyl ester. Alp had an estimated mol. wt of 32 Kda and the pI was determined at pH 7.9. The enzyme was fully inhibited by phenylmethyl sulphonyl fluoride, chymostatin and alpha-1-proteinase inhibitor, and it was largely inhibited by alpha-1-anti-chymotrypsin. Partial inhibition was observed with tosyl-phenylalanine chloromethyl ketone, but tosyl-lysine chloromethyl ketone was ineffective. Thus, Alp may be identical with the major chymotryptic activity of A. fumigatus, which has already been described. The N-terminal sequence of 25 amino acids revealed an 88% homology of Alp with the subtilisin-related proteinase of A. oryzae. Alp acted on casein over a broad range from pH 5.5 to 11.5 and also acts to a lesser extent on haemoglobin and serum albumin. The enzyme degraded elastin and a synthetic elastase substrate; hence, it may be identical with the previously described elastinolytic activity of the fungus. At pH 7.3 and a concentration of 1 microgram/ml, Alp was not toxic for Vero cells, but it efficiently detached such cells from a plastic surface. Specific antibodies against Alp were detected by enzyme immunoassay in the sera of patients and Alp-antigen was demonstrated by immunofluorescence in mycotic human lung. In addition, a second proteinase (Exalp) with extremely alkaline activity, and an aspartic proteinase of A. fumigatus are described.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Fungal / blood
  • Antigens, Fungal / analysis
  • Aspergillosis / immunology
  • Aspergillosis / microbiology*
  • Aspergillus fumigatus / enzymology*
  • Aspergillus fumigatus / immunology
  • Chromatography, Affinity
  • Chymotrypsin / metabolism
  • Guinea Pigs
  • Humans
  • Hydrogen-Ion Concentration
  • Immunoenzyme Techniques
  • Kinetics
  • Male
  • Middle Aged
  • Sequence Homology, Nucleic Acid
  • Serine Endopeptidases / immunology
  • Serine Endopeptidases / isolation & purification
  • Serine Endopeptidases / metabolism*
  • Serine Proteinase Inhibitors
  • Sodium Dodecyl Sulfate / pharmacology
  • Substrate Specificity
  • Vero Cells

Substances

  • Antibodies, Fungal
  • Antigens, Fungal
  • Serine Proteinase Inhibitors
  • Sodium Dodecyl Sulfate
  • Serine Endopeptidases
  • Chymotrypsin