Journal of Protein Chemistry

, Volume 14, Issue 1, pp 41–52

Isolation and partial characterization of basic proteinases from stem bromelain

  • Tibor Harrach
  • Klaus Eckert
  • Kai Schulze-Forster
  • Rolf Nuck
  • Detlef Grunow
  • H. Rainer Maurer
Article

Abstract

Crude bromelain extracts from pineapple stems (Ananas comosus) were fractionated by two-step FPLC-cation-exchange chromatography. At least eight basic proteolytically active components were detected. The two main components F4 and F5 together with the most active proteinase fraction F9 were characterized by SDS-PAGE, mass spectroscopy, multizonal cathodal electrophoresis, partial amino acid sequence, and monosaccharide composition analysis. F9 amounts to about 2% of the total protein and has a 15 times higher specific activity against the substratel-pyroglutamyl-l-phenylanalyl-l-leucine-p-nitroanilide (PFLNA) than the main component F4. The molecular masses of F4, F5, and F9 were determined to 24,397, 24,472, and 23,427, respectively, by mass spectroscopy. Partial N-terminal amino acid sequence analysis (20 amino acids) revealed that F9 differs from the determined sequence of F4 and F5 by an exchange at position 10 (tyrosine→serine) and position 20 (asparagine→ glycine). F4 and F5 contained fucose, N-acetylglucosamine, xylose, and mannose in ratio of 1.0∶2.0∶1.0∶2.0, but only 50% of the proteins seem to be glycosylated, whereas F9 was found to be unglycosylated. Polyclonal antibodies (IgG) against F9 detected F4 and F5 with tenfold reduced reactivity. ThepH optimum of F4 and F5 was betweenpH4.0 and 4.5 and for F9 close to neutralpH. The kinetic parameters for PFLNA hydrolysis were similar for F4 (Km 2.30 mM,kcat 0.87 sec−1 and F5 (Km 2.42 mM,kcat 0.68 sec−1), and differed greatly from F9 (Km 0.40 mM,kcat 3.94 sec−1).

Key words

Bromelain basic proteinases amino acid sequence analysis FPLC mass spectroscopy monosaccharide composition analysis pH optimum 

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Copyright information

© Plenum Publishing Corporation 1995

Authors and Affiliations

  • Tibor Harrach
    • 1
  • Klaus Eckert
    • 1
  • Kai Schulze-Forster
    • 1
  • Rolf Nuck
    • 2
  • Detlef Grunow
    • 2
  • H. Rainer Maurer
    • 1
  1. 1.Institut für Pharmazie, Abteilung Pharmazeutische BiochemieFreie Universität BerlinBerlinGermany
  2. 2.Institut für Molekularbiologie und BiochemieFreie Universität BerlinBerlinGermany

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