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Tryptase from rat skin: purification and properties.

V J Braganza1, W H Simmons

  • 1Department of Molecular and Cellular Biochemistry, Stritch School of Medicine, Loyola University of Chicago, Maywood, Illinois 60153.

Biochemistry
|May 21, 1991
PubMed
Summary

Researchers purified rat skin tryptase, a serine protease, revealing its unique substrate specificity and structural properties. This enzyme exhibits distinct characteristics compared to other known tryptases, offering insights into protease function.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Protease research

Background:

  • Tryptases are serine proteases implicated in various physiological and pathological processes.
  • Understanding the specific properties of tryptases from different tissues is crucial for elucidating their roles.
  • Rat skin tryptase has not been extensively characterized previously.

Purpose of the Study:

  • To purify and characterize rat skin tryptase.
  • To investigate its substrate specificity, kinetic properties, and structural features.
  • To compare rat skin tryptase with other known tryptases.

Main Methods:

  • Purification using ammonium sulfate fractionation and sequential affinity chromatography (agarose-glycyl-glycyl-p-aminobenzamidine and concanavalin A-agarose).

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  • Enzyme activity assays using specific substrates (BzArgOEt and H-D-Ile-Pro-Arg-p-nitroanilide).
  • Characterization of thermal stability, inhibition profiles, substrate specificity, molecular weight (gel filtration, SDS-PAGE), isoelectric focusing, and N-terminal amino acid sequencing.

    • Main Results:

    • Rat skin tryptase was purified 13,000-fold with a specific activity of 170 mumol/min mg-1.
    • The enzyme demonstrated thermal lability, requiring 1 M NaCl for stability and storage; heparin did not provide stabilization.
    • Substrate specificity studies indicated an extended binding site, with hydrolysis of specific bonds in [Arg8]vasopressin, neurotensin, and insulin B-chain; no general proteinase activity was observed.
    • Molecular weight was 145,000 (gel filtration) with subunits of 34,000 or 30,000 (electrophoresis), reduced by N-glycanase treatment, suggesting glycosylation.
    • Multiple isoelectric forms (pI 4.5-4.9) were observed.
    • N-terminal sequencing revealed homology to other tryptases (72-80%) but lower homology to bovine trypsin (40%).

    Conclusions:

    • Rat skin tryptase is a distinct serine protease with unique substrate preferences and structural characteristics.
    • Its properties suggest a specialized role, differing from other characterized tryptases and trypsin.
    • The findings provide a foundation for further investigation into the physiological functions of rat skin tryptase.