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Comp Biochem Physiol B Biochem Mol Biol. 2010 Feb;155(2):186-94. doi: 10.1016/j.cbpb.2009.11.004.

Characterization of cold-adapted Atlantic cod (Gadus morhua) trypsin I--kinetic parameters, autolysis and thermal stability.

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  • 1Department of Biochemistry, Science Institute University of Iceland, Dunhaga 3, 107 Reykjavík, Iceland.

Abstract

Atlantic cod trypsin I is a highly active cold-adapted protease. This study aimed at further characterization of this enzyme with respect to kinetic parameters, sites of autolysis and stability. For that purpose, trypsin I was purified by anion exchange chromatography. Its purity and identity was verified by SDS-PAGE analysis and mass spectrometry. Concomitantly, another cod trypsin isozyme, trypsin X, previously only described from its cDNA sequence was detected in a separate peak from the ion exchange chromatogram. There was a stepwise increase in the catalytic efficiency (k(cat)/K(m)) of cod trypsin I obtained with substrates containing one to three amino acid residues. As expected, the activity of trypsin I was maintained for longer periods of time at 15 degrees C than at higher temperatures. The residues of the trypsin I molecule most sensitive to autolysis were identified using Edman degradation. Eleven autolytic cleavage sites were detected within the trypsin I molecule. Unfolding experiments demonstrated that autolysis is a contributing factor in the stability of trypsin I. In addition, the data shows that cod trypsin I is less stable towards thermal unfolding than its mesophilic bovine analogue.

PMID:
19913635
[PubMed - indexed for MEDLINE]
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