Purification and characterization of two chymotrypsin-like proteases from the pyloric caeca of rainbow trout oncorhynchus-mykiss

Magnus M. Kristjansson, Henrik Hauch Nielsen

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    Two chymotrypsins, called chymotrypsin I and II, were purified from the pyloric caeca of rainbow trout, by (NH4)2SO4 fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). The approximate molecular weights of chymotrypsin I and II were 28,200 (.+-.1200) and 28,800 (.+-.900), respectively, as determined by SDS-PAGE and their isoelectric points were about 5. The pH optima of the enzymes were centered around nine, when assayed for succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Such-AAPF-NA) as substrate and both enzymes were unstable at pH values below 5. The amidase activity of both enzymes increased with temperature up to about 55.degree. C. Chymotrypsin I was found to be more heat stable than chymotrypsin II, an effect most likely explained by strong calcium binding of the former. The trout chymotrypsins were significantly more active than bovine .alpha.-chymotrypsin when assayed against Suc-AAPF-NA at 25.degree. C and casein at low temperatures (10-20.degree. C), indicating an adaptation of the activities of the trout chymotrypsins to the habitation temperatures of the fish.
    Original languageEnglish
    JournalComparative Biochemistry and Physiology - Part B: Biochemistry & Molecular Biology
    Volume101
    Issue number1-2
    Pages (from-to)247-253
    Number of pages7
    ISSN1096-4959
    Publication statusPublished - 1992

    Keywords

    • Amino Acid Sequence
    • Animals
    • Calcium
    • Caseins
    • Catalysis
    • Cattle
    • Chromatography, Gel
    • Chymotrypsin
    • Electrophoresis, Polyacrylamide Gel
    • Enzyme Stability
    • Hydrogen-Ion Concentration
    • Intestines
    • Molecular Sequence Data
    • Salmon
    • Serine Endopeptidases
    • Serine Proteinase Inhibitors
    • Temperature
    • EC 3.4.21.- Serine Endopeptidases
    • EC 3.4.21.1 Chymotrypsin
    • SY7Q814VUP Calcium
    • DIAMMONIUM SULFATE FRACTIONATION ION-EXCHANGE CHROMATOGRAPHY HYDROPHOBIC INTERACTION CHROMATOGRAPHY THERMOREGULATION
    • Pisces Vertebrata Chordata Animalia (Animals, Chordates, Fish, Nonhuman Vertebrates, Vertebrates) - Osteichthyes [85206]
    • 9014-01-1D: PROTEASES7783-20-2: DIAMMONIUM SULFATE
    • 07508, Ecology: environmental biology - Animal
    • 10062, Biochemistry studies - Nucleic acids, purines and pyrimidines
    • 10802, Enzymes - General and comparative studies: coenzymes
    • 10804, Enzymes - Methods
    • 10806, Enzymes - Chemical and physical
    • 14004, Digestive system - Physiology and biochemistry
    • 23012, Temperature - Thermoregulation
    • Biochemistry and Molecular Biophysics
    • Environmental Sciences
    • Ingestion and Assimilation
    • Digestive System
    • Ecology
    • Enzymology
    • Physiology

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