Purification, kinetical and molecular characterizations of a serine collagenolytic protease from greenshore crag (Carcinus maenas) digestive gland.
A serine collagenolytic protease was purified from a water soluble fraction of greenshore crab digestive gland by acidic precipitation, gel filtration on a Sephadex G-50 column, ion-exchange chromatography on a Fractogel TSK DEAE column, immobilized metal ion affinity chromatography (IMAC) on IDA (Cu2+) Sepharose 6B and ion-exchange chromatography on Hyper D column. The molecular mass of the monomeric Carcinus serine collagenase (CSC) was estimated to be 23,000 by SDS PAGE and its isoelectric point was found to be 4.0. The CSC is optimally active at pH 7 and 30 degrees C and is stable over a month at room temperature. The CSC activity is strongly inhibited by PMSF, 3,4-DCI, soybean trypsin inhibitor, alpha 1 proteinase inhibitor and elastatinal. The CSC hydrolyzes native collagen (Type I and III). CSC N-terminal sequence is similar to shrimp chymotrypsin-like protease and crab collagenolytic protease sequences. Kinetic parameters of the CSC were determined using some peptidyl-p-nitroanilides. The catalytic efficiency (kcat/Km) is Leu > Phe > Ala.
Roy
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Laboratoire Biochimie et Molécules Marines, VP/BM, IFREMER, Nantes, France. proy@ifremer.fr.
Colas
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B
Durand
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D000595:Amino Acid Sequence; D000818:Animals; D003386:Brachyura; D003374:Coumarins / Q000494:pharmacology; D004064:Digestive System / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D049934:Isocoumarins; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D014105:Tosyl Compounds / Q000494:pharmacology; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000494:pharmacology; D000515:alpha 1-Antitrypsin / Q000494:pharmacology
D003374:Coumarins; D049934:Isocoumarins; D009842:Oligopeptides; D015842:Serine Proteinase Inhibitors; D014105:Tosyl Compounds; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D000515:alpha 1-Antitrypsin; C003614:4-toluenesulfonyl fluoride; C046235:3,4-dichloroisocoumarin; C015516:succinyl-trialanine-4-nitroanilide; D008666:Metalloendopeptidases; C044194:microbial metalloproteinases; C010718:elastatinal