Kinetic and structural properties of two isoforms of trypsin isolated from the viscera of Japanese anchovy, Engraulis japonicus.
Two isoforms of anchovy trypsin (aT-I and aT-II) were purified from the visceral extracts by (NH4)2SO4 fractionation followed by affinity chromatography, gel filtration, and ion-exchange chromatography. The homogeneity of the purified preparation was evidenced by both native- and SDS-PAGE, and further by gelatin zymography. Identities of aT-I and aT-II as trypsins were established by N-terminal amino acid sequencing, which matched exactly to the corresponding stretches of their respective amino acid sequences obtained by molecular cloning [Ahsan et al. (2000), Marine Biotechnol., in press]. Both isoforms were completely inhibited by serine protease inhibitors as well as by specific trypsin inhibitors. The purified anchovy trypsins showed considerably higher catalytic efficiencies (kcat/Km) than bovine trypsin as measured toward benzoyl-arginine p-nitroanilide (BAPA) and benzoyl-arginine ethyl ester (BAEE) at 25 degrees C; in particular, aT-II was 35 times more efficient than its mammalian counterpart against BAPA. This was due mainly to a dramatic decrease of Km values for anchovy trypsins, which are indicative of an evolutionary response toward increased substrate binding at suboptimal temperatures in the marine environment.
Ahsan
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MN
Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan.
Watabe
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000976:Antipain / Q000494:pharmacology; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism; D001586:Benzoylarginine Nitroanilide / Q000378:metabolism; D004795:Enzyme Stability; D005399:Fishes; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014357:Trypsin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000494:pharmacology; D014781:Viscera / Q000201:enzymology
D007527:Isoenzymes; D007976:Leupeptins; D014361:Trypsin Inhibitors; D000976:Antipain; D001586:Benzoylarginine Nitroanilide; D001120:Arginine; C009727:benzoylarginine ethyl ester; D014357:Trypsin; C032854:leupeptin