Purification and characterization of two anionic trypsins from the hepatopancreas of carp |
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| Authors: | Min-Jie Cao Kiyoshi Osatomi Miho Suzuki Kenji Hara Katsuyasu Tachibana and Tadashi Ishihara |
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| Institution: | *Laboratory of Marine Biochemistry, Graduate School of Marine Science and Engineering, Nagasaki University, Nagasaki, Nagasaki 852-8521, Japan |
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| Abstract: | SUMMARY: Two trypsins, designated as trypsin A and trypsin B, have been purified from the hepatopancreas of carp. The purification procedures consisted of ammonium sulfate fractionation, and chromatographies on DEAE-Sephacel, Ultrogel AcA54 and Q-Sepharose. Trypsin A was purified to homogeneity with the molecular mass of approximately 28 kDa, while trypsin B gave two close bands of 28.5 kDa and 28 kDa on sodium dodecylsulfate polyacrylamide gel electrophoresis both under reducing and non-reducing conditions. On native-PAGE, both trypsin A and trypsin B showed a single band. Trypsin A and trypsin B revealed optimum temperature of 40°C and 45°C, respectively, and shared the same optimum pH 9.0 using Boc-Phe-Ser-Arg-MCA as substrate. Both enzymes were effectively inhibited by trypsin inhibitors and their susceptibilities were similar. The NH2-terminal amino acid sequences of trypsin A and trypsin B were determined to 37th and 40th amino acid residue, respectively. Their sequences were very homologous, but not identical to that of a trypsin-type serine proteinase from carp muscle and these of other trypsins. Immunoblotting test using the antibody raised against trypsin A cross-reacted with trypsin B positively. |
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| Keywords: | amino acid sequence carp hepatopancreas immunoblotting purification serine proteinase trypsin |
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