A thermostable trypsin from freshwater fish Japanese dace (Tribolodon hakonensis): a comparison of the primary structures among fish trypsins.

Trypsin from Japanese dace (Tribolodon hakonensis) (JD-T) living in freshwater (2-18 °C) was purified. JD-T represented typical fish trypsin characteristics regarding the effects of protease inhibitor, calcium-ion, and pH. For the effect of temperature, JD-T quite resembled to the trypsins from tropical-zone marine fish and freshwater fish (the catfish cultured in Thailand), i.e., the optimum temperature was 60 °C, and it was stable below 60 °C at pH 8.0 for 15 min incubation. From the data, it seemed that the trypsin from freshwater fish is thermostable in spite of the fact that their habitat temperatures are low. So, we determined the primary structure of JD-T to discuss its thermostability-structure relationship. JD-T possessed basic structural features of fish trypsin such as the catalytic triad, the Asp189 residue for substrate specificity, 12 Cys residues forming six disulfide-bridges, and the calcium-ion-binding loop. On the other hand, the contents of charged amino acid residues in whole JD-T molecule (16.2%) and N-terminal region (13.8%) were similar to those of tropical-zone marine fish and other freshwater fish trypsins. Then, JD-T conserved the five amino acid residues (Glu70, Asn72, Val75, Glu77, and Glu80) coordinate with calcium-ion, and the proportion of negatively charged amino acids to charged amino acids in the calcium-ion-binding region of JD-T (75.0%) was equivalent to those of tropical-zone marine fish and freshwater fish trypsins. Therefore, it was suggested that the high thermostability of JD-T are stemmed from these structural specificities.

Molecular and immunological characterization of ß'-component (Onc k 5), a major IgE-binding protein in chum salmon roe.

Salmon roe has a high allergic potency and often causes anaphylaxis in Japan. The major allergic protein of salmon roe is ß'-component, which is a 35kDa vitellogenin fragment consisting of two subunits. To elucidate structural information and immunological characteristics, ß'-component and the subunit components were purified from chum salmon (Onchorhincus keta) roe and vitellogenin-encoding mRNA was used to prepare ß'-component subunit-encoding cDNA. This was PCR-amplified, cloned and sequenced and the deduced amino acid sequence compared with partial sequences of ß'-component obtained by peptide mapping. The recombinant ß'-component subunit was produced by bacterial expression in Escherichia coli and its IgE-binding ability was measured by ELISA using the sera of a patient allergic to salmon roe. This was then compared with that of the native ß'-component with and without carboxymethylation. Following successful cloning of the cDNA encoding the ß'-component subunit, 170 amino acid residues were deduced and matched with the amino acid sequences of 121 and 88 residues in the 16kDa and 18kDa subunits, respectively. The sequences of both ß'-component subunits were almost identical, and the predicted secondary structure of the ß'-component showed a high content of ß-pleated sheets and no α-helices. There was no difference in IgE-binding ability between the native and recombinant ß'-component subunits at the same protein concentration, regardless of carboxymethylation. In conclusion, ß'-component is a homodimer protein composed of two isoform subunits having the same level of IgE-binding ability and, therefore, allergenic identity.

Purification and characteristics of trypsin from masu salmon (Oncorhynchus masou) cultured in fresh-water.

Trypsin from the pyloric ceca of masu salmon (Oncorhynchus masou) cultured in fresh water was purified by a series of chromatographies including Sephacryl S-200, Sephadex G-50 and diethylaminoethyl cellulose to obtain a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. The molecular mass of the purified trypsin was estimated to be approximately 24,000 Da by SDS-PAGE. The enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and N ( alpha )-p-tosyl-L: -lysine chloromethyl ketone. Masu salmon trypsin was stabilized by calcium ion. The optimum pH of the masu salmon trypsin was around pH 8.5, and the trypsin was unstable below pH 5.0. The optimum temperature of the masu salmon trypsin was around 60 degrees C, and the trypsin was stable below 50 degrees C, like temperate-zone and tropical-zone fish trypsins. The N-terminal 20 amino acid sequence of the masu salmon trypsin was IVGGYECKAYSQPHQVSLNS, and its charged amino acid content was lower than those of trypsins from frigid-zone fish and similar to those of trypsins from temperate-zone and tropical-zone fish. In the phylogenetic tree, the masu salmon trypsin was classified into the group of the temperate-zone fish trypsin.

Crystal Structure of the 3.8-MDa Respiratory Supermolecule Hemocyanin at 3.0 Å Resolution.

Molluscan hemocyanin, a copper-containing oxygen transporter, is one of the largest known proteins. Although molluscan hemocyanins are currently applied as immunotherapeutic agents, their precise structure has not been determined because of their enormous size. Here, we have determined the first X-ray crystal structure of intact molluscan hemocyanin. The structure unveiled the architecture of the 3.8-MDa supermolecule composed of homologous functional units (FUs), wherein the dimers of FUs hierarchically associated to form the entire cylindrical decamer. Most of the specific inter-FU interactions were localized at narrow regions in the FU dimers, suggesting that rigid FU dimers formed by specific interactions assemble with flexibility. Furthermore, the roles of carbohydrates in assembly and allosteric effect, and conserved sulfur-containing residues in copper incorporation, were revealed. The precise structural information obtained in this study will accelerate our understanding of the molecular basis of hemocyanin and its future applications.