Biochemical characterization of trypsins from the hepatopancreas of Japanese sea bass (Lateolabrax japonicus).

A cationic trypsin (trypsin A) and an anionic trypsin (trypsin B) were highly purified from the hepatopancreas of the Japanese sea bass (Lateolabrax japonicus) by ammonium sulfate precipitation, column chromatographies of DEAE-Sepharose and Sephacryl S-200 HR. Purified trypsins revealed single band on SDS-PAGE and their molecular masses were 21 kDa and 21.5 kDa, respectively. Trypsins A and B exhibited maximal activity at 40°C, and shared the same optimal pH at 9.0 using Boc-Phe-Ser-Arg-MCA as the substrate. The two trypsins were stable up to 45°C and in the pH range from 7.0 to 11.0. Trypsin inhibitors such as Pefabloc SC, PMSF and benzamidine are effective to these two enzymes and their susceptibilities were similar. Apparent K(m)s of trypsins A and B were 1.12 and 0.7 µM and k(cat)s of them were 72.08 and 67.79 S(-1) for Boc-Phe-Ser-Arg-MCA, respectively. The N-terminal amino acid sequences of the two trypsins were determined to the 24th residues, which were highly identical to trypsins from other species of fish while trypsins A and B only shared 45.8% identity. The digestive effect of the two trypsins on native shrimp muscular proteins indicated their effectiveness in the degradation of food proteins.

Identification of an aminopeptidase from the skeletal muscle of grass carp (Ctenopharyngodon idellus).

Aminopeptidases play important roles in turnover of proteins, metabolism of hormones and neurotransmission, cell maturation and immunological regulations. In the present study, an aminopeptidase was purified to homogeneity from the skeletal muscle of grass carp by ammonium sulfate fractionation and sequential chromatographic steps, including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite and Phenyl-Sepharose. The purified enzyme revealed a molecular mass of approximately 105 kDa both on SDS-PAGE and on gel filtration of Superdex 200. The enzymatic activity toward synthetic substrates was optimal at 40°C and pH 7.0-7.5. Metal-chelating agents such as EDTA and EGTA effectively inhibited the enzyme activity while inhibitors to serine, asparatic and cysteine proteinases did not show much effect, suggesting its belonging to metalloproteinase family. A specific aminopeptidase inhibitor bestatin was most effective in suppressing the enzymatic activity and performed in a competitive fashion. The enzymatic activity was slightly enhanced by metal ions of Mg2+ and Mn2+ while inhibited to different extents by Co2+, Cu2+, Zn2+ and Ca2+. Sulfhydryl reagent was necessary to maintain its activity. Purified enzyme demonstrated amidolytic activity most effectively against synthetic aminopeptidase substrate Leu-methylcoumarylamide (MCA) while N-terminal-blocked substrates and myofibrillar proteins were not hydrolyzed. The enzyme purified in the present study was quite possibly a leucine aminopeptidase (LAP) and functions during muscular protein metabolism.

Glucose-6-phosphate isomerase is an endogenous inhibitor to myofibril-bound serine proteinase of crucian carp (Carassius auratus).

Glucose-6-phosphate isomerase (GPI) was purified to homogeneity from the skeletal muscle of crucian carp ( Carassius auratus ) by ammonium sulfate fractionation, column chromatographies of Q-Sepharose, SP-Sepharose, and Superdex 200 with a yield of 8.0%, and purification folds of 468. The molecular mass of GPI was 120 kDa as estimated by gel filtration, while on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two subunits (55 and 65 kDa) were identified, suggesting that it is a heterodimer. Interestingly, GPI revealed specific inhibitory activity toward a myofibril-bound serine proteinase (MBSP) from crucian carp, while no inhibitory activity was identified toward other serine proteinases, such as white croaker MBSP and crucian carp trypsin. Kinetic analysis showed that GPI is a competitive inhibitor toward MBSP, and the K(i) was 0.32 microM. Our present results indicated that the multifunctional protein GPI is an endogenous inhibitor to MBSP and may play a significant role in the regulation of muscular protein metabolism in vivo.

[Exploration and classification in the amount of die stone of working cast model of denture restoration].

PURPOSE: The objective of this study was to survey the amount of working cast model of denture restoration, and to calculate the amount of die stone powder of each working cast model, then to classify the working cast model, with the expectation to perfuse the working cast model in clinic according to this classification, and to achieve the goal of increasing quality of working cast model and saving materials. METHODS: 311 working cast models were selected to perfuse with three kinds of tray including small size, middle size and large size randomly in clinic. The amount of die stone powder of working cast model was calculated, and the data were analyzed using SPSS 13.0 software package. The mean values of the die stone powder was used to establish a classification, which was verified clinically. RESULTS: The results showed that the working cast model of denture restoration could be divided into three kinds: small group (< or = 45 g), middle group (46-55 g) and large group (56-65 g). The samples for verification were all in the size of this classification of the amount of die stone powder. CONCLUSIONS: There is a close relationship between the amount of die stone powder of working cast model and the size of the tray, the dental arch. By clinical application of the classification, quantitative control of standard water powder ratio, improvement of model qualities and saving of materials can be achieved.

Purification and characterisation of trypsins from the pyloric caeca of mandarin fish (Siniperca chuatsi).

Two trypsins of anionic form (trypsin A) and cationic form (trypsin B) from the pyloric caeca of mandarin fish (Siniperca chuatsi) were highly purified by a series of chromatographies, including DEAE-Sephacel, Sephacryl S-200 HR, Q-Sepharose or SP-Sepharose. Purified trypsins revealed a single band on native-PAGE. The molecular weights of trypsin A and B were 21kDa and 21.5kDa, respectively, as estimated by SDS-PAGE, both under reducing and non-reducing conditions. Zymography analysis showed that both trypsins were active in degrading casein. Trypsin A and B exhibited maximal activity at 35°C and 40°C, respectively, and shared the same optimal pH of 8.5, using Boc-Phe-Ser-Arg-MCA as substrate. The two trypsins were stable up to 45°C and in the pH range from 4.5 to 11.0. Trypsin inhibitors are effective on these two enzymes and their susceptibilities were similar. Both trypsins were activated by metal ions such as Ca(2+) and Mg(2+) and inactivated by Fe(2+), Zn(2+), Mn(2+), Cu(2+), Al(3+), Ba(2+) and Co(2+) to different degrees. Apparent Km values of trypsin A and B were 2.18µM and 1.88µM, and Kcat values were 81.6S(-1) and 111.3S(-1) for Boc-Phe-Ser-Arg-MCA, respectively. Immunoblotting analysis using anti-common carp trypsin A positively cross-reacted with the two enzymes, suggesting their similarity. The N-terminal amino acid sequence of trypsin B was determined as IVGGYECEAH, which is highly homologous with trypsins from other species of fish.