Shizunori Ikeda

Enzymatic characterization of anionic trypsin of the catfish (Parasilurus asotus).

An anionic trypsin, isolated from the pancreatic extract of the catfish (Parasilurus asotus), had a pH optimum of 8.3 for the hydrolysis of N-tosyl-L-arginine methyl ester. The enzyme was most stable at pH 6.0-8.5, and was stabilized by calcium ions. The enzyme was inhibited by typical trypsin inhibitors including some serine proteinase inhibitors. Km and kcat values of the enzyme for N-tosyl-L-arginine methyl ester and N-tosyl-L-lysine methyl ester were quite similar to those of bovine cationic trypsin.

Comparison of muscle proteinase activity among fish species

The effects of temperature during storage, portion of muscle and growth stage of fish on the activity level of carp muscle acid (cathepsin D, EC 3.4.23.5), neutral and alkaline proteinases were examined. Icing storage, freezer storage and portion of muscle did not affect each proteinase activity (P less than 0.05), but acid proteinase activity was affected by growth stage (P less than 0.05) and the level decreased from small to large fish. The activity levels of three kinds of proteinases were compared among species (P less than 0.05) and the following order was obtained. Acid proteinase, white croaker = rainbow trout = carp greater than sardine = common mackerel, sardine greater than lizardfish, common mackerel = lizardfish. Neutral proteinase, rainbow trout greater than carp = white croaker greater than lizardfish = sardine = common mackerel. Alkaline proteinase, rainbow trout = sardine greater than white croaker = carp = common mackerel greater than lizardfish.

Distribution of pancreatic elastase and metalloproteinase in several species of fish

Abstract 1. 1. By CM-cellulose column chromatography, fifteen elastase-like enzyme preparations were separated from the activated pancreatic extracts of the gummy shark, red stingray, rainbow trout, carp, eel, bluefin tuna, yellowtail, sea bass, and angler. 2. 2. These preparations were characterized by using specific substrates and inhibitors. 3. 3. Pancreatic elastases, which belong to the group of serine proteinases, were found to be present in all the fishes examined. 4. 4. Among the fishes examined, pancreatic metalloproteinases with elastase activity were demonstrated only in the bluefin tuna and yellowtail.

Purification and characterization of a new metalloproteinase with elastolytic activity from the catfish pancreas.

An elastolytic enzyme was purified to homogeneity from the pancreas of the catfish Parasilurus asotus by chromatography on CM-cellulose column and affinity chromatography on (Ala)3-CH-Sepharose 4B column. The molecular weight of the enzyme was estimated to be 24 000 by SDS-polyacrylamide gel electrophoresis. The enzyme had elastolytic activity as well as activity toward Suc-(Ala)3-NA. The enzyme was inhibited by EDTA, EGTA, o-phenanthroline, dithiothreitol, and cysteine, but not by the serine proteinase inhibitors iPr2P-F and PhCH2SO2F. In addition, the enzyme was found to require Zn2+ for activity. These results indicate that the catfish enzyme belongs to the group of metalloproteinases and that it is a new type of pancreatic enzyme, unlike the pancreatic elastases which are serine proteinases.

Purification and some properties of two anionic trypsins from the eel (Anguilla japonica).

Two anionic enzymes, designated as trypsins 1 and 2, were purified from the pancreas of the eel Anguilla japonica by DEAE-cellulose column chromatography and Sephadex G-75 gel filtration. The final preparation of trypsin 1 was homogeneous but that of trypsin 2 still contained impurities. Both enzymes had similar pH optima of near 8.3 for the hydrolysis of N-tosyl-L-arginine methyl ester. Trypsin 1 was stabilized by calcium ions but the stability of trypsin 2 was not affected by calcium ions. Both enzymes were inhibited by typical trypsin inhibitors including serine proteinase inhibitors.

Distribution of trypsin and chymotrypsin, and their zymogens in digestive organs of the eel (Anguilla japonica)

Abstract 1. 1. Trypsinogen and chymotrypsinogen in the pancreatic extract of the eel (Anguilla japonica) were optimally activated by porcine enteropeptidase at 37°C and pH 8.0 in the presence of CaCl2. 2. 2. The method for measuring the potential activities of both zymogens was established on the basis of assays of the activities after the activation of the zymogens under the optimal conditions. 3. 3. In the fed and starved eel, the potential activities of both zymogens were found in the pancreas. 4. 4. These results indicate that the eel maintains digestive function of the pancreas even under undernourished conditions.

Intracellular distribution of fish muscle alkaline proteinase

1. Intracellular distribution of a muscle alkaline proteinase was investigated on four kinds of fish. 2. The total activity of the muscle proteinase of carp, Cyprinus carpio, was larger in both myofibrillar (Mf) and microsomal (Mic) fractions than the activity in mitochondrial, lysosomal, and supernatant fractions. The activity found in Mf fraction seemed to due to the Mic enzyme which was not separated from Mf fraction. 3. The relative specific activity was mostly found in Mic fraction in the species tested. 4. The results indicate that the distribution pattern of fish muscle alkaline proteinase is different from those of cathepsin D and acid phosphatase. 5. The Mic fraction hydrolyzed Mf and sarcoplasmic proteins. The rates were 40 and 55%, respectively, of the rate when casein was used as a substrate.

Purification and properties of carp (Cyprinus carpio) muscle calpain II (high-Ca2+-requiring form of calpain)

Calpain (Ca2+-dependent cysteine proteinase) was purified to apparent homogeneity from carp muscle by the method of DEAE-cellulose, hydroxylapatite and Ultrogel AcA 34 column chromatographies. The purified enzyme is classified as calpain II (high-Ca2+-requiring form of calpain) from the effects of Ca2+ concentration, pH and the antibiotics on the activity. Carp muscle calpain II was inhibited by rat liver calpastatin, the specific inhibitor for calpain. It is probable that the calpain-calpastatin system may play a biologically fundamental and common role in various cells, since the inhibitory effect of calpastatin on calpain from different tissues of different species is well conserved.

Some enzymic properties and digestive function of a pancreatic metalloproteinase in the catfish (Parasilurus asotus)

Abstract 1. 1. A pancreatic metalloproteinase, isolated from the catfish (Parasilurus asotus), had optimum pH and temperature of pH 7.5 and 40 to 45°C, respectively. 2. 2. The enzyme was most stable at pH 7.0 and 9.2 and below 30°C. 3. 3. The enzyme had high activities toward Suc-(Ala)3-NA, elastin, casein, and hemoglobin. 4. 4. The enzyme plays a role in the protein digestion in the anterior part of the intestine of the catfish.

Purification and some properties of a trypsin inhibitor from carp (Cyprinus carpio) muscle

A trypsin inhibitor was purified from carp muscle to apparent homogeneity by the successive chromatographies of DEAE-cellulose, DEAE-Sepharose CL-6B, Con A-Sepharose, Ultrogel AcA 44 and hydroxylapatite. The mol. wt of the inhibitor was estimated to be 58,000 by SDS-polyacrylamide gel electrophoresis or 50,000 by gel filtration. The inhibitor seemed to form a 1:1 stoichiometric complex with trypsin, alpha-chymotrypsin and elastase, respectively. Carp muscle trypsin inhibitor was likely to be identical with serum alpha 1-proteinase inhibitor judging from its glycoprotein nature, mol. wt and the inhibition stoichiometry.