Liliana Busconi

Identification of two alkaline proteases and a trypsin inhibitor from muscle of white croaker (Micropogon opercularis)

Extracts from white croaker skeletal muscle showed two alkaline proteases and a trypsin inhibitor when they were chromatographed in DEAE‐Sephacel. The activity against azocasein was maximal at pH 8.5 and 9.1 for proteases I and II, respectively. Both enzymes showed optimum activity at 60° C. The molecular masses were found to be 132 kDa for protease 1,363 kDa for protease II, and 65 kDa for the inhibitor. Protease I showed the characteristics of a trypsin‐like enzyme, and protease II those of a SH‐enzyme. These proteins may play important roles in mechanisms of cellular proteolysis.

Multicatalytic proteinase in fish muscle.

A partially active and a latent form of multicatalytic protease (MCP) were isolated from fish skeletal muscle. Both forms were inactive against protein substrates, but their activity against peptide substrates differed in one order of magnitude. The chymotrypsin-like activity of the partially active form was moderately stimulated by fatty acids and SDS, whereas its trypsin-like activity was inhibited by the same reagents. In contrast, both activities of the latent form were strongly stimulated by SDS. The chymotrypsin-like activity of the latent form was also stimulated by heating or high urea concentrations, whereas its trypsin-like activity did not change or was inhibited respectively by these treatments. These activation effects were irreversible. Pre-treatment of the latent form with SDS or urea in the absence of substrate led to its irreversible inactivation, whereas activation by pre-heating occurred in the presence or absence of substrate. These results suggest that MCP can exist in several active states with distinct properties. Studies on the distribution of MCP in fish tissues showed a much higher level of the enzyme in gonads than in any other tissue, suggesting a role of MCP in development.

Detection of a trypsin-like serine protease and its endogenous inhibitor in hake skeletal muscle

When dialyzed extracts from hake (Merluccius hubbsi) skeletal muscle were chromatographed in DEAE-Sephacel, an alkaline protease (37 degrees C, pH 8.5) and a trypsin inhibitor were isolated. The enzyme showed its maximal activity against azocasein in the range of pH between 7 and 9. The protease was able to hydrolyze the trypsin substrates Bz-Arg-OEt and Tos-Arg-OMe and did not cleave the chymotrypsin substrate Bz-Tyr-OEt. The enzyme was strongly inhibited by several serine protease inhibitors, whereas inhibitors of the other types of proteases scarcely affected it. The protease was able to degrade the major contractile and cytoskeletal constituent proteins of myofibrils and to accumulate acid-soluble products. The protease activity was completely suppressed by the addition of the trypsin inhibitor isolated from the same muscle. These results indicate that hake skeletal muscle contains a trypsin-like serine protease which might be involved in the catabolism of myofibrillar proteins, as well as in the proteolytic events that take place during post mortem storage of fish muscle.

Fish muscle cytoskeletal network: Its spatial organization and its degradation by an endogenous serine proteinase

The extraction of white croaker skeletal myofibrils with KI rendered a residue in which a network of longitudinal and transverse filaments could be observed by scanning electron microscopy. A trypsin-like serine proteinase isolated from the same muscle was able to produce a complete and rapid disruption of the network, while major myofibrillar proteins were only slightly modified. This fact suggests that the disassembly of the cytoskeletal network may be an early event in the proteolysis of myofibrils. Desmin was not attacked by the proteinase under the assayed conditions, which indicates that some other unidentified component of the network would be the primary target of the action of the enzyme on myofibrils.

Action of a serine proteinase from fish skeletal muscle on myofibrils.

The action of a serine proteinase from fish skeletal muscle on myofibrils was studied. The enzyme was able to destroy the structural integrity of myofibrils, and to degrade both their major contractile and cytoskeletal constituent proteins. Proteolysis could be completely prevented by the addition of a trypsin inhibitor isolated from the same muscle.

Purification and characterization of a latent form of multicalytic proteinase from fish muscle

1. A latent form of multicatalytic proteinase (MCP) was purified to apparent homogeneity from white croaker muscle by DEAE-Sephacel, Mono-Q, Sephacryl S-300 and second Mono-Q chromatographies. 2. The enzyme preparation was electrophoretically and immunologically similar to MCP purified from the same source by a different method (Folco et al., 1988b, Archs Biochem. Biophys. 267, 599-605) but showed much lower chymotrypsin- and trypsin-like activities. 3. These activities responded to sodium dodecyl sulphate (SDS), urea and heat treatments in different ways: SDS stimulated both activities, urea stimulated the former and inhibited the latter and heating stimulated the former and did not affect the latter. 4. The stimulation of chymotrypsin-like activity by the three treatments was irreversible. 5. Exposure of MCP to SDS or urea in the absence of substrate rapidly inactivated it, whereas heat activation took place irrespective of the presence of substrate. 6. The stimulating effect of SDS on chymotrypsin-like activity was lost in the presence of urea. 7. These results suggest that the enzyme may be activated by different mechanisms.

Activation of an alkaline proteinase from fish skeletal muscle by fatty acids and sodium dodecyl sulphate

1. Fish skeletal muscle contains an alkaline thiol proteinase with a temperature optimum of 60 degrees C and undetectable activity below 50 degrees C. 2. The present study shows that fatty acids and sodium dodecyl sulphate (SDS) shifted the temperature-activity curve of the enzyme toward the lower temperature side. 3. All unsaturated fatty acids tested strongly stimulated proteolytic activity at 37 degrees C, whereas myristic acid was the only saturated fatty acid that produced an important degree of activation. 4. These effects could be observed at millimolar concentrations of the reagents.

MULTICATALYTIC PROTEINASE IN FISH MUSCLE

A partially active and a latent form of multicatalytic protease (MCP) were isolated from fish skeletal muscle. Both forms were inactive against protein substrates, but their activity against peptide substrates differed in one order of magnitude. The chymotrypsin-like activity of the partially active form was moderately stimulated by fatty acids and SDS, whereas its trypsin-like activity was inhibited by the same reagents. In contrast, both activities of the latent form were strongly stimulated by SDS. The chymotrypsin-like activity of the latent form was also stimulated by heating or high urea concentrations, whereas its trypsin-like activity did not change or was inhibited respectively by these treatments. These activation effects were irreversible. Pre-treatment of the latent form with SDS or urea in the absence of substrate led to its irreversible inactivation, whereas activation by pre-heating occurred in the presence or absence of substrate. These results suggest that MCP can exist in several active states with distinct properties. Studies on the distribution of MCP in fish tissues showed a much higher level of the enzyme in gonads than in any other tissue, suggesting a role of MCP in development.

Distribution of multicatalytic proteinase in fish tissues

Abstract 1. 1. MCP with similar immunoreactivity in Ouchterlony double-diffusion test and Western blot analysis was found in various white croaker tissues. 2. 2. The MCP content in different tissues was evaluated by immunoassay. The enzyme level was much higher in gonad than in any other organ tested, suggesting an important role of MCP in the process leading to embryonic development.