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Multicatalytic proteinase in fish muscle
E J Folco1, L Busconi, C B Martone
1Instituto Nacional de Tecnología Industrial, Centro de Investigaciones de Tecnología Pesquera, Mar del plata, Argentina.
Abstract:
Proteinase II, a high-molecular-mass proteinase previously identified in white croaker skeletal muscle, was purified to apparent homogeneity by DEAE-Sephacel, phenyl-Sepharose CL 4B, and Sephacryl S-300 chromatographies. Under denaturing conditions, the enzyme dissociated into a cluster of subunits with Mr ranging from 18,000 to 26,000 and a large subunit with a Mr 60,000. The proteinase was able to hydrolyze N-terminal-blocked 4-methyl-7-coumarylamide substrates having either an aromatic amino acid (chymotrypsin-like activity) or an arginine residue (trypsin-like activity) adjacent to the fluorogenic group. The trypsin-like activity of the enzyme was inhibited by fatty acids and sodium dodecyl sulfate, whereas the chymotrypsin-like activity was stimulated by those compounds but inhibited by nonionic and cationic detergents. Several thiol reagents inhibited both proteinase II activities. However, leupeptin and Cu2+ strongly inhibited its trypsin-like activity but only slightly affected its chymotrypsin-like activity. Dithiothreitol stimulated both activities, but at different extents and in different concentration ranges. These results suggest that the enzyme is multicatalytic, having at least two different active sites.
Insights
White croaker skeletal muscle proteinase II was purified and found to be multicatalytic. This enzyme exhibits both trypsin-like and chymotrypsin-like activities, suggesting distinct active sites.
Area of Science:
- Biochemistry
- Enzymology
- Proteomics
Background:
- Proteinase II, a high-molecular-mass enzyme, was previously identified in white croaker skeletal muscle.
- Understanding the catalytic properties and subunit composition of this proteinase is crucial for elucidating its biological function.
Purpose of the Study:
- To purify Proteinase II to homogeneity.
- To characterize its subunit composition and enzymatic activities.
- To investigate the enzyme's substrate specificity and the effects of various inhibitors and activators.
Main Methods:
- Purification using DEAE-Sephacel, phenyl-Sepharose CL 4B, and Sephacryl S-300 chromatography.
- Analysis of subunit composition under denaturing conditions using molecular weight determination.
- Enzymatic assays using N-terminal-blocked 4-methyl-7-coumarylamide substrates.
- Inhibition and activation studies with fatty acids, detergents, thiol reagents, leupeptin, Cu2+, and dithiothreitol.
Main Results:
- Proteinase II was purified to apparent homogeneity.
- The enzyme consists of subunits with Mr ranging from 18,000 to 26,000 and a large subunit of Mr 60,000.
- Proteinase II demonstrated both chymotrypsin-like and trypsin-like activities.
- Differential inhibition and stimulation patterns observed with various agents suggest distinct active sites for each activity.
Conclusions:
- Proteinase II is a multicatalytic enzyme with at least two distinct active sites.
- The enzyme's complex subunit structure and dual catalytic activities highlight its unique biochemical properties.
- Further research is warranted to fully elucidate the structure-function relationship of Proteinase II.