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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.
Archives of Biochemistry and Biophysics
|December 1, 1988
Summary
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.