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Purification and partial characterization of a myofibril-bound serine protease from ostrich skeletal muscle
Shonisani C Tshidino1, Jason Krause, Abayomi P Adebiyi
1Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, Port Elizabeth 6031, South Africa.
Abstract:
A myofibril-bound serine protease (MBSP) was partially purified from ostrich (Struthio camelus) skeletal muscle. MBSP was dissociated from the myofibrillar fraction by ethylene glycol treatment at pH 8.5, followed by partial purification via Toyopearl Super Q 650 S and p-aminobenzamidine column chromatographies. Ostrich MBSP revealed a major protein band of approximately 21 kDa on SDS-PAGE, showing proteolytic activity after casein zymography. Optima pH and temperature of ostrich MBSP were 8 and 40 degrees C, respectively. Substrate specificity analysis revealed that the enzyme cleaved synthetic fluorogenic substrates at the carboxyl side of arginine residues. Kinetic parameters (K(m) and V(max) values) were calculated from Lineweaver-Burk plots. The kinetic characteristics of ostrich MBSP were compared to values obtained for commercial bovine trypsin in this study, as well as those obtained for MBSP from mouse and various fish species. The results suggest that ostrich MBSP is a tryptic-like serine protease. Ostrich MBSP exhibited low sequence identity to commercial bovine trypsin (44%), MBSP from lizard fish skeletal muscle (33%) and trypsinogen from ostrich pancreas (22%).
Insights
Researchers purified a myofibril-bound serine protease (MBSP) from ostrich skeletal muscle, identifying it as a tryptic-like enzyme with specific activity and low sequence similarity to other proteases.
Area of Science:
- Biochemistry
- Enzymology
- Proteomics
Background:
- Myofibril-bound serine proteases (MBSP) play roles in muscle function.
- Characterization of MBSP from diverse species aids understanding of protease evolution and function.
Purpose of the Study:
- To partially purify and characterize a myofibril-bound serine protease (MBSP) from ostrich skeletal muscle.
- To compare the properties of ostrich MBSP with other serine proteases.
Main Methods:
- Partial purification of MBSP from ostrich skeletal muscle using ethylene glycol treatment and column chromatographies (Toyopearl Super Q 650 S, p-aminobenzamidine).
- SDS-PAGE for molecular weight determination (~21 kDa).
- Casein zymography for proteolytic activity assessment.
- Determination of optimal pH (8) and temperature (40°C).
- Substrate specificity analysis using fluorogenic substrates.
- Kinetic parameter (K(m), V(max)) calculation via Lineweaver-Burk plots.
- Sequence identity comparison with bovine trypsin and other MBSP/trypsinogens.
Main Results:
- Ostrich MBSP was purified, showing a ~21 kDa band and proteolytic activity.
- Optimal activity at pH 8 and 40°C.
- Enzyme cleaved substrates at the carboxyl side of arginine residues, indicating tryptic-like activity.
- Low sequence identity (44%) to bovine trypsin.
Conclusions:
- Ostrich skeletal muscle contains a tryptic-like serine protease (MBSP) with distinct biochemical properties.
- The findings contribute to the comparative enzymology of muscle proteases across species.
