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Myofibril-bound serine protease and its endogenous inhibitor in mouse: extraction, partial characterization and
Marcela P Sangorrín1, Celina B Martone, Jorge J Sánchez
1Instituto de Investigaciones Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Casilla de Correo 1245, 7600, Mar del Plata, Argentina. msangorr@uncoma.edu.ar
Abstract:
The protein content of muscle is determined by the relative rates of synthesis and degradation. The balance between this process determines the number of functional contractile units within each muscle cell. Myofibril-bound protease, protease M previously reported in mouse skeletal muscle could be solubilized from the myofibrillar fraction by salt and acid treatment and partially purified by Mono Q and Superose 12 chromatography. Isolated protease M activity in vitro on whole myofibrils resulted in myosin, actin, troponin T, alpha-actinin and tropomyosin degradation. Protease M is serine type and was able to hydrolyze trypsin-type synthetic substrates but not those of chymotrypsin type. In gel filtration chromatography, protease M showed Mr 120.0 kDa. The endogenous inhibitor (MHPI) is a glycoprotein (110.0 kDa) that efficiently blocks the protease M-dependent proteolysis of myofibrillar proteins in a dose-dependent way, as shown by electrophoretic analysis and synthetic substrates assays. Protease M-Inhibitor system would be implicated in myofibrillar proteins turnover.
Insights
Muscle protein content relies on synthesis and degradation rates. A newly purified protease M enzyme degrades key myofibrillar proteins, suggesting its role in muscle protein turnover.
Area of Science:
- Muscle physiology
- Protein biochemistry
- Enzymology
Background:
- Muscle protein content is regulated by synthesis and degradation rates.
- This balance dictates the number of functional contractile units in muscle cells.
- Myofibril-bound protease M was previously identified in mouse skeletal muscle.
Purpose of the Study:
- To solubilize and partially purify protease M from mouse skeletal muscle myofibrils.
- To characterize the activity and properties of protease M.
- To investigate the role of protease M and its inhibitor in myofibrillar protein turnover.
Main Methods:
- Solubilization using salt and acid treatment.
- Partial purification via Mono Q and Superose 12 chromatography.
- In vitro activity assays on whole myofibrils and synthetic substrates.
- Gel filtration chromatography for molecular weight determination.
- Electrophoretic analysis to assess protein degradation and inhibitor function.
Main Results:
- Protease M was solubilized and partially purified.
- Protease M degraded myosin, actin, troponin T, alpha-actinin, and tropomyosin.
- Protease M is a serine protease with trypsin-like activity and a molecular weight of 120.0 kDa.
- An endogenous glycoprotein inhibitor (MHPI, 110.0 kDa) effectively blocked protease M activity.
- MHPI demonstrated dose-dependent inhibition of protease M-dependent proteolysis.
Conclusions:
- A protease M-inhibitor system is implicated in myofibrillar protein turnover.
- This system likely plays a role in regulating muscle protein content.
- Further research into this protease system could reveal therapeutic targets for muscle-related disorders.