Is there a protease that preferentially cleaves the M-line in partially dehydrated muscle?
M Yamaguchi1, M Muguruma, T Sako
1Muscle Biology Laboratory, Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA.
Abstract:
When a partially dehydrated muscle fibre bundle (PDM, 65% H(2)O, pH 5.5, at 4 °C) was treated with a supernatant fraction (M-line-cleaving fraction: MCF) of muscle homogenate for 5 hr, the M-lines disappeared. MCF was extracted from rabbit skeletal muscles by homogenization with 15 mM HCl containing 0.5 M NaCl (pH 3.7), fractionated with 25-65% (NH(4))(2)SO(4) and clarified by Sephadex G-75. Rabbit psoas PDM was obtained with an osmotic dehydration sheet and glycerinated. One end of the bisected fibre bundle was incubated with 10 mM Na-acetate (pH 5.5), 1 mM EDTA, 5 mM β-mercaptoethanol (β-MCE), 150 mM KCl, 10 mM NaN(3) with MCF at 25 °C for 5 hr, the muscle being stretched and relaxed several times. The other end was incubated in the same solution, except that MCF was omitted (control). Electron microscopy showed the myofibrils broken down at the M-line in the presence of MCF. The myofilaments were closely packed near the Z-line and flared out at both ends near the centre of the sarcomere (bow-tie shape). Thus, the Z-line is not the only target of proteases and structural decomposition can also occur at the M-line under specific conditions. An M-line cleaving protease may exist in the MCF muscle extract.
Insights
A novel M-line cleaving fraction (MCF) from rabbit skeletal muscle causes M-line disappearance in partially dehydrated muscle fiber bundles. This indicates structural decomposition can occur at the M-line, suggesting the existence of specific M-line proteases.
Area of Science:
- Muscle physiology
- Biochemistry
- Protease research
Background:
- Muscle fiber structure and integrity are crucial for function.
- Proteolytic activity in muscle is well-documented, primarily targeting Z-lines.
- The M-line's role in sarcomere stability and potential degradation pathways are less understood.
Purpose of the Study:
- To investigate the effect of a specific muscle homogenate fraction on M-line integrity.
- To determine if M-line degradation can occur independently of Z-line degradation.
- To identify potential proteases responsible for M-line breakdown.
Main Methods:
- Preparation of partially dehydrated muscle fiber bundles (PDM) from rabbit psoas muscle.
- Extraction and fractionation of muscle homogenate to obtain an M-line cleaving fraction (MCF).
- Incubation of PDM with MCF and subsequent analysis using electron microscopy.
Main Results:
- Treatment with MCF led to the disappearance of M-lines in PDM.
- Electron microscopy revealed myofibril breakdown at the M-line, with myofilaments exhibiting a 'bow-tie' shape.
- Control samples without MCF showed intact M-lines.
Conclusions:
- The M-line is susceptible to proteolytic degradation under specific conditions.
- A novel M-line cleaving protease activity is present in the MCF.
- These findings expand the understanding of sarcomere decomposition pathways beyond Z-line targeting.
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