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Updated: Aug 24, 2026
Evaluation of Protein–Protein Interactions using an On-Membrane Digestion Technique
Published on: July 19, 2019
Effects of autolysis on properties of mu- and m-calpain
Hongqi Li1, Valery F Thompson, Darrel E Goll
1Muscle Biology Group, University of Arizona, Tucson, AZ 85721-0038, USA.
Abstract:
Although the biochemical changes that occur during autolysis of mu- and m-calpain are well characterized, there have been few studies on properties of the autolyzed calpain molecules themselves. The present study shows that both autolyzed mu- and m-calpain lose 50-55% of their proteolytic activity within 5 min during incubation at pH 7.5 in 300 mM or higher salt and at a slower rate in 100 mM salt. This loss of activity is not reversed by dialysis for 18 h against a low-ionic-strength buffer at pH 7.5. Proteolytic activity of the unautolyzed calpains is not affected by incubation for 45 min at ionic strengths up to 1000 mM. Size-exclusion chromatography shows that ionic strengths of 100 mM or above cause dissociation of the two subunits of autolyzed calpains and that the dissociated large subunits (76- or 78-kDa) aggregate to form dimers and trimers, which are proteolytically inactive. Hence, instability of autolyzed calpains is due to aggregation of dissociated heavy chains. Autolysis removes the N-terminal 19 (m-calpain) or 27 (mu-calpain) amino acids from the large subunit and approximately 90 amino acids from the N-terminus of the small subunit. These regions form contacts between the two subunits in unautolyzed calpains, and their removal leaves only contacts between domain IV in the large subunit and domain VI in the small subunit. Although many of these contacts are hydrophobic in nature, ionic-strength-induced dissociation of the two subunits in the autolyzed calpains indicates that salt bridges have an important, possibly indirect, role in the domain IV/domain VI interaction.
Insights
Autolyzed calpains lose significant proteolytic activity in high salt due to subunit dissociation and heavy chain aggregation. This instability is linked to altered inter-subunit contacts after autolysis.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Calpains are calcium-dependent proteases involved in various cellular processes.
- Autolysis is a key regulatory mechanism for calpain activity.
- Limited understanding exists regarding the properties of autolyzed calpain molecules.
Purpose of the Study:
- To investigate the properties and stability of autolyzed mu- and m-calpain.
- To elucidate the molecular mechanisms underlying the loss of proteolytic activity in autolyzed calpains.
- To characterize the role of ionic strength and subunit interactions in calpain stability.
Main Methods:
- Incubation of autolyzed and unautolyzed calpains at varying salt concentrations and pH.
- Assay of proteolytic activity.
- Size-exclusion chromatography to analyze subunit dissociation and aggregation.
- Analysis of N-terminal deletions resulting from autolysis.
Main Results:
- Autolyzed mu- and m-calpain lost 50-55% activity in high salt (≥300 mM), with slower loss in 100 mM salt.
- Activity loss was irreversible by dialysis.
- Unautolyzed calpains remained active across a wide ionic strength range (up to 1000 mM).
- High ionic strength induced dissociation of autolyzed calpain subunits, leading to aggregation of heavy chains and loss of activity.
- Autolysis removes N-terminal regions crucial for inter-subunit contacts.
Conclusions:
- Instability of autolyzed calpains is attributed to salt-induced dissociation of subunits and subsequent aggregation of heavy chains.
- The altered inter-subunit contacts after autolysis, particularly between domain IV and VI, are sensitive to ionic strength.
- Salt bridges play a significant role in maintaining the interaction between calpain subunits.
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