Do the calpains degrade actin, α‐actinin, or myosin?

Proteolytic activity of the calpains is characterized by their unique and very limited subsite specificity. The initial report describing purification and partial characterization of the calpains found that m‐calpain did not cleave either actin or myosin, the two major proteins in striated muscle, and did not cleave α‐actinin, the major protein in Z‐disks, although it rapidly and completely removed Z‐disks. Since then, different studies have reported that the calpains degrade myosin heavy chain (MHC), actin, and α‐actinin. Therefore, we have re‐examined the effects of the two calpains, μ‐ and m‐calpain, on these three proteins. At high ratios of protease to substrate (~1:25 w/w), both μ‐ and m‐calpain slowly cleave the 200‐kDa MHC to fragments of 185‐, 172‐ or 145‐kDa. Both calpains cleave the 25‐kDa myosin light chain 1 to a fragment of 22‐kDa ; this cleavage occurs only in myofibrils and not when the calpains are incubated with purified myosin. Similarly, both calpains cleave the 100‐kDa α‐actinin polypeptide to fragments of 98‐, 80‐, 79‐, and 76‐kDa when incubated with myofibrils; purified α‐actinin is degraded more slowly to 80‐ and 76‐kDa fragments. Over 90% of the 100‐kDa α‐actinin polypeptide is in the 100‐/98‐kDa form after 120 min at 25 °C and calpain to myofibril ratio of 1:25 w/w. Neither calpain cleaves actin regardless or whether the actin is in the purified state or in myofibrils. Supported by the NRI, NIH, and the MDA.