The effects of a milli(m)-calpain isolated from the white muscle of sea bass (Dicentrarchus labrax L) and commercial cathepsins B, D and L, used in combination on the myofibrillar and sarcoplasmic proteins were examined. Protein digestion was first performed by the endogenous m-calpain, (luring 2 h before the addition of a mixture of cathepsins B, D and L and a further incubation up to 22 h. Calpain degraded a 27 kDa sarcoplasmic component as well as myosin heavy chain, a-actinin, desmin and a 32 kDa component from the myofibrillar fraction. A 97 kDa component and the assumed creatine kinase-aldolase doublet were degraded during the incubation of sarcoplasmic proteins with the cathepsin mixture while, among the myofibrillar proteins, myosin, actin, tropomyosin, and the 22 kDa component were digested by the same enzymatic preparation. Myofibril-bound a-actinin and tropomyosin were released into the soluble fraction by m-calpain and the cathepsins mixture. Some smaller fragments were also produced. This appears to be the result of the cumulative action of each protease, as already investigated in our previous studies, except for the degradation of the sarcoplasmic 97 kDa component. It was only degraded if the cathepsins were mixed; this indicates that the three cathepsins functioned synergistically on muscle proteins. No difference in the proteolytic effect of cathepsins was observed, whether calpain was acting first or not. This work showed that calpain did not modify any muscle protein prior to later hydrolysis by the catheptic proteinases. (C) 2004 Elsevier Ltd. All rights reserved.