EFFECT OF A MYOFIBRIL-BOUND SERINE PROTEINASE ON THE DEG OF GIANT PROTEIN TITIN AND NEBULIN

Abstract

A simplified method has been adapted for the extraction and purification of the massive myofibrillar protein titin from the skeletal muscle of sea bream to homogeneity, and a specific polyclonal antibody against titin was generated in rat. The effect of myofibril-bound serine proteinase (MBSP) on the degradation of titin and nebulin from sea bream (Sparus latus Houttuyn) was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by Western blot. Analysis revealed that both titin and nebulin could be degraded by MBSP, with nebulin degrading faster whether at the optimum temperature of MBSP (55C) and cold storage temperature (4C). These results suggested that MBSP is an important enzyme not only involved in the degradation of myofibrillar proteins such as myosin heavy chain, α-actinin, actin and tropomyosin, but also giant proteins titin and nebulin during the postmortem stage. PRACTICAL APPLICATIONS Titin and nebulin are giant proteins with molecular masses of 3,000 kDa and 600–800 kDa, respectively. Until now, few research concerning the degradation of these two proteins of fish has been reported. In the present study, a simplified method was adapted to purify native titin, and a specific polyclonal antibody against it was prepared. The degradation effect of a myofibril-bound serine proteinase on titin and nebulin at two different temperatures (55C, 4C) was investigated. It is expected that the specific antibody against titin may be used to detect the degradation of titin during the postmortem stage of fish effectively.