Effect of nitric oxide and calpastatin on the inhibition of μ-calpain activity, autolysis and proteolysis of myofibrillar proteins

The aim of this study was to investigate the dual effect of the nitric oxide donor NOR-3 and calpastatin on mu-calpain activity, autolysis, and proteolytic ability. mu-Calpain and calpastatin were purified and allocated to the following five treatments: mu-calpain, mu-calpain + calpastatin, mu-calpain + NOR-3, mu-calpain + calpastatin + NOR-3, and mu-calpain + NOR-3 + calpastatin. mu-Calpain autolysis and the activity against purified myofibrils was initiated by addition of calcium. Results showed that NOR-3 could induce mu-calpain S-nitrosylation and effectively block the activity via the inhibition of mu-calpain autolysis. Calpastatin inhibited mu-calpain activity in a dose-dependent manner. The combined treatment of NOR-3 and calpastatin exerted a further inhibitory effect on mu-calpain activity, autolysis and proteolysis which was affected by the addition order of NOR-3 and calpastatin. Our data suggest that S-nitrosylation may play a regulatory role in mediating mu-calpain activity in the presence of calpastatin.