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 µ-calpain activity, autolysis, and proteolytic ability. µ-Calpain and calpastatin were purified and allocated to the following five treatments: µ-calpain, µ-calpain + calpastatin, µ-calpain + NOR-3, µ-calpain + calpastatin + NOR-3, and µ-calpain + NOR-3 + calpastatin. µ-Calpain autolysis and the activity against purified myofibrils was initiated by addition of calcium. Results showed that NOR-3 could induce µ-calpain S-nitrosylation and effectively block the activity via the inhibition of µ-calpain autolysis. Calpastatin inhibited µ-calpain activity in a dose-dependent manner. The combined treatment of NOR-3 and calpastatin exerted a further inhibitory effect on µ-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 µ-calpain activity in the presence of calpastatin.