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[摘要]：Nitric oxide (NO center dot) is synthesized in skeletal muscle and its production increases during contractile activity. Although myosin is the most abundant protein in muscle, it is not known whether myosin is a target of NO center dot or NO center dot derivatives. In the present study, we have shown that exercise increases protein S-nitrosylation in muscle, and, among contractile proteins, myosin is the principal target of exogenous SNOs (S-nitrosothiols) in both skinned skeletal muscle fibres and differentiated myotubes. The reaction of isolated myosin with S-nitrosoglutathione results in S-nitrosylation at multiple cysteine thiols and produces two populations of protein-bound SNOs with different stabilities. The less-stable population inhibits the physiological ATPase activity, without affecting the affinity of myosin for actin. However, myosin is neither inhibited nor S-nitrosylated by the NO center dot donor diethylamine NONOate, indicating a requirement for transnitrosylation between low-mass SNO and myosin cysteine thiols rather than a direct reaction of myosin with NO center dot or its auto-oxidation products. Interestingly, alkylation of the most reactive thiols of myosin by N-ethylmaleimide does not inhibit formation of a stable population of protein-SNOs, suggesting that these sites are located in less accessible regions of the protein than those that affect activity. The present study reveals a new link between exercise and S-nitrosylation of skeletal muscle contractile proteins that may be important under (patho)physiological conditions. |
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