Nogueira Leonardo, Figueiredo-Freitas Cicero, Casimiro-Lopes Gustavo, Magdesian Margaret H, Assreuy Jamil, Sorenson Martha M
Instituto de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, RJ 21941-590, Brazil.
Biochem J. 2009 Nov 11;424(2):221-31. doi: 10.1042/BJ20091144.
Nitric oxide (NO*) 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* or NO* 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* 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* 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.
一氧化氮（NO*）在骨骼肌中合成，其生成量在收缩活动期间会增加。尽管肌球蛋白是肌肉中含量最丰富的蛋白质，但尚不清楚肌球蛋白是否是NO或NO衍生物的作用靶点。在本研究中，我们发现运动可增加肌肉中的蛋白质S-亚硝基化，并且在收缩蛋白中，肌球蛋白是去皮肤骨骼肌纤维和分化的肌管中外源性SNOs（S-亚硝基硫醇）的主要作用靶点。分离的肌球蛋白与S-亚硝基谷胱甘肽反应会导致多个半胱氨酸硫醇发生S-亚硝基化，并产生两种具有不同稳定性的与蛋白质结合的SNOs群体。稳定性较差的群体抑制生理ATP酶活性，而不影响肌球蛋白对肌动蛋白的亲和力。然而，NO供体二乙胺NONOate既不抑制肌球蛋白，也不会使其发生S-亚硝基化，这表明需要低质量SNO与肌球蛋白半胱氨酸硫醇之间进行转亚硝基化反应，而不是肌球蛋白与NO或其自氧化产物直接反应。有趣的是，N-乙基马来酰亚胺对肌球蛋白最具反应性的硫醇进行烷基化并不抑制稳定的蛋白质-SNOs群体的形成，这表明这些位点位于蛋白质中比影响活性的位点更难接近的区域。本研究揭示了运动与骨骼肌收缩蛋白S-亚硝基化之间的新联系。这一联系在（病理）生理条件下可能很重要。
