Somerville L L, Wang K
Clayton Foundation Biochemical Institute, Department of Chemistry, University of Texas, Austin 78712-1096.
Arch Biochem Biophys. 1988 Apr;262(1):118-29. doi: 10.1016/0003-9861(88)90174-9.
Titin and nebulin are two major protein components of a cytoskeletal matrix that coexists with thick and thin filaments within the sarcomere of a wide range of striated muscles. Purified titin and nebulin from mouse diaphragm muscle are similar in size, in relative abundance, and in amino acid composition to analogous proteins from other mammals or avians. Phosphate analysis of these nucleic-acid-free proteins indicated that both proteins contain substantial amounts of protein-bound phosphate: about 12 mol of phosphate per mole of titin subunit and 11 mol of phosphate per mole of nebulin subunit. Incubation of intact, excised mouse diaphragm with radioactive inorganic phosphate resulted in significant incorporation of radiophosphate into titin and nebulin. The identification of titin and nebulin phosphorylation was facilitated by a simple salt fractionation and nuclease digestion procedure that effectively separated titin and nebulin from radiolabeled nucleic acids. Such in vivo phosphorylation studies indicated that approximately 2 mol of phosphate per titin subunit and 5 to 7 mol of phosphate per nebulin subunit were incorporated within 5 h of incubation. The incorporation nearly doubled when the beta-adrenergic agonist, isoproterenol, or a phosphodiesterase inhibitor, theophylline, was present in the medium. For both proteins, phosphorylation occurred mainly on serine residues. Nebulin also appears to possess a smaller number of threonine sites. Taken together, our data indicate that a small proportion (20 to 40%) of the steady-state titin phosphates are rapidly turning over. In contrast, most of the nebulin phosphates (50 to 100%) are readily exchanged. The modulation of turnover by external stimuli that increase cytosolic cAMP raises the possibility that at least a portion of the multiple phosphorylation sites of titin and nebulin may be involved in the functional regulation of the sarcomere matrix.
肌联蛋白和伴肌动蛋白是细胞骨架基质的两种主要蛋白质成分，它们与多种横纹肌肌节内的粗肌丝和细肌丝共存。从小鼠膈肌中纯化得到的肌联蛋白和伴肌动蛋白在大小、相对丰度和氨基酸组成上与其他哺乳动物或鸟类的类似蛋白质相似。对这些不含核酸的蛋白质进行的磷酸盐分析表明，这两种蛋白质都含有大量与蛋白质结合的磷酸盐：每摩尔肌联蛋白亚基约含12摩尔磷酸盐，每摩尔伴肌动蛋白亚基约含11摩尔磷酸盐。用放射性无机磷酸盐孵育完整的、切除的小鼠膈肌，结果放射性磷酸盐大量掺入肌联蛋白和伴肌动蛋白中。通过简单的盐分级分离和核酸酶消化程序促进了肌联蛋白和伴肌动蛋白磷酸化的鉴定，该程序有效地将肌联蛋白和伴肌动蛋白与放射性标记的核酸分离。此类体内磷酸化研究表明，在孵育5小时内，每摩尔肌联蛋白亚基掺入约2摩尔磷酸盐，每摩尔伴肌动蛋白亚基掺入5至7摩尔磷酸盐。当培养基中存在β-肾上腺素能激动剂异丙肾上腺素或磷酸二酯酶抑制剂茶碱时，掺入量几乎翻倍。对于这两种蛋白质，磷酸化主要发生在丝氨酸残基上。伴肌动蛋白似乎也有较少数量的苏氨酸位点。综合来看，我们的数据表明，稳态下一小部分（20%至40%）的肌联蛋白磷酸盐在快速周转。相比之下，大部分伴肌动蛋白磷酸盐（50%至100%）易于交换。通过增加胞质环磷酸腺苷的外部刺激对周转的调节增加了这样一种可能性，即肌联蛋白和伴肌动蛋白的多个磷酸化位点中至少有一部分可能参与肌节基质的功能调节。
