Identification, phosphorylation, and dephosphorylation of a second site for myosin light chain kinase on the 20,000-dalton light chain of smooth muscle myosin.
Identification, phosphorylation, and dephosphorylation of a second site for myosin light chain kinase on the 20,000-dalton light chain of smooth muscle myosin.
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DOI：
10.1016/s0021-9258(17)42425-2
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发表时间：
1986-01
期刊：
影响因子：
--
通讯作者：
M. Ikebe;D. Hartshorne;M. Elzinga
中科院分区：
文献类型：
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作者：
M. Ikebe;D. Hartshorne;M. Elzinga
在相对较高浓度的肌球蛋白轻链激酶中，肌球蛋白的20,000道尔顿轻链上的第二个位置被磷酸化(Ikebe，M.和HartShorne，D.J.(1985)J.Biol)。化学。260、10027-10031)。在这一交流中，确定了该位点，并描述了与其磷酸化和去磷酸化相关的动力学。用α-胰凝乳酶对火鸡肌球蛋白双磷酸化的20,000道尔顿轻链进行酶解，用反相色谱分离磷酸化的多肽。根据氨基酸分析和部分序列测定，第二个磷酸化位点是苏氨酸18。该位点不同于蛋白激酶C磷酸化的苏氨酸残基。在分离的轻链中，丝氨酸19和苏氨酸18的磷酸化过程遵循单一指数规律，表明是一个随机过程，尽管其磷酸化速率有很大的不同。对于分离的轻链，丝氨酸19和苏氨酸18的kcat/Km分别为550和0.2min-1微米-1。在肌球蛋白完整的情况下，丝氨酸19的磷酸化是双相的；快相和慢相的kcat/Km值分别为22.5和7.5min-1微米-1。相比之下，完整肌球蛋白中苏氨酸18的磷酸化是一个随机的但明显较慢的过程，kcat/Km=0.44分钟-1微米-1。双磷酸化肌球蛋白(约4摩尔磷酸/摩尔肌球蛋白)和分离的轻链(约2摩尔磷酸盐/摩尔轻链)的去磷酸化遵循随机过程，丝氨酸19和苏氨酸18位点的去磷酸化发生的速率相似。
At relatively high concentrations of myosin light chain kinase, a second site on the 20,000-dalton light chain of smooth muscle myosin is phosphorylated (Ikebe, M., and Hartshorne, D. J. (1985) J. Biol. Chem. 260, 10027-10031). In this communication the site is identified and kinetics associated with its phosphorylation and dephosphorylation are described. The doubly phosphorylated 20,000-dalton light chain from turkey gizzard myosin was hydrolyzed with alpha-chymotrypsin and the phosphorylated peptide was isolated by reverse phase chromatography. Following amino acid analyses and partial sequence determinations the second site of phosphorylation is shown to be threonine 18. This site is distinct from the threonine residue phosphorylated by protein kinase C. The time courses of phosphorylation of serine 19 and threonine 18 in isolated light chains follow a single exponential indicating a random process, although the phosphorylation rates differ considerably. The values of kcat/Km for serine 19 and threonine 18 for isolated light chains are 550 and 0.2 min-1 microM-1, respectively. With intact myosin, phosphorylation of serine 19 is biphasic; kcat/Km values are 22.5 and 7.5 min-1 microM-1 for the fast and slow phases, respectively. In contrast, phosphorylation of threonine 18 in intact myosin is a random, but markedly slower process, kcat/Km = 0.44 min-1 microM-1. Dephosphorylation of doubly phosphorylated myosin (approximately 4 mol of phosphate/mol of myosin) and isolated light chains (approximately 2 mol of phosphate/mol of light chain) follows a random process and dephosphorylation of the serine 19 and threonine 18 sites occurs at similar rates.