Different phosphorylated forms of myosin in contracting tracheal smooth muscle.
Different phosphorylated forms of myosin in contracting tracheal smooth muscle.
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发表时间：
1986-05
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通讯作者：
Anthony Persechini;K. Kamm;J. Stull
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作者：
Anthony Persechini;K. Kamm;J. Stull
钙调蛋白依赖性肌球蛋白轻链激酶磷酸化每个肌球蛋白分子上的两个轻链亚基。我们已经开发了一种方法来测量平滑肌中肌球蛋白的非磷酸化，单磷酸化和二磷酸化形式。在焦磷酸盐存在下，通过非变性聚丙烯酰胺凝胶电泳在组织提取物中分离出四条蛋白带。免疫印迹表明，三种形式（指定为M，MP和MP2）与兔抗血清制备的纯化磷酸化轻链（P-轻链）从牛气管平滑肌。有证据表明，M，MP，和MP2代表nonphosphorylated，monophosphorylated，和diphosphorylated肌球蛋白，分别，和其他蛋白带可能是细丝蛋白。在牛气管条神经刺激1.0至3.5 s和速冻中测量不同磷酸化形式的肌球蛋白的形成。在未受刺激的肌肉中未检测到MP或MP2;直接测量的P-轻链磷酸化程度为0.02 +/- 0.01 mol磷酸盐/mol P-轻链。在2.5秒刺激后，获得0.63 +/- 0.06 mol磷酸盐/mol P-轻链和0.40 +/- 0.06 mol MP2/肌球蛋白总量的最大值。在1.0至3.5 s的连续神经刺激期间，P-轻链磷酸化程度（直接测量或计算）与MP2相对量之间的关系与随机磷酸化过程一致。
Calmodulin-dependent myosin light chain kinase phosphorylates two light chain subunits on each myosin molecule. We have developed a method for measuring nonphosphorylated, monophosphorylated, and diphosphorylated forms of myosin in smooth muscle. Four protein bands were separated in tissue extracts by nondenaturing polyacrylamide gel electrophoresis in the presence of pyrophosphate. Immunoblots demonstrated that three forms (designated M, MP, and MP2) reacted with rabbit antisera prepared against the purified phosphorylatable light chain (P-light chain) from bovine tracheal smooth muscle. Evidence was obtained that M, MP, and MP2 represented nonphosphorylated, monophosphorylated, and diphosphorylated myosin, respectively, and that the other protein band was probably filamin. The formation of different phosphorylated forms of myosin was measured in bovine trachealis strips neurally stimulated from 1.0 to 3.5 s and quick-frozen. There was no detectable MP or MP2 in unstimulated muscles; the extent of P-light chain phosphorylation measured directly was 0.02 +/- 0.01 mol of phosphate/mol of P-light chain. After 2.5-s stimulation, maximal values of 0.63 +/- 0.06 mol of phosphate/mol of P-light chain and 0.40 +/- 0.06 MP2/myosintotal were obtained. During continuous neural stimulation from 1.0 to 3.5 s, the relationship between the extent of P-light chain phosphorylation (measured directly or calculated) and the relative amount of MP2 is consistent with a random phosphorylation process.