Ikeda M, Deery W J, Nielsen T B, Ferdows M S, Field J B
Endocrinology. 1986 Aug;119(2):591-9. doi: 10.1210/endo-119-2-591.
Cultured dog thyroid cells incubated with [32P] phosphate contain at least two phosphoproteins of 19 and 21 kDalton (K), as determined by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Myosin light chain appears to be a component of the 19K and 21K phosphoproteins by the following criteria: 1) coextraction with myosin heavy chain from Triton-insoluble cytoskeletons with KCl-ATP, 2) coisolation with myosin heavy chain by immunoprecipitation, and 3) purification of undenatured myosin with pyrophosphate-agarose gel electrophoresis. The phosphorylation state of these proteins is decreased by incubation of cells with TSH. In the basal state, the 19K and 21K proteins from Triton-insoluble cytoskeleton fractions contain 0.86 +/- 0.07 (+/- SE) mol phosphate/mol protein, which is reduced to 0.34 +/- 0.03 in TSH-treated cells. TSH-induced dephosphorylation occurs in 1 min with 2.5 mU/ml TSH and reaches a maximum at 15 min. This TSH effect appears to be mediated by cAMP, since it is mimicked by (Bu)2cAMP, forskolin, cholera toxin, and prostaglandin E1 and is potentiated by isobutylmethylxanthine. Carbamylcholine, ionophore A23187, and norepinephrine, which inhibit TSH stimulation of cAMP, have no effect on basal phosphorylation of the 19K and 21K proteins, but do inhibit the effect of TSH.
用[32P]磷酸盐孵育培养的犬甲状腺细胞，通过一维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和放射自显影测定，含有至少两种分子量分别为19和21千道尔顿（kDalton，K）的磷蛋白。肌球蛋白轻链似乎是19K和21K磷蛋白的一个组成部分，依据以下标准：1）与肌球蛋白重链一起从Triton不溶性细胞骨架中用KCl-ATP共提取；2）通过免疫沉淀与肌球蛋白重链共分离；3）用焦磷酸-琼脂糖凝胶电泳纯化未变性的肌球蛋白。用促甲状腺激素（TSH）孵育细胞会降低这些蛋白质的磷酸化状态。在基础状态下，来自Triton不溶性细胞骨架组分的19K和21K蛋白质含有0.86±0.07（±标准误）摩尔磷酸盐/摩尔蛋白质，在TSH处理的细胞中减少到0.34±0.03。用2.5 mU/ml TSH处理1分钟即可发生TSH诱导的去磷酸化，并在15分钟时达到最大值。这种TSH效应似乎是由环磷酸腺苷（cAMP）介导的，因为它可被双丁酰环磷腺苷（Bu）2cAMP、福斯可林、霍乱毒素和前列腺素E1模拟，并且可被异丁基甲基黄嘌呤增强。氨甲酰胆碱、离子载体A23187和去甲肾上腺素抑制TSH对cAMP的刺激，对19K和21K蛋白质的基础磷酸化没有影响，但确实抑制TSH的作用。
