Calcium and calmodulin-enhanced in vitro phosphorylation of hen brain cold-stable microtubules and spinal cord neurofilament triplet proteins after a single oral dose of tri-o-cresyl phosphate.
Calcium and calmodulin-enhanced in vitro phosphorylation of hen brain cold-stable microtubules and spinal cord neurofilament triplet proteins after a single oral dose of tri-o-cresyl phosphate.
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单次口服三邻甲苯基磷酸酯后，钙和钙调蛋白增强了母鸡脑冷稳定微管和脊髓神经丝三联体蛋白的体外磷酸化。
DOI：
10.1073/pnas.83.16.6174
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发表时间：
1986
影响因子：
11.1
通讯作者：
Abou-Donia,MB
中科院分区：
文献类型：
--
作者：
Suwita,E;Lapadula,DM;Abou-Donia,MB
在出现迟发性神经毒性后，评估了单次口服750 mg/kg磷酸三邻甲苯酯（TOCP）对母鸡脑微管制剂和脊髓神经丝内源性磷酸化的影响。蛋白磷酸化与[γ-32 P]ATP进行了分析，通过一维和二维凝胶电泳，放射自显影，和显微光密度。TOCP处理增强了粗制鸡脑胞质溶胶中微管蛋白的Ca 2+和钙调蛋白依赖性磷酸化（α-微管蛋白为160%，β-微管蛋白为140%）和冷稳定微管（α-和β-微管蛋白分别为165%和155%）。微管相关蛋白2（MAP-2）磷酸化在所研究的脑组分中也增加--即，脑细胞质（145%），冷稳定微管（133%）和冷不稳定微管（328%）。有显着增加磷酸化的70 kDa的蛋白质在脑细胞质和冷稳定的微管部分。TOCP还以Mg 2+依赖的方式刺激70 kDa（119%）和160 kDa（129%）的脊髓蛋白的磷酸化。加入Ca 2+和钙调蛋白进一步增强了这些70-kDa（563%）和160-kDa（221%）蛋白质以及52-，59-和210-kDa蛋白质的磷酸化，分别高达126%，160%和196%。进行双向电泳以鉴定这些蛋白质。在脑和脊髓制备物中，它们被确认为α-和β-微管蛋白（52和59 kDa），在脊髓制备物中被确认为神经丝三联体蛋白（70、160和210 kDa）。脑内70-kDa蛋白不是神经丝蛋白。使用金黄色葡萄球菌V8蛋白酶的肽图谱显示，在对照组和TOCP处理的母鸡中，脑和脊髓细胞骨架蛋白具有相同的磷酸肽模式，这表明磷酸化位点不太可能被TOCP处理改变。
The effect of a single 750-mg/kg oral dose of tri-o-cresyl phosphate (TOCP) on the endogenous phosphorylation of brain microtubule preparations and spinal cord neurofilaments was assessed in hens after the development of delayed neurotoxicity. Protein phosphorylation with [gamma-32P]ATP was analyzed by one-dimensional and two-dimensional gel electrophoresis, autoradiography, and microdensitometry. TOCP treatment enhanced the Ca2+- and calmodulin-dependent phosphorylation of tubulin in crude chicken brain cytosol (160% for alpha-tubulin and 140% for beta-tubulin) and cold-stable microtubules (165% and 155% for alpha- and beta-tubulin, respectively). Microtubule-associated protein 2 (MAP-2) phosphorylation was also increased in brain fractions studied--i.e., brain cytosol (145%), cold-stable microtubules (133%), and cold-labile microtubules (328%). There was significant increase in phosphorylation of a 70-kDa protein in the brain cytosol and in the cold-stable microtubule fractions. TOCP also stimulated the phosphorylation of spinal cord proteins of 70 kDa (119%) and 160 kDa (129%) in a Mg2+-dependent manner. Addition of Ca2+ and calmodulin further enhanced the phosphorylation of these 70-kDa (563%) and 160-kDa (221%) proteins as well as of 52-, 59-, and 210-kDa proteins by as much as 126%, 160%, and 196%, respectively. Two-dimensional electrophoresis was carried out to identify these proteins. They were confirmed as alpha- and beta-tubulin (52 and 59 kDa) in brain and spinal cord preparations and the neurofilament triplet proteins (70, 160, and 210 kDa) in the spinal cord preparation. The 70-kDa protein in brain was not neurofilament in origin. Peptide mapping using Staphylococcus aureus V8 protease showed the brain and spinal cord cytoskeletal proteins have identical phosphopeptide patterns in control and TOCP-treated hens, indicating that it was unlikely that the phosphorylation sites were altered by TOCP treatment.