Cyclic AMP and amine effects on phosphorylation of specific protein in abdominal ganglion of Aplysia californica; localization and kinetic analysis.
Cyclic AMP and amine effects on phosphorylation of specific protein in abdominal ganglion of Aplysia californica; localization and kinetic analysis.
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环AMP和胺对海兔腹部神经节特定蛋白磷酸化的影响
DOI：
10.1002/neu.480050604
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发表时间：
1974
期刊：
影响因子：
--
通讯作者：
S. Barondes
中科院分区：
文献类型：
--
作者：
I. Levitan;C. J. Madsen;S. Barondes
用章鱼或5-羟色胺孵育体重120-260克的海兔腹神经节10分钟后，cAMP(环腺苷3‘，5’-单磷酸)水平显著升高，这种升高分别被酚妥拉明和甲基丝氨酸所阻断。与章鱼胺孵育21小时，cAMP水平也会升高，但与5-羟色胺孵育时间延长则不会。这两种胺在与神经节孵育21小时后，都增加了32P或33P对表观分子量为118,000的特定蛋白质的掺入。对这种磷蛋白的选择性作用先前被酚妥拉明(对章鱼胺效应)或甲基丝氨酸(对5-羟色胺效应)所阻断，并可通过在介质中加入二丁酰环腺苷来模拟。
需要超过15小时的孵育才能产生稳定的磷化蛋白图谱，在该图谱中，可以在聚丙烯酰胺凝胶上可靠地识别5个磷化蛋白峰。随着孵育时间的缩短，神经节之间在结合放射性磷酸盐的蛋白质方面存在显著的差异。低于15小时的孵育不能证明二丁酰cAMP对特定蛋白磷酸化的影响。
在与放射性磷酸盐和二丁酰cAMP孵育21小时后，从神经节中取出的大量已鉴定的大型细胞中，没有观察到对分子量为118,000的磷酸蛋白的特异性影响。在结缔神经和袋状细胞团中也没有观察到这种影响，但在这些解剖后残留的神经节部分一直存在。在亚细胞分离时，这种现象在粗核部分中没有观察到，但在粗线粒体组分中持续存在。这些结果与二丁基cAMP引起的磷蛋白代谢的改变可能定位于包含该神经节内所有突触接触的神经束的可能性一致。
Incubation of abdominal ganglia, obtained from Aplysia californica weighing 120--260 g, with octopamine or serotonin led to marked increases in cAMP (cyclic adenosine 3′,5′-monophosphate) levels after 10 min of incubation; these increases were blocked by phentolamine and methysergide, respectively. Incubation for 21 hr with octopamine also produced an elevation in cAMP levels but prolonged incubation with serotonin did not. Both these amines increased incorporation of 32P or 33P into a specific protein with apparent molecular weight of 118,000, upon incubation with the ganglion for 21 hr. The selective effect on this phosphoprotein was previously shown to be blocked by phentolamine (for octopamine effect) or methysergide (for serotonin effect) and could be mimicked by addition of dibutyryl cAMP to the medium.
Incubation for more than 15 hr was required to generate a stable phosphoprotein pattern in which 5 phosphoprotein peaks could be reliably identified on polyacrylamide gels. With shorter incubations there was marked variability between ganglia with respect to the proteins which incorporated radioactive phosphate. The dibutyryl cAMP effect on phosphorylation of specific protein could not be demonstrated with incubations shorter than 15 hr.
The specific effect on the phosphoprotein with molecular weight 118,000 was not observed in a number of large identified cells removed from the ganglion after 21 hr of incubation with radioactive phosphate and dibutyryl cAMP. The effect was also not observed in connective nerves and the bag cell clusters, but was consistently present in that portion of the ganglion remaining after these dissections. Upon subcellular fractionation the phenomenon was not observed in a crude nuclear fraction but was consistently present in a crude mitochondrial fraction. All these results are consistent with the possibility that the change in phosphoprotein metabolism produced by dibutryl cAMP may be localized in the neuropil, which contains all the synaptic contacts in this ganglion.