Walaas S I, Perdahl-Wallace E, Winblad B, Greengard P
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York.
J Mol Neurosci. 1989;1(2):105-16. doi: 10.1007/BF02896894.
Protein phosphorylation systems regulated by cyclic adenosine 3',5'-monophosphate (cyclic AMP), or calcium in conjunction with calmodulin or phospholipid/diacylglycerol, have been studied by phosphorylation in vitro of particulate and soluble fractions from human postmortem brain samples. One-dimensional or two-dimensional gel electrophoretic protein separations were used for analysis. Protein phosphorylation catalyzed by cyclic AMP-dependent protein kinase was found to be highly active in both particulate and soluble preparations throughout the human CNS, with groups of both widely distributed and region-specific substrates being observed in different brain nuclei. Dopamine-innervated parts of the basal ganglia and cerebral cortex contained the phosphoproteins previously observed in rodent basal ganglia. In contrast, calcium/phospholipid-dependent and calcium/calmodulin-dependent protein phosphorylation systems were less prominent in human postmortem brain than in rodent brain, and only a few widely distributed substrates for these protein kinases were found. Protein staining indicated that postmortem proteolysis, particularly of high-molecular-mass proteins, was prominent in deeply located, subcortical regions in the human brain. Our results indicate that it is feasible to use human postmortem brain samples, when obtained under carefully controlled conditions, for qualitative studies on brain protein phosphorylation. Such studies should be of value in studies on human neurological and/or psychiatric disorders.
通过对人死后脑样本的颗粒和可溶部分进行体外磷酸化，研究了由环磷酸腺苷（cAMP）或钙与钙调蛋白或磷脂/二酰基甘油共同调节的蛋白质磷酸化系统。采用一维或二维凝胶电泳蛋白质分离法进行分析。发现cAMP依赖性蛋白激酶催化的蛋白质磷酸化在整个人中枢神经系统的颗粒和可溶制剂中均具有高活性，在不同脑核中观察到广泛分布和区域特异性的底物群。基底神经节和大脑皮层中多巴胺支配的部分含有先前在啮齿动物基底神经节中观察到的磷蛋白。相比之下，钙/磷脂依赖性和钙/钙调蛋白依赖性蛋白质磷酸化系统在人死后脑中不如在啮齿动物脑中突出，仅发现了这些蛋白激酶的少数广泛分布的底物。蛋白质染色表明，死后蛋白水解，特别是高分子量蛋白质的水解，在人脑深部的皮质下区域很明显。我们的结果表明，在精心控制的条件下获取的人死后脑样本可用于脑蛋白质磷酸化的定性研究。此类研究对人类神经和/或精神疾病的研究应具有价值。
