Hirano A A, Greengard P, Huganir R L
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York.
J Neurochem. 1988 May;50(5):1447-55. doi: 10.1111/j.1471-4159.1988.tb03029.x.
The rat CNS contains high levels of tyrosine-specific protein kinases that specifically phosphorylate the tyrosine-containing synthetic peptide poly(Glu80,Tyr20). The phosphorylation of this peptide is rapid and occurs with normal Michaelis-Menten kinetics. Using this peptide to assay for enzyme activity, we have measured the protein tyrosine kinase activity in homogenates from various regions of rat CNS. A marked regional distribution pattern was observed, with high activity present in cerebellum, hippocampus, olfactory bulb, and pyriform cortex, and low activity in the pons/medulla and spinal cord. The distribution of protein tyrosine kinase activity was examined in various subcellular fractions of rat forebrain. The majority of the activity was associated with the particulate fractions, with enrichment in the crude microsomal (P3) and crude synaptic vesicle (LP2) fractions. Moreover, the subcellular distribution of pp60csrc, a well-characterized protein tyrosine kinase, was examined by immunoblot analysis using an affinity-purified antibody specific for pp60csrc. The subcellular distribution of pp60csrc paralleled the overall protein tyrosine kinase activity. In addition, using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were demonstrated on immunoblots of homogenates from the various regions and the subcellular fractions. The immunoblots revealed numerous phosphotyrosine-containing proteins that were present in many of the CNS regions examined and were associated with specific subcellular fractions. The differences in tyrosine-specific protein kinase activity, and in phosphotyrosine-containing proteins, observed in various regional areas and subcellular fractions may reflect specific functional roles for protein tyrosine kinase activity in mammalian brain.
大鼠中枢神经系统含有高水平的酪氨酸特异性蛋白激酶，这些激酶能特异性地磷酸化含酪氨酸的合成肽聚（Glu80，Tyr20）。该肽的磷酸化迅速，且符合正常的米氏动力学。利用此肽检测酶活性，我们测定了大鼠中枢神经系统不同区域匀浆中的蛋白酪氨酸激酶活性。观察到明显的区域分布模式，小脑、海马体、嗅球和梨状皮质活性高，脑桥/延髓和脊髓活性低。我们检测了大鼠前脑不同亚细胞组分中蛋白酪氨酸激酶活性的分布。大部分活性与颗粒组分相关，在粗微粒体（P3）和粗突触小泡（LP2）组分中富集。此外，使用对pp60csrc特异的亲和纯化抗体，通过免疫印迹分析检测了特征明确的蛋白酪氨酸激酶pp60csrc的亚细胞分布。pp60csrc的亚细胞分布与总体蛋白酪氨酸激酶活性平行。另外，使用对磷酸酪氨酸特异的抗体，在来自不同区域和亚细胞组分的匀浆免疫印迹上证实了蛋白酪氨酸激酶的内源性底物。免疫印迹显示，在所检测的许多中枢神经系统区域中存在大量含磷酸酪氨酸的蛋白质，且这些蛋白质与特定的亚细胞组分相关。在不同区域和亚细胞组分中观察到的酪氨酸特异性蛋白激酶活性以及含磷酸酪氨酸蛋白质的差异，可能反映了蛋白酪氨酸激酶活性在哺乳动物脑中的特定功能作用。
