Study on physiological function of a brainspecific 14 kDa protein (PNP 14)

从牛大脑皮层中分离纯化了一种分子量为14 kDa的脑特异性蛋白。在本工作中，PNP 14的一级结构被确定。该蛋白质由134个氨基酸残基组成（分子量= 14，122 Da）。我们发现该蛋白在体内和体外都被磷酸化。体外实验表明，Ca*，钙调蛋白依赖性蛋白激酶II负责Ser 118的磷酸化，酪氨酸激酶如EGF受体和pp 60 β<v-src>催化PNP 14的酪氨酸磷酸化。EGF受体磷酸化的PNP 14的磷酸化位点为Tyr 39。PNP 14与磷脂酰肌醇3-激酶或pp 60形成复合物<c-src>。用免疫组织化学和原位杂交方法研究PNP 14在大鼠脑内的定位及其mRNA表达。PNP 14在小脑皮质呈阳性反应。在小脑延髓区未检测到PNP 14免疫反应。在大脑中，免疫反应性遍及整个大脑皮质，尤其是在第五层，海马和纹状体也有免疫反应。电镜观察显示，突触前轴突终末的胞质基质中有免疫反应。PNP 14 mRNA主要表达于大脑皮质、海马和大脑皮质颗粒层。这些结果表明，PNP 14在突触前轴突终末中起重要作用，其功能受磷酸化反应的调节。

A novel brain-specific protein with a molecular mass of 14 kDa was found and purified from bovine crebral cortex. In the present work, the primary structure of PNP 14 was determined. This protein is composed of 134 amino acid residues (molecularmass = 14,122 Da). We found that the protein was phosphorylated in*2+*vivo as well as in vitro. In vitro experiments showed that Ca*, calmodulin-dependent protein kinase II is responsible for the phosphorylation of Ser118, and tyrosine kinases such as EGF receptors and pp60^<v-src> catalyze tyrosine phosphorylation of PNP 14. The phosphorylation site of PNP 14 phosphorylated by EGF receptors was Tyr39. PNP 14 formed complex with phosphtidylinositol 3-kinase or pp60^<c-src>. Localization of PNP 14 and its mRNA expression in rat brain were investigated by immunohistochemistry and in situ hybridization. PNP 14 immunoreactivity was detected in cerebellar cortex. No PNP 14 immunoreactivity was detected in cerebellar medulla. In cerebrum, the immunoreactivity was found throughout cerebral cortex, especially in the layr V.It was also found in hippocampus and striatum. Electron microscopic observation revealed that the immunoreactivity was detected in the cytoplasmic matrix in the presynaptic axon terminals. PNP 14 mRNA was observed in granular layr of cerbellar cortex, hippocampus and cerebral cortex. These findings suggested that PNP 14 play important role in presynaptic axon terminals and the function is regulated by phosphorylation reaction.