Identification and characterization of proteins working in the stimulus-serection coupling.

NAP-22是从牛和大鼠脑中纯化的酸性膜蛋白。该蛋白在组织的不溶性部分中被回收，只有一部分NAP-22被含有Triton X-100的溶液溶解，一旦被溶解，该蛋白具有非常亲水的性质，其物理化学性质如热稳定性，酸性等电点，在2.5%高氯酸溶液中的溶解度，sds -聚丙烯酰胺凝胶电泳的异常行为显示其与肉酰基化的富丙氨酸c激酶底物相似（MARCKS,p87）。p80)和GAP-43（神经调节素，F1, pp46, p57, B-50）。免疫印迹分析显示NAP-22在脑组织中主要表达。背根神经节细胞和坐骨神经节细胞提取物中也检测到NAP-22。这意味着NAP-22在神经组织中普遍表达。出生后大脑中这种蛋白质的含量增加，3-5周龄时约占总蛋白质的0.8%。然后，更多的内容逐渐减少，在成年期达到0.4%左右。NAP-22在发育过程中的表达变化表明，该蛋白不仅是维持突触功能所必需的，而且是支持神经突形成或突触发生所必需的。利用特异性单克隆抗体对NAP-22进行免疫组化定位，发现其定位于突触区，尤其是突触前膜和突触囊泡。本研究制备了含有Triton不溶性部分的NAP-22，并利用SDS-PAGE、2d凝胶电泳、western blotting和部分氨基酸测序对该部分的蛋白质成分进行了分析。直径100- 300nm的小囊泡是该组分的主要成分。在TIC中发现了GAP-43（神经调节素）、src和fyn激酶、三聚体G蛋白（Go、Gi和Gs）和一些gpi锚定蛋白（N-CAM、ty -1）的定位，尽管富集程度因蛋白质而异。髓鞘膜产生少量蛋白质。从生长锥和成人脑中观察到的tic的蛋白质成分变化不大。考虑到已经确定的生长锥中GAP-43的定位，三聚体G蛋白、src激酶和GAP-43可能参与突触传递，以及gpi锚定蛋白在该部分的恢复，该复合物似乎不仅在分泌过程中发挥重要作用，而且在神经元细胞极性的建立和维持中发挥重要作用。少

NAP-22 is an acidic membrane protein purified from bovine and rat brain. This protein was recovered in the insoluble fraction of the tissue and only some part of NAP-22 was solubilized with a solution containing Triton X-100, Once solubilized, this protein was very hydrophilic and its physicochemical properties such as heat stability, acidic isoelectric point, solubility in a 2.5% perchloric acid solution, and an anomalous behavior in SDS-polyacrylamide gel electrophoresis showed its resemblance to myristoylated alanine rich C-kinase substrate (MARCKS,p87, p80) and GAP-43 (neuromodulin, F1, pp46, p57, B-50). An immunoblotting assay showed a predominant expression of NAP-22 in brain. NAP-22 was also detected in the extracts of dorsal root ganglion cells and sciatic nerve ganglion cells. This means a general expression of NAP-22 in the nervous tissue. The content of this protein in brain increases after birth to the level of about 0.8% of total protein at the age of 3-5 weeks old. The co … More ntent then decreases gradually reaching about 0.4% at the adulthood. This change of expression of NAP-22 during development suggests that this protein is necessary not only to maintain the synaptic function but also to support either neurite formation or synaptogenesis. The immunohistochemical localization of NAP-22 studied using a specific monoclonal antibody showed its localization at the synaptic region, especially at the presynaptic membrane and at the synaptic vesicle. In this study, NAP-22 containing Triton insoluble fraction was prepared and protein components in this fraction were analyzed using SDS-PAGE,2-D gel electrophoresis, western blotting, and partial amino acid sequencing.Small vesicles having 100-300 nm diameters were the main components in this fraction. Localization of GAP-43 (neuromodulin) , src and fyn kinases, trimeric G proteins (Go, Gi and Gs) , and some GPI-anchored proteins (N-CAM,Thy-1) in TIC was shown, although the degree of enrichment differed from protein to protein. Little amount of protein was derived from myelin membrane. Little change in the protein components was observed between the TICs from growth cone and from adult brain. Considering the well established localization of GAP-43 in the growth cone, possible participation of trimeric G proteins, src kinase, and GAP-43 in the synaptic transmission, and the recovery of GPI-anchored proteins in this fraction, this complex seems to have an important role not only in the course of secretion but also in the establishment and maintenance of neuronal cell polarity. Less