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

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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