Nielander H B, Van Rozen A J, Schrama L H, Kasparaitis M, Oestreicher A B, Gispen W H, Schotman P
Division of Molecular Neurobiology, Rudolf Magnus Institute, Utrecht, The Netherlands.
J Mol Neurosci. 1991;3(2):85-93. doi: 10.1007/BF02885529.
B-50 (= GAP-43, F1, and P-57 or neuromodulin) is a nervous tissue-specific, growth-associated protein, localized in the presynaptic membrane. Phosphorylation by protein kinase C at Ser41 appears to play a role in B-50/calmodulin interaction and neurotransmitter release. Previous studies have shown that digestion of the phosphorylated protein with S. aureus V8 protease (SAP) resulted consecutively in 28- and 15-kDa phospho fragments, the latter containing all incorporated phosphate. These proteolytic products of digestion with SAP have frequently been used to identify B-50 in various systems. Therefore we were interested to find out the location of these fragments in the rat B-50 molecule. For this purpose, the rat cDNA for B-50 was used to generate full-length and truncated cRNAs for cell-free translation. B-50 and B-50 peptides were either N-terminally labeled with [35S]methionine (residues 1 and 5) as a tracer, or they were phosphorylated in vitro by protein kinase C. SAP digestion of the immunoprecipitated, 35S-labeled translation products produced similar 28- and 15-kDa fragments as were obtained from 32P-labeled B-50, indicating that these fragments are N-terminal. Relative mobilities of the N-terminal B-50 fragments of known length were used as internal standards for the calculation of the length of SAP and phospho fragments. Comparing the 35S- and 32P-labeled products, four SAP sites at Glu12, Glu28, Glu65, and Glu132 could be deduced. The latter two sites are in accordance with sequence data of C-terminal fragments from the literature. All available data could be fitted into one scheme.
B - 50（= GAP - 43、F1、P - 57或神经调节蛋白）是一种神经组织特异性的生长相关蛋白，定位于突触前膜。蛋白激酶C在丝氨酸41处的磷酸化似乎在B - 50/钙调蛋白相互作用和神经递质释放中起作用。先前的研究表明，用金黄色葡萄球菌V8蛋白酶（SAP）消化磷酸化蛋白会依次产生28 kDa和15 kDa的磷酸化片段，后者包含所有掺入的磷酸盐。这些用SAP消化产生的蛋白水解产物经常被用于在各种系统中鉴定B - 50。因此，我们有兴趣弄清楚这些片段在大鼠B - 50分子中的位置。为此，使用大鼠B - 50的cDNA生成用于无细胞翻译的全长和截短的cRNA。B - 50和B - 50肽要么在N端用[35S]甲硫氨酸（第1和第5位残基）作为示踪剂进行标记，要么在体外被蛋白激酶C磷酸化。对免疫沉淀的、35S标记的翻译产物进行SAP消化，产生了与从32P标记的B - 50获得的类似的28 kDa和15 kDa片段，表明这些片段是N端的。已知长度的N端B - 50片段的相对迁移率被用作计算SAP和磷酸化片段长度的内标。比较35S和32P标记的产物，可以推断出在Glu12、Glu28、Glu65和Glu132处有四个SAP位点。后两个位点与文献中C端片段的序列数据一致。所有可用数据都可以拟合到一个方案中。
