PURIFICATION AND CHARACTERIZATION OF FETAL SPECIFIC SYNAPTOSOMAL 50KDa PROTEIN

胎儿特异性突触体 50KDa 蛋白的纯化和表征

• 批准号: 03670572
• 负责人: TAKEDA Minoru
• 金额: $ 1.28万
• 依托单位: SHOWA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03670572/
• 关键词: FETAL BRAIN SPECIFIC PROTEIN; AGING; SYNAPSE; 50KDa PROTEIN; CELL DIFFERENTIATION; AXON ELONGATION; HYDROPHOBE AMINO ACIDS; BRAIN DISORDER BY AGING; 中枢神経; 一次構造

In order to study changes of CNS functions and disorders by aging, we tried to find qualitative and quantitative changes of synaptosomal proteins by aging. The fetal specific 50K protein was purified from lyophilisate of ultracentrifugation sup of rat P_2 fraction after hypotonic and ultrasonic treatment.The fetal 50K protein was separated on preparative SDS-PAGE and extracted from gels into 0.1% SDS solution. The extract was further applied on Sephadex G-75 column pre-equilibrated with 20 mM imidazole-HC1 pH 7.0 containing 0.1% SDS. The fetal 50K protein was recovered after void volume fraction. At this step, the 50K protein was purified as a single band on SDS-PAGE. The partial amino acid sequence of one peptide fragment obtained by HPLC after trypsin digestion was as follow ; N-F(A)ILVFLE(A)PFNMDFVW-. This sequence showed partial homology with polyoma viral DNA binding protein by computer search. When the fetal 50K was added in culture medium of neurobalstoma cells, ^3H-thymidine uptake was increased 2 times at 10^<-8>M.

为了研究衰老对中枢神经系统功能和疾病的影响，我们试图从定性和定量上发现突触体蛋白在衰老过程中的变化。从大鼠P_2部位经低渗和超声处理后的超离心液冻干液中纯化出胎儿特异性50K蛋白。在制备SDS- page上分离胎儿50K蛋白，从凝胶中提取到0.1% SDS溶液中。提取液进一步应用于Sephadex G-75柱，用20 mM咪唑- hc1 pH 7.0含0.1% SDS预平衡。空体积分数后回收胎儿50K蛋白。在此步骤中，50K蛋白在SDS-PAGE上被纯化为单个条带。经胰蛋白酶消化后的高效液相色谱法得到的肽段部分氨基酸序列如下：N-F (A) ILVFLE (A) PFNMDFVW -。经计算机检索，该序列与多瘤病毒DNA结合蛋白部分同源。将胎儿50K添加到神经瘤细胞的培养基中，在10^<-8>M时，^ 3h -胸苷的摄取增加了2倍。

项目成果

竹田 稔他7名: "ラット胎児脳シナプトソ-ム画分の50Kタンパク" 神経化学. 29. 476-477 (1991)

Minoru Takeda 等 7 人：“大鼠胎儿脑突触体部分中的 50K 蛋白”《神经化学》29. 476-477 (1991)。