Alcázar A, Martín M E, Soria E, Rodríguez S, Fando J L, Salinas M
Departamento de Investigación, Hospital Ramón y Cajal, Madrid, Spain.
J Neurochem. 1995 Aug;65(2):754-61. doi: 10.1046/j.1471-4159.1995.65020754.x.
Eukaryotic initiation factor 2B, or guanine nucleotide-exchange factor, has been purified for the first time from the brain by a novel procedure that allows the purification of initiation factor 2 as well and uses a salt wash postmicrosomal supernatant as starting material. The procedure includes a three-part chromatographic step in heparin-Sepharose and in SP-5PW and diethylaminoethyl-5PW ion-exchange high-performance chromatographies. The purification of the factors was followed by measuring activity in the guanine nucleotide-exchange assay and the capacity of initiation factor 2 to form a ternary complex with the initiation form of methionyl-tRNA and GTP. The method yields guanine nucleotide-exchange factor (75%) and highly purified initiation factor 2 (> 95%), which are separated in the last step. The exchange factor from the brain is a multimeric protein with five subunits of molecular masses of 82, 65, 52, 42, and 30 kDa; it stimulates ternary complex formation in the presence of GDP, and this activity is inhibited by N-ethylmaleimide. A 37-kDa protein that copurifies with initiation factors is characterized in this study as a new calmodulin-binding protein (p37); it is highly phosphorylated by casein kinase activities and can comigrate with the alpha subunit of initiation factor 2 under standard sodium dodecyl sulfate electrophoresis conditions.
真核生物起始因子2B，即鸟嘌呤核苷酸交换因子，首次通过一种新方法从大脑中纯化得到。该方法也能纯化起始因子2，并以盐洗微粒体后上清液作为起始材料。该方法包括在肝素琼脂糖、SP-5PW和二乙氨基乙基-5PW离子交换高效液相色谱中的三步色谱步骤。通过在鸟嘌呤核苷酸交换测定中测量活性以及起始因子2与甲硫氨酰-tRNA起始形式和GTP形成三元复合物的能力来跟踪因子的纯化过程。该方法可得到鸟嘌呤核苷酸交换因子（75%）和高度纯化的起始因子2（>95%），它们在最后一步被分离。大脑中的交换因子是一种多聚体蛋白，由分子量分别为82、65、52、42和30 kDa的五个亚基组成；它在GDP存在下刺激三元复合物的形成，并且这种活性被N-乙基马来酰亚胺抑制。在本研究中，一种与起始因子共纯化的37 kDa蛋白被鉴定为一种新的钙调蛋白结合蛋白（p37）；它被酪蛋白激酶活性高度磷酸化，并且在标准十二烷基硫酸钠电泳条件下可与起始因子2的α亚基共迁移。
