Van der Mast C, Thomas A, Goumans H, Amesz H, Voorma H O
Eur J Biochem. 1977 May 16;75(2):455-64. doi: 10.1111/j.1432-1033.1977.tb11548.x.
By means of affinity chromatography of lysates from Krebs II ascites cells and rabbit reticulocytes on Sepharose-heparin an active fraction of initiation factors has been obtained. The fraction is eluted from the column at 350 mM KCl using a linear gradient and displays a number of activities, i.e. binding of Met-tRNAfMet to form a ternary complex with GTP; transferring this complex to 40-S subunits in an A-U-G-independent step and finally coupling of the 40-S initiation complex to the 60-S subunit, a reaction which is completely A-U-G-dependent. Moreover, MettRNA is bound into the P-site as is indicated by its puromycin sensitivity. The method is very suitable for large-scale preparation. Further purification and characterization of the factors have been carried out on DEAE-cellulose and phosphocellulose columns. Evidence is presented that the polysomes present in a lysate that has been passed through the Sepharose-heparin column can only complete their nascent chains, initiation of new polypeptides is completely dependent on addition of initiation factors.
通过用琼脂糖-肝素对克雷布斯II腹水细胞和兔网织红细胞的裂解物进行亲和层析，获得了起始因子的活性部分。该部分用线性梯度在350 mM KCl浓度下从柱上洗脱下来，并表现出多种活性，即甲硫氨酰-tRNAfMet与GTP结合形成三元复合物；在不依赖A-U-G的步骤中将该复合物转移到40-S亚基上，最后将40-S起始复合物与60-S亚基偶联，这一反应完全依赖A-U-G。此外，甲硫氨酰-tRNA如对嘌呤霉素敏感所表明的那样结合到P位点。该方法非常适合大规模制备。已在DEAE-纤维素柱和磷酸纤维素柱上对这些因子进行了进一步的纯化和特性鉴定。有证据表明，通过琼脂糖-肝素柱的裂解物中存在的多核糖体只能完成其新生链，新多肽的起始完全依赖于起始因子的添加。
