Masullo M, Raimo G, Parente A, Gambacorta A, De Rosa M, Bocchini V
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli, Italy.
Eur J Biochem. 1991 Aug 1;199(3):529-37. doi: 10.1111/j.1432-1033.1991.tb16151.x.
The elongation factor 1 alpha (aEF-1 alpha) was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus by chromatographic procedures utilising DEAE-Sepharose, hydroxyapatite and FPLC on Mono S. The purified protein binds [3H]GDP at a 1:1 molar ratio and it is essential for poly(Phe) synthesis in vitro; it also binds GTP but not ATP. These findings indicate that aEF-1 alpha is the counterpart of the eubacterial elongation factor Tu (EF-Tu). Purified aEF-1 alpha is a monomeric protein with a relative molecular mass of 49,000 as determined by SDS/PAGE and by gel filtration on Sephadex G-100; its isoelectric point is 9.1. The overall amino acid composition did not reveal significant differences when compared with the amino acid composition of eubacterial EF-Tu from either Escherichia coli or Thermus thermophilus, of eukaryotic EF-1 alpha from Artemia salina or of archaebacterial EF-1 alpha from Methanococcus vannielii. The close similarities between the average hydrophobicity and the numbers of hydrogen-bond-forming or non-helix-forming residues suggest that common structural features exist among the factors compared. aEF-1 alpha shows remarkable thermophilic properties, as demonstrated by the rate of [3H]GDP binding which increases with temperature, reaching a maximum at 95 degrees C; it is also quite heat-resistant, since after a 6-h exposure at 60 degrees C and 87 degrees C the residual [3H]GDP-binding ability was still 90% and 54% of the control, respectively. The affinity of aEF-1 alpha for GDP and GTP was also evaluated. At 80 degrees C Ka' for GDP was about 30-fold higher than Ka' for GTP; at the same temperature Kd' for GDP was 1.7 microM and Kd' for GTP was 50 microM; these values were 300-fold and 100-fold higher, respectively, than those reported for E. coli EF-Tu at 30 degrees C; compared to the values at 0 degree C of EF-Tu from E. coli and T. thermophilus or EF-1 alpha from A. salina, pig liver and calf brain, smaller differences were observed with eukaryotic factors.(ABSTRACT TRUNCATED AT 400 WORDS)
通过利用DEAE-琼脂糖、羟基磷灰石和Mono S上的快速蛋白质液相色谱（FPLC）等色谱方法，从嗜热嗜酸古细菌嗜热栖热菌中纯化出了均一的延伸因子1α（aEF-1α）。纯化后的蛋白质以1:1的摩尔比结合[3H]GDP，并且对于体外多聚（苯丙氨酸）合成至关重要；它也结合GTP，但不结合ATP。这些发现表明aEF-1α是真细菌延伸因子Tu（EF-Tu）的对应物。通过SDS/PAGE和Sephadex G-100凝胶过滤测定，纯化后的aEF-1α是一种相对分子质量为49,000的单体蛋白；其等电点为9.1。与来自大肠杆菌或嗜热栖热菌的真细菌EF-Tu、来自卤虫的真核EF-1α或来自万氏甲烷球菌的古细菌EF-1α的氨基酸组成相比，总体氨基酸组成没有显示出显著差异。平均疏水性以及形成氢键或非螺旋形成残基的数量之间的密切相似性表明，所比较的这些因子之间存在共同的结构特征。aEF-1α表现出显著的嗜热特性，如[3H]GDP结合速率随温度升高而增加，在95℃时达到最大值；它也相当耐热，因为在60℃和87℃下暴露6小时后，残留的[3H]GDP结合能力分别仍为对照的90%和54%。还评估了aEF-1α对GDP和GTP的亲和力。在80℃时，GDP的Ka'比GTP的Ka'高约30倍；在相同温度下，GDP的Kd'为1.7μM，GTP的Kd'为50μM；这些值分别比30℃时大肠杆菌EF-Tu报道的值高300倍和100倍；与大肠杆菌和嗜热栖热菌的EF-Tu或卤虫、猪肝和小牛脑的EF-1α在0℃时的值相比，真核因子之间的差异较小。（摘要截短至400字）
