Nilsson L, Nygård O
Eur J Biochem. 1985 Apr 15;148(2):299-304. doi: 10.1111/j.1432-1033.1985.tb08839.x.
Tryptic cleavage of EF-2, molecular mass 93 kDa, produced an 82-kDa polypeptide and a 10-kDa fragment, which was further degraded. By a slower reaction the 82-kDa polypeptide was gradually split into a 48-kDa and a 34-kDa fragment. Similarly, treatment with chymotrypsin resulted in the formation of an 82-kDa polypeptide and a small fragment. In contrast to the tryptic 82-kDa polypeptide the corresponding chymotryptic cleavage product was relatively resistant to further attack. The degradation of the 82-kDa polypeptide with either trypsin or chymotrypsin was facilitated by the presence of guanosine nucleotides, indicating a conformational shift in native EF-2 upon nucleotide binding. No effect was observed in the presence of ATP, indicating that the effect was specific for guanosine nucleotides. After affinity labelling of native EF-2 with oxidized [3H]GTP and subsequent trypsin treatment the radioactivity was recovered in the 48-kDa polypeptide showing that the GTP-binding site was located within this part of the factor. Correspondingly, tryptic degradation of EF-2 labelled with [14C]NAD+ in the presence of diphtheria toxin showed that the site of ADP-ribosylation was within the 34-kDa polypeptide. By cleavage with the tryptophan-specific reagent N-chlorosuccinimide the site of ADP-ribosylation could be located at a distance of 40-60 kDa from the GTP-binding site and about 4-11 kDa from the nearest terminus.
分子量为93 kDa的延伸因子2（EF-2）经胰蛋白酶切割后，产生了一个82 kDa的多肽和一个10 kDa的片段，该10 kDa片段进一步降解。通过较慢的反应，82 kDa的多肽逐渐被切割成一个48 kDa和一个34 kDa的片段。同样，用胰凝乳蛋白酶处理也导致形成一个82 kDa的多肽和一个小片段。与胰蛋白酶切割产生的82 kDa多肽不同，相应的胰凝乳蛋白酶切割产物对进一步的攻击相对具有抗性。胰蛋白酶或胰凝乳蛋白酶对82 kDa多肽的降解在鸟苷核苷酸存在时会加速，这表明核苷酸结合后天然EF-2的构象发生了变化。在ATP存在的情况下未观察到影响，这表明该效应对鸟苷核苷酸具有特异性。用氧化的[³H]GTP对天然EF-2进行亲和标记并随后用胰蛋白酶处理后，放射性在48 kDa多肽中被检测到，这表明GTP结合位点位于该因子的这一部分内。相应地，在白喉毒素存在下用[¹⁴C]NAD⁺标记EF-2后进行胰蛋白酶降解表明，ADP核糖基化位点位于34 kDa多肽内。通过用色氨酸特异性试剂N-氯代琥珀酰亚胺切割，ADP核糖基化位点可定位在距GTP结合位点40 - 60 kDa处，距最近的末端约4 - 11 kDa处。
