May M J, Hartley M R, Roberts L M, Krieg P A, Osborn R W, Lord J M
Department of Biological Sciences, University of Warwick, Coventry, UK.
EMBO J. 1989 Jan;8(1):301-8. doi: 10.1002/j.1460-2075.1989.tb03377.x.
When recombinant ricin A chain transcripts are translated in a rabbit reticulocyte lysate the ribosomes are rapidly inactivated as shown by their inability to support translation of yeast preproalpha factor or chicken lysozyme transcripts added subsequently. In contrast, ribosomes which have translated transcripts encoding non-toxic polypeptides such as ricin B chain, readily translate the second transcript under identical conditions. Ribosome inactivation is accompanied by a highly specific modification of 28S rRNA which occurs at the same position as the N-glycosidic cleavage of an adenine residue and which is thought to cause inactivation of the ribosomes. Protein synthesis by wheat germ ribosomes was not inhibited under the conditions which inhibit reticulocyte ribosomes confirming earlier observations that plant cytoplasmic ribosomes are much less sensitive to inhibition by ricin A chain than are mammalian ribosomes. Using the same assay we have shown that deleting an internal hexapeptide, which shares homology with hamster elongation factor-2, completely abolishes catalytic activity. Deleting a second pentapeptide conserved between ricin A chain and the ribosome-inactivating plant toxin trichosanthin, had no effect. Deleting the first nine residues from the N-terminus of A chain did not affect toxicity whereas deleting a further three residues inactivated the polypeptide. Point mutations which individually converted arginine 48 and arginine 56 of ricin A chain to alanine residues or which deleted arginine 56 were also without effect on the catalytic activity of the toxin.
当重组蓖麻毒素A链转录本在兔网织红细胞裂解物中进行翻译时，核糖体迅速失活，这表现为它们无法支持随后添加的酵母前原α因子或鸡溶菌酶转录本的翻译。相比之下，已经翻译了编码无毒多肽（如蓖麻毒素B链）转录本的核糖体，在相同条件下能够顺利翻译第二种转录本。核糖体失活伴随着28S rRNA的高度特异性修饰，该修饰发生在与腺嘌呤残基的N-糖苷键切割相同的位置，并且被认为会导致核糖体失活。在抑制网织红细胞核糖体的条件下，小麦胚芽核糖体的蛋白质合成并未受到抑制，这证实了早期的观察结果，即植物细胞质核糖体对蓖麻毒素A链抑制的敏感性远低于哺乳动物核糖体。使用相同的检测方法，我们发现删除一个与仓鼠延伸因子-2具有同源性的内部六肽，会完全消除催化活性。删除蓖麻毒素A链和核糖体失活植物毒素天花粉蛋白之间保守的第二个五肽，没有效果。从A链的N端删除前九个残基不影响毒性，而再删除三个残基则会使该多肽失活。将蓖麻毒素A链的精氨酸48和精氨酸56分别突变为丙氨酸残基，或者删除精氨酸56的点突变，对毒素的催化活性也没有影响。
