Everse J, Lappi D A, Beglau J M, Lee C L, Kaplan N O
Proc Natl Acad Sci U S A. 1977 Feb;74(2):472-6. doi: 10.1073/pnas.74.2.472.
Studies on the structure-function relationship of diphtheria toxin are reported. New methods are described for the preparation of pure intact ("unnicked") toxin and for the preparation of the individual A and B chains. A biological assay method for the B chain is also presented, as well as a method for the labelling of "nicked" (one peptide bond broken) diphtheria toxin with 131I such that the label is confined to only one of the two polypeptide chains. Alterations of diphtheria toxin with specific reagents reveal that modifications of the tryptophan, methionine, and arginine residues did not result in a significant loss in toxicity, whereas treatment of the toxin with omicron-phthalaldehyde or by photooxidation with rose bengal results in a complete loss of the toxic activity. Modification of tyrosine by iodination results in active toxin, whereas modification by tetranitromethane causes a loss in activity. Preliminary results also indicate that the isolated A chain is about an order of magnitude more active in incorporating adenosine diphosphoribose into translocase (elongation factor 2) than whole or nicked toxin is under identical conditions. The observed structural properties are discussed in view of the functional activity of diphtheria toxin in cell-free systems as well as in cell cultures. Evidence is presented indicating that the B chain binds to membranes: it inhibits the action of nicked toxin on HeLa cells.
本文报道了对白喉毒素结构-功能关系的研究。描述了制备纯完整（“未切割”）毒素以及制备单个A链和B链的新方法。还介绍了一种用于B链的生物学检测方法，以及一种用131I标记“切割”（一个肽键断裂）白喉毒素的方法，使得标记仅局限于两条多肽链中的一条。用特定试剂对白喉毒素进行改造后发现，色氨酸、甲硫氨酸和精氨酸残基的修饰不会导致毒性显著丧失，而用邻苯二甲醛处理毒素或用孟加拉玫瑰红进行光氧化会导致毒性活性完全丧失。通过碘化修饰酪氨酸会产生活性毒素，而用四硝基甲烷修饰则会导致活性丧失。初步结果还表明，在相同条件下，分离出的A链在将腺苷二磷酸核糖掺入转位酶（延伸因子2）方面的活性比完整或切割毒素高约一个数量级。结合白喉毒素在无细胞系统以及细胞培养中的功能活性，对观察到的结构特性进行了讨论。有证据表明B链与膜结合：它抑制切割毒素对HeLa细胞的作用。
