Bruce C, Baldwin R L, Lessnick S L, Wisnieski B J
Department of Microbiology, University of California, Los Angeles 90024.
Proc Natl Acad Sci U S A. 1990 Apr;87(8):2995-8. doi: 10.1073/pnas.87.8.2995.
The cytotoxic mechanism of diphtheria toxin (DTx) is associated with its ability to inhibit protein synthesis by ADP-ribosylation of elongation factor 2. Although DTx intoxication leads to internucleosomal DNA cleavage and cell lysis, these events do not occur when protein synthesis is inhibited by alternative treatments (e.g., cycloheximide). Here we show that endonucleolytic degradation of DNA is an intrinsic activity of DTx and also of the crossreactive mutant protein CRM197. Assays using DNA-impregnated gels as well as linear and supercoiled DNA in solution revealed not only that CRM197 has nuclease activity but also that its specific activity is actually significantly greater than that of the wild-type molecule. Since CRM197 contains a single amino acid substitution that renders it incapable of ADP-ribosylation, we propose that the active sites for ADP-ribosyltransferase and nuclease activities are distinct.
白喉毒素（DTx）的细胞毒性机制与其通过对延伸因子2进行ADP核糖基化来抑制蛋白质合成的能力有关。虽然DTx中毒会导致核小体间DNA裂解和细胞裂解，但当通过其他处理方式（如环己酰亚胺）抑制蛋白质合成时，这些事件不会发生。在此我们表明，DNA的内切核酸酶降解是DTx以及交叉反应性突变蛋白CRM197的固有活性。使用DNA浸渍凝胶以及溶液中的线性和超螺旋DNA进行的测定不仅揭示了CRM197具有核酸酶活性，而且其比活性实际上显著高于野生型分子。由于CRM197包含一个使其无法进行ADP核糖基化的单氨基酸取代，我们提出ADP核糖基转移酶和核酸酶活性的活性位点是不同的。
