title: Characterization of the deoxyribonuclease activity of diphtheria toxin.
abstract: Having discovered that the A domain of diphtheria toxin exhibits intrinsic nuclease activity (Chang, M. P., Baldwin, R. L., Bruce, B., and Wisnieski, B. J. (1989) Science 246, 1165-1168), we proceeded to examine the requirements for optimal enzymic expression. In vitro assays with linear double-stranded DNA demonstrated that optimal activity occurs at pH 7.5 and 37 degrees C. A characterization of the stringent cation-dependence of the reaction revealed increasing activity with increasing Mn2+ up to 30 mM. In contrast, activity levels with Ca2+ or Zn2+ alone peaked at 100 microM and with Mg2+ alone at 1 mM. The Zn2(+)- and Mg2(+)-stimulated activities appear to be dependent on trace amounts of Ca2+. Indeed, inclusion of 2 mM Ca2+ plus 3 mM Mg2+ in the reaction buffer promoted a high level of DNA cleavage even though very little cleavage was seen with either cation alone at 2-3 mM. Addition of 20-200 mM NaCl or KCl caused progressive inhibition. Detection of diphtheria toxin nuclease activity under physiologically relevant conditions suggests that it may be operative in vivo and supports our contention that diphtheria toxin-induced cytolysis is not a simple consequence of protein synthesis inhibition, but rather the final step in a cytolytic pathway linked to chromosomal integrity.
authors.affiliation: Department of Microbiology, University of California, Los Angeles 90024.
authors.forename: L T
authors.initials: LT
authors.lastname: Nakamura
authors.forename: B J
authors.initials: BJ
authors.lastname: Wisnieski
mesh_terms: D002413:Cations, Divalent; D003851:Deoxyribonucleases; D004167:Diphtheria Toxin / Q000378:metabolism*; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D002413:Cations, Divalent; D004167:Diphtheria Toxin; D008345:Manganese; D003851:Deoxyribonucleases