title: Localization of diphtheria toxin nuclease activity to fragment A.
abstract: We describe a series of experiments that aimed to establish whether nuclease activity is actually associated with diphtheria toxin (DTx) and its A subunit (DTA), as we originally reported (M. P. Chang, R. L. Baldwin, C. Bruce, and B. J. Wisnieski, Science 246:1165-1168, 1989). Here we show that (i) trypsinization of DTx does indeed produce nucleolytically active DTA, (ii) reduction of electroeluted, unreduced, cleaved DTx (58 kDa) yields nuclease-active DTA (24 kDa), and (iii) fractionation of DTx and DTA by anion-exchange chromatography leads to coelution of nuclease activity with both forms of the toxin, even though each form elutes at a distinct salt concentration. In addition, we show that Escherichia coli-derived DTA also expresses nuclease activity. These studies confirm our initial assertion that the nuclease activity observed in DTx preparations is intrinsic to the DTA portion of DTx.
authors.affiliation: Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Los Angeles 90024.
authors.forename: S L
authors.initials: SL
authors.lastname: Lessnick
authors.forename: J B
authors.initials: JB
authors.lastname: Lyczak
authors.forename: C
authors.initials: C
authors.lastname: Bruce
authors.forename: D G
authors.initials: DG
authors.lastname: Lewis
authors.forename: P S
authors.initials: PS
authors.lastname: Kim
authors.forename: M L
authors.initials: ML
authors.lastname: Stolowitz
authors.forename: L
authors.initials: L
authors.lastname: Hood
authors.forename: B J
authors.initials: BJ
authors.lastname: Wisnieski
mesh_terms: D002852:Chromatography, Ion Exchange; D003851:Deoxyribonucleases / Q000737:chemistry*; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D004167:Diphtheria Toxin; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003851:Deoxyribonucleases