title: Specific chemical cleavage of diphtheria toxin with hydroxylamine. Purification and characterization of the modified proteins.
abstract: Specific chemical cleavage of diphtheria toxin with hydroxylamine was performed to remove peptides of 10 and 7 kDa from the carboxyl terminus. The resulting modified proteins of 51 and 48 kDa (HA51DT and HA48DT, respectively) were purified and characterized with respect to structural and biological properties. The 51-kDa toxin binds to ATP-agarose, as does intact diphtheria toxin, while HA48DT does not bind to the nucleotide matrix. Neither modified toxin binds to the membranes of diptheria toxin-sensitive cells, and, consequently, neither is toxic. However, when covalently linked to a membrane binding moiety, both HA51DT and HA48DT are toxic. Cell-killing ability during a short exposure time indicated that concanavalin A (Con A) derivatives of diphtheria toxin and HA51DT are equally toxic, ConA HA48DT being somewhat less toxic, while the conjugate of ConA to A-chain kills a small number of cells only at inordinately high concentration (1 microM). We have thus separated the cell membrane binding function of diphtheria toxin from its membrane permeation function by removing specific small peptides from the carboxyl terminus. These modified toxins may have applications in the preparation of highly potent hybrid toxins.
authors.affiliation: Department of Animal Science, University of Wyoming, Laramie 82071.
authors.forename: D A
authors.initials: DA
authors.lastname: Myers
authors.forename: C L
authors.initials: CL
authors.lastname: Villemez
mesh_terms: D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003208:Concanavalin A / Q000494:pharmacology; D004167:Diphtheria Toxin / Q000378:metabolism*; D006801:Humans; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis
chemical_list: D004167:Diphtheria Toxin; D006898:Hydroxylamines; D010446:Peptide Fragments; D003208:Concanavalin A; D019811:Hydroxylamine