Isolation, purification, and characterization of fragment B, the NH2-terminal half of the heavy chain of tetanus toxin.
Fragment B, the N-terminal half of the heavy chain, an important domain of the tetanus neurotoxin molecule, was isolated for the first time. Tetanus toxin (composed of three domains, A, B, and C) was prepared from culture filtrates. Fragment A-B, derived from the toxin treated mildly with papain, was used for the isolation of fragment B. Fragment A-B obtained was dissociated into fragments A and B by reduction with 100 mM dithiothreitol and treatment with 2 M urea. Fragment B was separated from fragment A by ion-exchange column chromatography on a Mono Q column equilibrated with 20 mM Tris hydrochloride buffer (pH 7.6), containing 1 mM dithiothreitol and 2 M urea, in a fast-protein liquid chromatography system by elution with a linear gradient of 0 to 0.5 M NaCl. Fragment B was obtained in two forms having molecular weights of 48,000 +/- 2,000, which were indistinguishable by sodium dodecyl sulfate-gel electrophoresis or antigenic specificity, but distinguishable on polyacrylamide gel electrophoresis without sodium dodecyl sulfate and on isoelectric focusing (pI 6.7 and 7.3). The recovery of fragment B was 50 to 72% of that of fragment A-B on a molar basis. Purified fragment B was not toxic to mice on intravenous or intramuscular injection at doses of up to 100 micrograms, but was found to form channels (ca. 2.3 pS) in a lipid bilayer membrane by a patch clamp technique. The role of domain B of the tetanus toxin molecule in the mechanism of action of the toxin is discussed.
Matsuda
M
M
Department of Tuberculosis Research I, Osaka University, Japan.
Lei
D L
DL
Sugimoto
N
N
Ozutsumi
K
K
Okabe
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004229:Dithiothreitol; D000939:Epitopes; D005779:Immunodiffusion; D007473:Ion Channels / Q000648:ultrastructure; D007526:Isoelectric Point; D008051:Lipid Bilayers; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D013744:Tetanus Toxin / Q000276:immunology / Q000302:isolation & purification* / Q000633:toxicity; D014508:Urea
D000939:Epitopes; D007473:Ion Channels; D008051:Lipid Bilayers; D010446:Peptide Fragments; D013744:Tetanus Toxin; D014508:Urea; D004229:Dithiothreitol