title: Intermediates in translocation of diphtheria toxin across the plasma membrane.
abstract: Active diphtheria toxin consists of two parts, fragments A and B. Fragment A has enzymatic activity and inhibits protein synthesis. Fragment B binds to cellular receptors, and upon exposure to low pH it inserts into the membrane and facilitates translocation of the A fragment into the cytosol, concomitantly with formation of cation-selective channels. Reduction of the interfragment disulfide bridge is required for release of fragment A and intoxication. In cells treated with N-ethylmaleimide (NEM), which inhibits reduction of the disulfide bridge, fragment A was translocated to the cytosol but not released from fragment B. In the presence of NEM a peptide larger than fragment A was protected against extracellularly added Pronase. This peptide (M(r) approximately 24,000) was released to the supernatant fraction of saponin-treated cells. This indicates that fragment A, which is 21 kDa, is covalently attached via a disulfide bond to an N-terminal (M(r) approximately 3,000) piece of fragment B. The 24-kDa fragment disappeared upon reduction, and the 21-kDa fragment A appeared instead. NEM did not prevent channel activity by fragment B in the context of full-length toxin, demonstrating that channel formation occurs in spite of inhibited reduction of the disulfide bond. Thus, channel formation is not dependent on release of fragment A from the toxin-receptor complex.
authors.affiliation: Institute for Cancer Research, Norwegian Radium Hospital, Montebello.
authors.forename: I H
authors.initials: IH
authors.lastname: Madshus
authors.forename: A
authors.initials: A
authors.lastname: Wiedlocha
authors.forename: K
authors.initials: K
authors.lastname: Sandvig
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism*; D004167:Diphtheria Toxin / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D005033:Ethylmaleimide / Q000737:chemistry / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000737:chemistry; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D011402:Pronase / Q000494:pharmacology; D011994:Recombinant Proteins; D012503:Saponins / Q000494:pharmacology; D014709:Vero Cells
chemical_list: D004167:Diphtheria Toxin; D004220:Disulfides; D007473:Ion Channels; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012503:Saponins; D011402:Pronase; D005033:Ethylmaleimide