Elimination of the disulphide bridge in fragment B of diphtheria toxin: effect on membrane insertion, channel formation, and ATP binding.
Active diphtheria toxin consists of two disulphide-linked fragments, termed A and B. Fragment B, which contains an internal disulphide bridge, facilitates translocation of the enzymatically active fragment A to the cytosol of eukaryotic cells. In this process cation-selective channels are formed. An in vitro translated full-length mutant lacking the internal disulphide bridge (A-58**) was functionally indistinguishable from its disulphide-containing counterpart (A-58) with respect to trypsin sensitivity, receptor binding, A-fragment translocation, and channel formation. In contrast, the B fragment of A-58** (B-36**) was slightly less trypsin resistant than the S-S-containing B fragment, B-36, and was approximately 300-fold less efficient than B-36 in permeabilizing cells. When first dialysed and then reconstituted with A fragment, B fragment without disulphide bridge yielded a less-active toxin than did wild-type B fragment. We conclude that the disulphide bridge in fragment B is not necessary for toxicity, as earlier believed, and that channel formation may play a role in membrane translocation.
Stenmark
H
H
Institute for Cancer Research, Norwegian Radium Hospital, Montebello, Oslo, Norway.
Olsnes
S
S
Madshus
I H
IH
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability; D003353:Corynebacterium diphtheriae / Q000235:genetics*; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D004220:Disulfides / Q000737:chemistry*; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014709:Vero Cells
D004167:Diphtheria Toxin; D004220:Disulfides; D010446:Peptide Fragments; D015222:Sodium Channels; C029795:diphtheria toxin fragment A; C039171:diphtheria toxin fragment B; D000255:Adenosine Triphosphate; D012964:Sodium; D014357:Trypsin