Receptor-mediated transport of the hybrid protein ricin-diphtheria toxin fragment A with subsequent ADP-ribosylation of intracellular elongation factor II.
A hybrid protein of ricin and the enzymatically active fragment A of diphtheria toxin (toxin A) has been synthesized and purified. The diphtheria toxin A fragment of the hybrid protein is shown to enter the cytosol compartment of HeLa cells, its presence assayed by the fall of intracellular elongation factor II (EF-2) and the rise of ADP-ribosylated EF-2. Hybrid entrance to HeLa cells is blocked by lactose which blocks receptor-mediated entry of ricin but not by NH4Cl which blocks the transport of diphtheria toxin. It is concluded that the diphtheria toxin fragment A moiety of the hybrid enters the cell cytosol via the ricin receptor-mediated transport system. The kinetics of intracellular ADP-ribosylation of EF-2 by diphtheria toxin have also been studied. Ribosylation is preceded by a toxin dose-dependent lag period. The data suggest that the time constant responsible for the lag period is in the transport step. Models consistent with these data are discussed.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004167:Diphtheria Toxin / Q000494:pharmacology*; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000096:biosynthesis; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000187:drug effects; D012276:Ricin / Q000494:pharmacology*
D004167:Diphtheria Toxin; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D009702:Nucleoside Diphosphate Sugars; D010445:Peptide Elongation Factors; D000246:Adenosine Diphosphate Ribose; D012276:Ricin; D009713:Nucleotidyltransferases