Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes.
Whereas there is biological evidence that the reductive cleavage of disulfide bonds is critical for the activation of endocytosed macromolecules such as toxins, immunotoxins, and other drug carriers, virtually nothing is known about the specifics of this cleavage. To study this process, a model compound was synthesized in which a radioiodinated tyramine was linked through a disulfide bond to an undegradable carrier, poly(D-lysine), known to be efficiently endocytosed. Cultured Chinese hamster ovary cells were pulse-labeled with this probe, and the disulfide cleavage was measured as released acid-soluble radioactivity at different times of chase. Pulse-labeled cells were also subjected to subcellular fractionation to identify intracellular structures associated with disulfide cleavage. Cleavage began without lag, amounted to about approximately 7% of the initial cell-bound radioactivity in the first hour and continued for more than 6 h. It was abolished in the presence of N-ethylmaleimide. When sulfhydryl groups present at the cell surface were blocked with cell-impermeant sulfhydryl reagent, the initial phase of disulfide cleavage was inhibited, indicating that cleavage began at the cell surface. A long-lasting intracellular phase of disulfide cleavage began after about approximately 30 min of chase. Subcellular fractionation and kinetic analysis indicated that neither lysosomes nor endosomes were participating in that phase, leaving the Golgi apparatus as the most probable site of endocytic disulfide cleavage.
Feener
E P
EP
Department of Pathology, Boston University School of Medicine, Massachusetts 02118.
Shen
W C
WC
Ryser
H J
HJ
D000818:Animals; D002460:Cell Line; D003538:Cystamine / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism*; D004705:Endocytosis; D006868:Hydrolysis; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism*; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism; D015388:Organelles / Q000378:metabolism*; D011107:Polylysine / Q000031:analogs & derivatives* / Q000378:metabolism; D013997:Time Factors; D014439:Tyramine / Q000031:analogs & derivatives* / Q000378:metabolism
D004220:Disulfides; C066290:iodotyramine-3-(propionyldithio)propionic acid-poly(lysine); C066289:methotrexate-3-(aminoethyldithio)propionic acid-poly(lysine); D011107:Polylysine; D003538:Cystamine; D014439:Tyramine; D008727:Methotrexate