Abstract
A previous study on cleavage of disulfide bonds in endocytosed model compounds had shown that an initial phase of cleavage was totally inhibited by membrane-impermeant sulfhydryl inhibitors and thus was mediated by cell surface sulfhydryls (Feener, E. P., Shen, W.-C., and Ryser, H. J.-P. (1990) J. Biol. Chem. 265, 18780-18785). This paper uses the same inhibitors (5,5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuriphenylsulfonic acid) to examine the role of surface sulfhydryls in the cytotoxicity of diphtheria toxin (DT). Since the interchain disulfide of endocytosed DT must be cleaved prior to translocation of chain A from endosomes to cytoplasm, it was postulated that surface sulfhydryls might mediate the cleavage of that disulfide bond as well. Both sulfhydryl blockers did indeed markedly inhibit DT cytotoxicity. This effect was not due to inactivation of unbound DT, inhibition of receptor-mediated endocytosis, or impairment of acidification of endosomes. We conclude that cell surface sulfhydryls susceptible to blockage by 5,5'-dithiobis(2-nitro-benzoic acid) and p-chloromercuriphenylsulfonic acid are required for the cytotoxicity of DT and, most likely, for the reductive cleavage of DT's interchain disulfides. Ricin cytotoxicity was not decreased; this is consistent with the view that ricin reaches the cytoplasm from a late endocytic structure and with the finding that endocytosed disulfides are also cleaved in a cell fraction containing elements of the Golgi apparatus (Feener, E. P., Shen, W.-C., and Ryser, H. J.-P. (1990) J. Biol. Chem. 265, 18780-18785).
Highlights
From the Departmentof Pathology, Boston University School of Medicine, Boston, Massachusetts 02118 of primary vesicles, it appeared likely that they would continue to act during the first minutes that follow vesiculation
This paper uses the same inhibi- chains andhence a reduction of the interchain disulfide prior tors (5,5'-dithiobis(2-nitrobenzoicacid) and p-chloromercuriphenylsulfonicacid) to examine the role of surface sulfhydryls in the cytotoxicity of diphtheria toxin (DT)
Must be cleavedprior to translocation of chain A from endosomesto cytoplasm, it was postulated that surface sulfhydryls might mediate the cleavage of that disulfide bond as well
Summary
Monolayers of CHO cells were exposed to concentrations of 0, 100, and 500 rig/ml DT for 2 h at 37 “C in the presence or absence of the sulfhydryl inhibitors. After washing, they were reincubated in a medium containing radioactive amino acids to measure the toxin’s effect on protein biosynthesis. In the absence of inhibitors, 100 and 500 ng of DT caused a marked, dose-dependent decrease of amino acid incorporation (Table I). Addition of 1.0 mM DTNB almost totally prevented this inhibition (93.5 and 85.9%, respectively), and 0.1 mM
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
