Abstract
Ricin translocation was demonstrated (using both fluorescence- and radiolabel-based assays) across the membrane of endosomes purified from mouse lymphocytes. Selectivity of the process was shown by the absence of translocation activity of transferrin and horseradish peroxidase used as membrane-bound and fluid-phase endosome labels, respectively. Endocytosed 125I-ricin translocation was found to be strictly ATP- (Km approximately 4 mM) and temperature-dependent, with up to 30% endosomal 125I-ricin appearing in the external medium after 2 h at 37 degrees C. No treatments neutralizing the acidic endosome pH (ammonium chloride, nigericin, chloroquine) significantly impaired ricin translocation, and the pH gradient across the endosome membrane is not required for this process. Chase experiments showed that the ability of 125I-ricin to translocate increases with its depth in the endocytic system (i.e. plasma membrane << early endosomes < late endosomes). Both A and B ricin chains displayed translocation ability as demonstrated by the results of our assay on ricin, ricin B, transferrin-ricin A, and transferrin-ricin B conjugates. Biological activity of both ricin chains is preserved after translocation as shown by the inhibitory effect of the A chain on cell-free protein synthesis and the binding of the B chain to lactose-agarose.
Highlights
Fluorescence- and radiolabel-basedassays) across the The process of cell intoxication by DT is well known.After membrane of endosomes purified from mouse lymphoe-ndocytosis of this toxin, the low pH (5-5.5) encountered in cytes
The rest of the Golgi apparatus is disconferrin-ricin B conjugates. Biological activity of both nected from the TGN in the presence of brefeldin A (BFA) [12,13,14], and ricin chainsis preserved after translocation as shown bytheinhibitory effect ofthe A chain on cell-free protein synthesis andthebindingofthe B chain to lactose-agarose
A likely hypothesis is that ricin translocation requires a cellular component whose synthesis is blocked by BFA [15].This agrees with the absence of any protective effect of BFA against the cytotoxic effects of immunotoxins prepared by linking the ricin A chain to a monoclonal anti
Summary
B antibodies) and B chainswere provided by Dr P. Membranes were resuspended in 0.25 M sucrose, 1mM EDTA, 10 mM Tris, pH 8, and the label bound to vesicles presenting the original membrane orientation was scraped off using the following procedure: adding lactose up to 0.1 M, followed by warming for 2 min a t 37 "C, cooling to 2 "C,adding trypsin (0.3 mg/ml final concentration), leaving for 20 min at 2 "C, and terminating by the addition of soybean trypsin inhibitor (0.3mg/ml final concentration).Membranes were spun for 30 min a t 140,000 X g through a cushion of 20% sucrose before the translocation assay This lactose/trypsin treatment proved to be efficient on intact cells to displace all plasma membrane-bound conjugates.
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