Fate of injected 125I‐labeled cholera toxin taken up by rat liver in vivo

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Abstract
Subcellular fractionation techniques have been used to assess the localization of injected 125I-labeled cholera toxin (125I-CT) taken up by rat liver in vivo, and to determine whether internalization of the toxin is required for the generation of the active A1 peptide. The uptake of injected 125I-CT into the liver is maximal at 5 min (about 10% injected dose/g). At this time the radioactivity is for the most part recovered in the microsomal (P) fraction, but later on it progressively associates with the mitochondrial-lysosomal (ML) and supernatant fractions. The radioactivity is enriched 7-fold in plasma membranes at 5–15 min, and 15–60-fold in Golgi-endosome (GE) fractions at 15–60 min. On analytical sucrose gradients the radioactivity associated with the P fraction is progressively displaced from the region of 5′-nucleotidase (a plasma membrane marker) to that of galactosyltransferase (a Golgi marker). On Percoll gradients, however, it is displaced towards acid phosphatase (a lysosomal marker). Density-shift experiments, using Triton WR 1339, suggest that some radioactivity associated with the P fraction (at 30 min) and all the radioactivity present in the ML fraction (at 2 h) is intrinsic to ácid-phosphatase-containing structures, presumably lysosomes. Comparable experiments using 3,3′-diaminobenzidine cytochemistry indicate that the radioactivity present in GE fractions is separable from galactosyltransferase, and thus is presumably associated with endosomes. The fate of injected 125I-labeled cholera toxin B subunit differs from that of the whole toxin by a more rapid uptake (and/or clearance) of the ligand into subcellular fractions, and a greater accumulation of ligand in the ML fraction. Analysis of GE fractions by SDS/polyacrylamide gel electrophoresis shows that, up to 10 min after injection of 125I-CT, about 80% of the radioactivity is recovered as A subunit and 20% as B subunit, similarly to control toxin. Later on there is a time-dependent decrease in the amount of A subunit and, at least with the intermediate GE fraction, a concomitant appearance of A1 peptide (about 15% of the total at 60 min). In contrast the radioactivity associated with plasma membranes remains indistinguishable from unused toxin. It is concluded that, upon interaction with hepatocytes, 125I-CT (both subunits A and B) sequentially associates with plasma membranes, endosomes and lysosomes, and that endosomes may represent the major subcellular site at which the A1 peptide is generated.