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

Abstract

Subcellular fractionation techniques have been used to assess the localization of injected 251-labeled cholera toxin (1251-CT) taken up by rat liver in vivo, and to determine whether internalization of the toxin is required for the generation of the active Al peptide.The uptake of injected 1251-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 acid-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 1251-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 1251-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 Al peptide (about 15% of the total at 60min). In contrast the radioactivity associated with plasma membranes remains indistinguishable from unused toxin.It is concluded that, upon interaction with hepatocytes, 1251-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 Al peptide is generated.Cholera toxin (CT), a bacterial toxin which consists of two different non-covalently linked subunits, A and B [l -41, is known to exert its effects on cells by activation of adenylate cyclase [5, 61. The initial step in toxin action is the binding of its B subunit, composed of five identical peptides, to a specific cell-surface receptor, the ganglioside GM1 [7]. Subsequently the A subunit, which consists of two peptides linked by a single disulfide bond, Al and A2, is reduced, and the free Al peptide catalyses the activation of adenylate cyclase thro...

A Dipeptide Metalloendoprotease Substrate Completely Blocks the Response of Cells in Culture to Cholera Toxin

“…CT was radiolabeled with 125 I using the chloramine-T method described by Cuatrecasas (21) and as modified by Janicot and Desbuquois (22). Unreacted [ 125 I]iodine was removed using gel filtration on a Sephadex G-50 minicolumn using the centrifugation procedure of Tuszynski et al (23).…”

Fate of injected glucagon taken up by rat liver in vivo. Degradation of internalized ligand in the endosomal compartment

“…Livers were homogenized and submitted to subcellular fractionation by using established procedures with slight modifications [16]. To minimize the degradation of ['25Iliodoglucagon which occurs in homogenates during fractionation, N-ethylmaleimide (2 mM), bacitracin (1 mg/ml) and 1,10-phenanthroline (5 mM) were routinely included in the homogenization medium.…”

Degradation of insulin in isolated liver endosomes is functionally linked to ATP‐dependent endosomal acidification

“…Average activities of constituents in the homogenate were: 'Z51-insulin, FITC-GalBSA and FITC-dextran, 0.025%, 0.05% and 0.015% of injected doselmg protein, respectively; ATP-dependent acidification as measured using acridine orange or FITC-GalBSA, 11 YO and 3.8% ATP-dependent fluorescence quenching min-' (mg protein)-I, respectively; insulin-degrading activity at pH 7 and at pH 4.5, 0.3 and 0.0075 ng 1Z51-insulin degraded min-I (mg protein)-respectively. Results for galactosyltransferase, acid phosphatase and 5'-nucleotidase activitics in Golgi-endosomal fractions have been taken from a previous report of this laboratory [22] Constituent Specific activity in subcellular fractions . Each cxpcrimental point represents the mean f SEM of at least three determinations except for gel-filtration studies occurring above pH 7.…”

“…HRP-GalBSA (0.2 mg), FITC-GalBSA (0.8 -1 mg) and FITC-dextran (20 mg) were injected 10 min, 10 min and 3 h, respectively, prior to sacrifice endosomal fraction (density, 1.03-1.16 g cm-3) or light, intermediate and heavy Golgi-endosomal fractions (respective densities, 1.03-1.08 g ~m -~, 1.08-1.11 g cmp3 and 1.11-1.16 g cm-3) were prepared from the microsomal fraction by a modification [22] of the method of Ehrenreich et al [23]. In some experiments, the total Golgi-endosomal fraction was subfractionated by centrifugation in continuous sucrose [22] or Percoll [ l l , 221 density gradients; sucrose gradients were carried out after diaminobenzidine cytochemistry as described by Courtoy et al [24]. Following isolation, subcellular fractions were resuspended in ice-cold 0.25 M sucrose and either immediately assayed for insulin degradation and ATP-dependent acidification, or stored at -80 "C for the later determination of protein, ligand and marker enzyme concentrations.…”