Uptake of injected 125I-ricin by rat liver in vivo : subcellular distribution and characterization of the internalized ligand

• Published in: Biochemical journal 1992-05, Vol.284 (1), p.249-257
• Main Authors: FRENOY, J.-P, TURPIN, E, JANICOT, M, GEHIN-FOUQUE, F, DESBUQUOIS, B
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Cell physiology; Cell-Free System - metabolism; Fundamental and applied biological sciences. Psychology; Iodine Radioisotopes; Liver - metabolism; Male; Membrane and intracellular transports; Molecular and cellular biology; Rats; Rats, Inbred Strains; Ricin - metabolism; Subcellular Fractions - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2840249

Subcellular-fractionation techniques were used to characterize the endocytic pathway followed by ricin in rat liver in vivo and tentatively identify the site(s) at which the ricin interchain disulphide bridge is split. After injection of 125I-ricin, hepatic uptake of radioactivity was maximum at 30 min (40% of injected dose). At 5 min, about 80% of the radioactivity in the homogenate was recovered in the microsomal (P) fraction, but later on the recovery of the radioactivity in the mitochondrial-lysosomal (ML) fractions progressively increased (50% at 30 min) at the expense of that in the P fraction. Subfractionation of the P and ML fractions on analytical sucrose-density gradients revealed a time-dependent translocation of the radioactivity from low- to high-density endocytic structures, with median relative densities at 5 and 60 min of about 1.15 and 1.16 (P fraction) and 1.19 and 1.22 (ML fraction) respectively. The late distribution of the radioactivity in the ML fraction was similar to that of the lysosomal marker acid phosphatase. Studies with co-injected lactose and mannan showed that ricin was internalized mainly via the mannose receptor. In the presence of mannan, the late recovery of radioactivity in the ML fraction was decreased, and the distribution of the radioactivity associated with the P fraction was shifted toward lower densities (median relative density 1.13), indicating a different pathway of endocytosis. Analysis of the radioactivity associated with the ML and S fractions by SDS/PAGE revealed a time-dependent increase in the amount of intact A- and B-chains and low-molecular-mass products. When ML fractions containing partially processed ricin were incubated at 37 degrees C at pH 5 or at pH 7.2 in the presence of ATP, only low-molecular-mass products were generated. We conclude that internalized ricin associates with endocytic structures whose size and density of equilibration increase with time, and that, although detectable in these structures, reduction of the ricin interchain disulphide bridge occurs to a large extent in the cytosol.