The cyanogen bromide fragment I of asialoorosomucoid is transported more efficiently than asialoorosomucoid in rat hepatocytes.
Cultured rat hepatocytes internalized and degraded 125I-labeled asialoorosomucoid (125I-ASOR) through asialoglycoprotein receptor at rates about half that of its cyanogen bromide fragment I (125I-ASCNBr-I). Reduction and carboxymethylation of the fragment resulted in decreased rates of internalization and degradation which were still greater than those of 125I-ASOR. In the presence of 5 microM colchicine, degradation of all three ligands was inhibited. However, the intracellular level of 125I-ASOR at steady state remained unchanged, while those of the fragments increased continuously. Study of the binding of these ligands to hepatocytes at 4 degrees C indicated that there was no significant difference in binding parameters between ASOR, ASCNBr-I and RC-ASCNBr-I (reduced and carboxymet ASCNBr-I). Studies of the fate of these ligands preloaded in the cell at 37 degrees C indicated that a higher fraction of the internalized ASOR than of the fragments was released by diacytosis. In contrast to ASOR, diacytosis of the fragments was not enhanced by colchicine. Studies of the distribution of intracellular ligands by Percoll density gradient centrifugation indicated that they were internalized initially into two early endosomal compartments of d = 1.037 g/ml and d = 1.045 g/ml. In the presence of colchicine, accumulation of the ligands in a third endosomal compartment of d = 1.08-1.095 g/ml was revealed, while in the presence of leupeptin accumulation of the ligands in lysosomes was observed. The results of a kinetic analysis indicated that both cyanogen bromide fragments were transported to all these compartments more rapidly than was ASOR. It appears that they are internalized and degraded more rapidly than ASOR due to a more efficient sorting of the internalized ligand into the pathway of lysosomal degradation.
Chang
T M
TM
Department of Medicine, Genesee Hospital, Rochester, NY 14607.
Chakraborti
P
P
Chang
C H
CH
D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D001692:Biological Transport; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D003078:Colchicine / Q000494:pharmacology; D003488:Cyanogen Bromide; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008024:Ligands; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D009961:Orosomucoid / Q000031:analogs & derivatives* / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011971:Receptors, Immunologic / Q000378:metabolism
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D007976:Leupeptins; D008024:Ligands; D009961:Orosomucoid; D010446:Peptide Fragments; D011971:Receptors, Immunologic; C017030:asialoorosomucoid; D003488:Cyanogen Bromide; D003078:Colchicine