Degradation of [6-3H]- and [1-14C]glucosamine-labeled asialo-alpha 1-acid glycoprotein by the perfused rat liver.
We studied the degradation and metabolism of radioactive glucosamine-labeled asialo-alpha 1-acid glycoprotein by the perfused rat liver. Removal of 130 micrograms of the protein from the perfusate occurred with a T 1/2 of 10.3 min. Radioactivity associated initially with the microsomal fraction of the tissue homogenate and was then transferred to the lysosomes where hydrolysis of N-acetylglucosamine residues took place. Radioactivity left the lysosomal-rich fraction of the homogenate with a T 1/2 of 50 min. Final accumulation of radioactivity was in the supernatant and greater than 80% of this material was found to be UDP-N-acetylhexosamines. In those studies, 10 mumol of unlabeled glucosamine had been added to the perfusate to expand the intracellular pool of UDP-GlcNAc and prevent further metabolism of any radioactive N-acetylglucosamine released by the lysosomes. When this procedure was not done, greater than one-third of the lysosomally derived N-acetylglucosamine was reused by the liver to form new radioactive glycoproteins secreted into the perfusate. Overall hydrolysis of N-acetylglucosamine from the glycoprotein substrate was greater than 80% during 2 h of perfusion. However, leupeptin, a thiol cathepsin inhibitor, decreased the release of amino sugar by 50%, even though this compound had no effect on the activity of any lysosomal glycosidase in vitro. Large glycopeptides of molecular weight 35,000 accumulated in the lysosomes due to leupeptin. From these studies, we conclude that efficient digestion of the carbohydrate component of this glycoprotein in situ requires the concerted activity of both lysosomal glycosidases and proteases.
D000818:Animals; D001212:Asialoglycoproteins; D001692:Biological Transport; D002250:Carbon Radioisotopes; D005944:Glucosamine / Q000378:metabolism*; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D009961:Orosomucoid / Q000031:analogs & derivatives* / Q000378:metabolism; D010477:Perfusion; D051381:Rats; D011919:Rats, Inbred Strains; D014316:Tritium
D001212:Asialoglycoproteins; D002250:Carbon Radioisotopes; D007976:Leupeptins; D009961:Orosomucoid; C017030:asialoorosomucoid; D014316:Tritium; C032854:leupeptin; D005944:Glucosamine