Use of radioactive glucosamine in the perfused rat liver to prepare alpha 1-acid glycoprotein (orosomucoid) with 3H- or 14C-labelled sialic acid and N-acetylglucosamine residues.
1. A method was developed whereby [1-14C]glucosamine was used in a perfused rat liver system to prepare over 2 mg of alpha 1-acid glycoprotein with highly radioactive sialic acid and glucosamine residues. 2. The liver secreted radioactive alpha 1-acid glycoprotein over a 4-6 h period, and this glycoprotein was purified from the perfusate by chromatography on DEAE-cellulose at pH 3.6. 3. The sialic acid on the isolated glycoprotein had a specific radioactivity of 3.1 Ci/mol, whereas the glucosamine-specific radioactivity was 4.3 Ci/mole. The latter amino-sugar residues on the isolated protein were only 13-fold less radioactive than the initially added [1-14C]glucosamine. Orosomucoid with a specific radioactivity of 31.3 microCi/mg of protein was obtainable by using [6-3H]glucosamine. 4. The amino acid composition of the purified orosomucoid was comparable with that found by others for the same glycoprotein isolated from rat serum. A partial characterization of the carbohydrate structure was done by sequential digestion with neuraminidase, beta-D-galactosidase and beta-D-hexosaminidase. 5. Many other radioactive glycoproteins were found to be secreted into the perfusate by the liver. Thus this experimental system should prove useful for obtaining other serum glycoprotein with highly radioactive sugar moieties.
D000596:Amino Acids / Q000032:analysis; D000606:Amino Sugars / Q000032:analysis; D000818:Animals; D002250:Carbon Radioisotopes; D005944:Glucosamine / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000494:pharmacology; D066298:In Vitro Techniques; D007553:Isotope Labeling / Q000379:methods*; D008099:Liver / Q000378:metabolism*; D008297:Male; D009961:Orosomucoid / Q000378:metabolism*; D010477:Perfusion; D051381:Rats; D011919:Rats, Inbred Strains; D014316:Tritium
D000596:Amino Acids; D000606:Amino Sugars; D002250:Carbon Radioisotopes; D006023:Glycoproteins; D009961:Orosomucoid; D014316:Tritium; D006026:Glycoside Hydrolases; D005944:Glucosamine