Post-translational alterations in newly synthesized cartilage proteoglycans induced by the glutamine analogue 6-diazo-5-oxo-L-norleucine. Time course of inhibition and recovery.
Incorporation of [35S]sulphate by cultures of matrix-free cells from chick embryo sterna in the presence of the glutamine analogue 6-diazo-5-oxo-L-norleucine (0.58 mM) was inhibited in a time-dependent manner to less than 15% of that in control cultures after 2 h. Characterization of the major cartilage proteoglycan synthesized under these conditions showed that it contained few, if any, normal-sized chondroitin sulphate chains and only about half of the normal complement of substituted serine residues. Subsequent addition of D-glucosamine hydrochloride (final concn. 2 mM) resulted in a time-dependent recovery of [35S]sulphate incorporation to 90% of control cultures after 2 h, but restored the chondroitin sulphate chains to normal size within 15 min. On the basis of these results, it is concluded that a 2 h preincubation is necessary to deplete the chondrocytes of the endogenous supply of UDP-N-acetyl-D-glucosamine required for optimal glycoconjugate synthesis, and that this situation results in the synthesis of a chondroitin sulphate proteoglycan with significantly altered properties, owing to the paucity of glycosaminoglycan chains; however, this condition is completely reversible if the D-glucosamine pool is repleted.
Clark
C C
CC
Department of Orthopaedic Surgery, University of Pennsylvania School of Medicine, Philadelphia 19104-6081.
Richards
C F
CF
Iozzo
R V
RV
D053674:Aggrecans; D000818:Animals; D002356:Cartilage / Q000196:embryology / Q000378:metabolism*; D002478:Cells, Cultured; D002642:Chick Embryo; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism*; D002850:Chromatography, Gel; D003980:Diazooxonorleucine / Q000494:pharmacology*; D016326:Extracellular Matrix Proteins; D005944:Glucosamine / Q000494:pharmacology; D005973:Glutamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D037181:Lectins, C-Type; D011499:Protein Processing, Post-Translational; D011509:Proteoglycans; D013462:Sulfur Radioisotopes; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D053674:Aggrecans; D011508:Chondroitin Sulfate Proteoglycans; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D013462:Sulfur Radioisotopes; D003980:Diazooxonorleucine; D005973:Glutamine; D014537:Uridine Diphosphate N-Acetylglucosamine; D005944:Glucosamine