Biosynthesis of proteochondroitin sulfate: substrate requirements for formation of two independent species.
Formation of two species of [14C]proteochondroitin sulfate has previously been demonstrated with UDP-D-[14C]glucuronic acid and UDP-N-acetylgalactosamine as substrates with a microsomal preparation from chick-embryo epiphyseal cartilage. A large species of [14C]proteoglycan that appeared at an earlier stage of synthesis was excluded on Sepharose CL-2B, indicating that it was larger than proteoglycans found in cartilage matrix. Another newly synthesized, smaller [14C]proteoglycan species also formed was retarded on Sepharose CL-2B, and appeared to be at a later stage of synthesis. A 6-h pulse-chase experiment using UDP-[14C]GlcA and UDP-GalNAc followed by cold UDP-GlcA demonstrates that there was no conversion of the large [14C]proteoglycan to the small [14C]proteoglycan. Sulfation of the newly formed large and small [14C]proteoglycans with adenylyl sulfate 3'-phosphate also did not alter their chromatographic patterns, indicating that sulfation did not trigger any post-synthetic size modification. Synthesis with lower concentrations of the sugar nucleotides resulted in a disproportionate diminution in formation of the large proteoglycan. The apparent Km values for UDP-GlcA for the formation of large and small proteoglycans were 0.055 and 0.015 mM, respectively. Concentration requirements for UDP-GalNAc also showed a similar 4-fold difference. These results indicate that, even though the large proteoglycan was at an earlier stage of synthesis, it was not a precursor to the small proteoglycan, and that these proteoglycans represent two separately synthesized species.
D000818:Animals; D002250:Carbon Radioisotopes; D002642:Chick Embryo; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis* / Q000302:isolation & purification; D006132:Growth Plate / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism*; D011509:Proteoglycans / Q000096:biosynthesis*; D014316:Tritium; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism*; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
D002250:Carbon Radioisotopes; D011508:Chondroitin Sulfate Proteoglycans; D011509:Proteoglycans; D014539:Uridine Diphosphate Sugars; D014316:Tritium; D014535:Uridine Diphosphate Glucuronic Acid; D014536:Uridine Diphosphate N-Acetylgalactosamine