Biosynthesis of proteoglycans by chick limb bud chondrocytes

De Luca, S.; Caplan, A.I.; Hascall, V.C.

Journal of Biological Chemistry 253(13): 4713-4720


ISSN/ISBN: 0021-9258
PMID: 566270
Accession: 004843525

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Chick limb bud mesenchyme cells isolated from stage 23-24 embryos were grown in vitro under conditions which promote chondrogenesis. Proteoglycan synthesis is maximal on day 8 of culture. A series of day 8 cultures were pulsed for 10 min with medium containing [3H]serine and [35S]sulfate and subsequently chased with medium in the absence of the labeled precursors for times from 0-180 min. Proteoglycans were isolated from the cell-matrix layers by extraction with 4 M guanidinium chloride and then purified in direct dissociative density gradients by standard procedures. This pulse-chase protocol was designed to provide information on the sequential and temporal aspects of the biosynthesis of these complex macromolecules. At the end of the 10-min pulse the amount of 35S radioactivity in the monomer proteoglycan fraction was 50-60% of the value observed after the 10-min chase, with no appreciable increase observed at longer chase times. The 3H radioactivity in the proteoglycan monomer after the pulse was only about 10% of the maximal amount which was achieved later, by about the 25-min chase time. Sepharose 2B chromatography of the monomer fractions showed the same qualitative elution profiles for both 35S and 3H activity at all chase times. The profiles coincided with the hexuronic acid profiles observed for monomer samples isolated from the matrix of the day 8 cultures. Mixtures of the monomer preparations with hyaluronic acid were chromatographed on Sepharose 2B. The elution profiles indicated that, at the end of the pulse, about 40% of the total 3H activity and 60% of the total 35S activity were in macromolecules which were able to bind to hyaluronic acid. The proportion of the 3H activity increased to 60% bound by the 10-min chase while that for 35S activity remained unchanged. Sepharose 6B chromatography of trypsin digests of the proteoglycan monomers showed that at the end of the pulse the peptide regions covalently linked to the chondroitin sulfate chains contained only 38% of the total 3H activity. This proportion increased to 75% after 25-min chase. For papain digests at the end of the pulse, less than 10% of the total 3H activity present in the sample was associated with the chondroitin sulfate chains. This proportion increased to approximately 38% after the 25-min chase time. The polysaccharide attachment region is associated with the NH2 terminus if the core protein is a single polypeptide chain and the synthesis of a complete monomer proteoglycan takes between 20-35 min.