Abstract
Proteoglycans isolated from the Swarm rat chondrosarcoma were shown to contain 35 mol of phosphate/mol of proteoglycan. While 20% of this phosphate was released by digestion with dilute alkali in the presence of sodium borohydride and is presumably of the phosphoserine/phosphothreonine type, 78% of the phosphate copurified with the peptide-free chondroitin sulfate chains. When chondroitin sulfate chains purified by ethanol precipitation or Sephacryl S200 column chromatography were digested with chondroitinase AC and the digests chromatographed on Bio-Gel P-4, the phosphate co-migrated with a carbohydrate fragment that contained 2 glucuronic acid (one as delta 4,5-unsaturated sugar), 1-galactosamine, 2-galactose, and 1-phosphate residue/xylitol. A second fragment of similar composition but lacking phosphate was also recovered in a ratio of about 3 to 1 relative to the phosphorylated fragment. The phosphate in the chondroitin sulfate linkage region fragment had the alkaline phosphatase sensitivity as well as 31P NMR spectra of a monophosphate esterified to a secondary sugar alcohol. The phosphate was localized on the C-2 of the chain initiating xylose since these residues as xylitol showed a delayed release during acid hydrolysis and the xylitol was recovered intact after periodate oxidation. In the chondrosarcoma, 2-phosphoxylose appears to be a normal synthetic product since [32P]phosphate was readily incorporated into the proteoglycan and the incorporated isotope had similar biochemical properties as the unlabeled phosphate.
Highlights
Threefunctional domains: 1) a globular protein region ( M, z 6 X IO4) that interacts with hyaluronic acid and a stabilizing link protein to form the large extended aggregate complexes native to the tissue (1-3); 2) the region immediately adjacent that is substituted with 60% of the 50-80 keratan sulfate chains present in some cartilages and with a few chondroitin sulfate chains (4); and 3) the remaining portion of protein core that contains the majority of the 80-100 chondroitin
Characterization of the 32P-Proteoglycan-The [32P]phosphate in dissociatively prepared proteoglycan fractions eluted as two peaks from Sepharose CL-2B columns (Fig. 1).The
When chondroitinase AC digests of the 32P-proteoglycan monomer were chromatographed on Sepharose CL-GB, all the
Summary
( 6 )Chondroitin Sulfate Isolation-Preparative Sephacryl S200 column with a bed of 2.2 X 103 cm equilibrated and eluted with 0.25 M sodium acetate, 0.1 M potassium phosphate, pH7.0; the flow rate was 30 ml/h with 110fractions of 3.5 ml collected. ( d ) RechromatographyofChondroitinaseAC Oligosaccharides-Analytical Bio-Gel P-4 column with a bed of 1.1 X 190 cm was equilibrated and eluted with 0.5 M pyridine adjusted to pH 6.2 with glacial acetic acid; the flow rate was 5.25 ml/h and 130 fractions of1.4ml were collected with a sample volume of 0.7to 1.0 ml.
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