Abstract
Abstract A colorimetric procedure was developed for assaying the enzyme activity of chondroitinase AC from Flavobacterium heparinum and of chondroitinase ABC from Proteus vulgaris when chondroitin 4-sulfate is used as a substrate. The enzymatic digestion product, 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose, is oxidized with periodic acid to yield, among other products, formylpyruvic acid. The latter compound reacts with thiobarbituric acid to yield a chromophore with an absorption maximum at 549 nm. Both enzymes were used to remove chondroitin sulfate from bovine nasal cartilage proteoglycan. The protein-keratan sulfate core isolated after limit digestion of proteoglycan with chondroitinase AC contains about 44% protein, 38% keratan sulfate, and 18% of the enzymatically modified oligosaccharide attachment region between the chondroitin sulfate chains and protein core: gluco-4-enepyranosyluronic acid-(galactosyl)2-xylose. Limit digestion of proteoglycan with chondroitinase ABC leaves a residual disaccharide on the chondroitin sulfate chains in the core preparation. Both monomeric and aggregated preparations of proteoglycan were treated with chondroitinase AC and the resulting core fractions were studied in the ultracentrifuge. A single, fairly symmetrical peak with a sedimentation coefficient of 7.7 S was observed for the core prepared from monomeric proteoglycan. In addition to this monomeric peak, the core prepared from aggregated proteoglycan exhibited a faster sedimenting, broad peak with a sedimentation coefficient of about 18 S. The results indicate that the noncovalent interactions which are essential for the aggregation of proteoglycans do not require the presence of the chondroitin sulfate portion of the macromolecules.
Highlights
The results indicate that the noncovalent interactions which are essential for the aggregation of proteoglycans do not require the presence of the chondroitin sulfate portion of the macromolecules
This paper describes experiments in which subunit and complex were treated with the chondroitinase enzymes to prepare protein-keratan sulfate core preparations
The chondroitinases, apparently do not degrade the protein-keratan sulfate core structure of the subunit molecules into smaller molecules or subunits such as those suggested for the “monomers” observed in PPL3 preparations (5)
Summary
A calorimetric procedure was developed for assaying the enzyme activity of chondroitinase AC from Flavobacterium heparinum and of chondroitinase ABC from Proteus vulgaris when chondroitin 4-sulfate is used as a substrate. The proteinkeratan sulfate core isolated after limit digestion of proteoglycan with chondroitinase AC contains about 44% protein, 38% keratan sulfate, and 18% of the enzymatically modified oligosaccharide attachment region between the chondroitin sulfate chains and protein core: gluco-4-enepyranosyluronic acid-(galactosyl)2-xylose. Limit digestion of proteoglycan with chondroitinase ABC leaves a residual disaccharide on the chondroitin sulfate chains in the core preparation. Both monomeric and aggregated preparations of proteoglycan were treated with chondroitinase AC and the resulting core fractions were studied in the ultracentrifuge. The preponderance of chondroitin sulfate in the proteoglycan preparations makes it difficult to determine some of the physical and chemical properties of the proteill-keratan sulfate core structure of these macromolecules. The following paper (9) describes the physical properties of monomeric protein-keratan sulfate core, and of keratan sulfate prepared from it
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