The quantitative discrimination of corneal type I, but not skeletal type II, keratan sulfate in glycosaminoglycan mixtures by using a combination of dimethylmethylene blue and endo-beta-D-galactosidase digestion.
The quantitation of individual glycosaminoglycans in mixtures of polyanions using the dimethylmethylene blue (DMB) method described by R. W. Farndale, D. J. Buttle, and A. J. Barrett (1986, Biochim. Biophys. Acta 883, 173) is dependent on enzymatic hydrolysis by specific polysaccharidases. While using this method to examine the keratan sulfate (KS) of the intervertebral disc we found that digestion with commercially available keratanase decreased binding to DMB by less than 30%, whereas corneal KS was reduced by 85%. However, by preincubating the KS fractions with endo-beta-D-galactosidase prior to keratanase treatment the corneal KS could be completely digested and disc KS digestion increased to 60%. It is suggested that the resistance of the disc KS to these digestive procedures arises from branching and/or sites of multisulfation on the polysaccharide chain. Agarose gel electrophoresis and compositional analyses of the keratan sulfates supported such an interpretation.
Melrose
J
J
Raymond Purves Research Laboratories, University of Sydney, Royal North Shore Hospital of Sydney, St. Leonards, Australia.
Ghosh
P
P
D011508:Chondroitin Sulfate Proteoglycans / Q000032:analysis; D003315:Cornea / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004925:Escherichia / Q000201:enzymology; D005696:Galactosidases / Q000494:pharmacology*; D006025:Glycosaminoglycans / Q000032:analysis*; D006026:Glycoside Hydrolases; D006801:Humans; D007403:Intervertebral Disc / Q000032:analysis; D007632:Keratan Sulfate / Q000032:analysis*; D000071234:Lumican; D008751:Methylene Blue / Q000031:analogs & derivatives* / Q000494:pharmacology; D011549:Pseudomonas / Q000201:enzymology; D013329:Structure-Activity Relationship; D001616:beta-Galactosidase / Q000494:pharmacology*
D011508:Chondroitin Sulfate Proteoglycans; D006025:Glycosaminoglycans; C000604306:LUM protein, human; D000071234:Lumican; D007632:Keratan Sulfate; D005696:Galactosidases; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase; D008751:Methylene Blue; C016401:1,9-dimethylmethylene blue