Purification of Keratan Sulfate-endogalactosidase and its action on keratan sulfates of different origin.
A glycosidase which attacks corneal keratan sulfate was purified from extracts of Pseudomonas sp. IFO-13309. When corneal keratan sulfate was degraded by the purified enzyme, Sephadex G-50 chromatography indicated the presence of a number of oligosaccharides differing in size and sulfate content. The characterization of two major fractions of the oligosaccharides indicated that the point of enzyme attack is limited to the endo-beta-D-galactoside bonds in which nonsulfated D-galactose residues participate. The enzyme, unlike ordinary exo-beta-D-galactosidases, did not catalyze the hydrolysis of phenyl beta-D-galactoside. Moreover, beta-D-galactosyl-(1 leads to 3)-2-acetamido-2-deoxy-beta-D-glucosyl-(1 leads to 3)-beta-D-galactosyl-(1 leads to 4)-D-glucose ("lacto-N-tetraose") was completely refractory to the action of this enzyme, suggesting that a structure of the type, X-(1 leads to 3)-beta-D-galactosyl-(1 leads to 4)-Y, is not the only specificity-determining factor, i.e. neighboring sugars, X and Y, or even larger portions of substrate molecule must have an important effect. Compared with corneal keratan sulfate, keratan sulfates from human nucleus pulposus and shark cartilage were attacked at lower rates with a resultant production of oligosaccharides of relatively large size. The result is in agreement with the view that considerable variations exist in the structure of keratan sulfates of different origin, and further suggests that the enzyme may serve as a useful reagent in studying these variations.
D000368:Aged; D000818:Animals; D002356:Cartilage; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002854:Chromatography, Paper; D003315:Cornea; D004590:Electrophoresis, Paper; D005696:Galactosidases / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007633:Keratins; D007700:Kinetics; D008875:Middle Aged; D009844:Oligosaccharides / Q000032:analysis; D009928:Organ Specificity; D011549:Pseudomonas / Q000201:enzymology*; D012754:Sharks; D013431:Sulfates; D013997:Time Factors
D009844:Oligosaccharides; D013431:Sulfates; D007633:Keratins; D005696:Galactosidases