Preparation from keratin sulfate of substrates for the measurement of 2-acetamido-2-deoxy-D-glucose 6-sulfate sulfatase and (1 goes to 3)-N-acetyl-beta-D-glucosaminidase.
An extract of bacterial cells Pseudomonas sp. IFO-13309 grown on medium containing 0.1% bovine cornea keratan sulfate of low sulfate content degraded exhaustively bovine cornea keratan sulfate to give 2-acetamido-2-deoxy-beta-D-gluco-pyranosyl 6-sulfate-(1 goes to 3)-D-galactose, isolated by gel filtration on Sephadex G-25 and purified by preparative paper chromatography. This was reduced with sodium borotritide to give 2-acetamido-2-deoxy-beta-D-glucopyranosyl 6-sulfate-(1 goes to 3)-D-[1-3H]galactitol, purified by gel filtration on Sephadex G-15, which was an excellent substrate for the measurement of 2-acetamido-2-deoxy-D-glucose 6-sulfate sulfatase. The reduced, radioactive monosulfated disaccharide was desulfated with methanolic 70mM hydrogen chloride and purified by gel filtration on Sephadex G-15 to give O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1 goes to 3)-D-[1-3H]galactitol, which allowed the measurement of (1 goes to 3)-N-acetyl-beta-D-glucosaminidase. This enzyme may participate in the normal degradation of keratan sulfate.
D000118:Acetylglucosaminidase / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D003315:Cornea; D004187:Disaccharides; D006025:Glycosaminoglycans; D006596:Hexosaminidases / Q000378:metabolism*; D007632:Keratan Sulfate; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity; D013429:Sulfatases / Q000378:metabolism*
D004187:Disaccharides; D006025:Glycosaminoglycans; D007632:Keratan Sulfate; D013429:Sulfatases; C018719:N-acetylglucosamine-6-sulfatase; D006596:Hexosaminidases; D000118:Acetylglucosaminidase