Human liver N-acetylgalactosamine 6-sulphatase. Purification and characterization.
Human N-acetylgalactosamine 6-sulphatase (EC 3.1.6.14), which is involved in the lysosomal degradation of the glycosaminoglycans keratan sulphate and chondroitin 6-sulphate, was purified more than 130,000-fold in 2.8% yield from liver by an eight-step column procedure. One major form was identified with a pI of 5.7 and a native molecular mass of 62 kDa by gel filtration. When analysed by SDS/PAGE, dithioerythritol-reduced enzyme contained polypeptides of molecular masses 57 kDa, 39 kDa and 19 kDa, whereas non-reduced enzyme contained a major polypeptide of molecular mass 70 kDa. It is proposed that active enzyme contains either the 57 kDa polypeptide or disulphide-linked 39 kDa and 19 kDa polypeptides. Minor amounts of other enzyme forms separated during the chromatofocusing step and the Blue A-agarose step were not further characterized. Purified N-acetylgalactosamine 6-sulphatase was inactive towards 4-methylumbelliferyl sulphate, but was active, with pH optima of 3.5-4.0, towards 6-sulphated oligosaccharide substrates. Km values of 12.5 and 50 microM and Vmax. values of 1.5 and 0.09 mumol/min per mg were determined with oligosaccharide substrates derived from chondroitin 6-sulphate and keratan sulphate respectively. Sulphate, phosphate and chloride ions were inhibitors of enzyme activity towards both substrates, with 50 microM-Na2SO4 giving 50% inhibition towards the chondroitin 6-sulphate trisaccharide substrate.
Bielicki
J
J
Department of Chemical Pathology, Adelaide Children's Hospital, South Australia.
Hopwood
J J
JJ
D000328:Adult; D002811:Chondroitinsulfatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography; D002850:Chromatography, Gel; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D013379:Substrate Specificity
D002811:Chondroitinsulfatases; D004229:Dithiothreitol