Human liver N-acetylglucosamine-6-sulphate sulphatase. Purification and characterization.
Human N-acetylglucosamine-6-sulphate sulphatase was purified at least 50,000-fold to homogeneity in 78% yield from liver with a simple three-step four-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, chromatofocusing and Cu2+-chelating Sepharose chromatography. In all, four forms were isolated and partially characterized. Forms A and B, both with a pI greater than 9.5 and representing 30% and 60% respectively of the recovered enzyme activity, were separated by hydroxyapatite chromatography of the enzyme preparation obtained from the Cu2+-chelating Sepharose step. Both forms A and B had native molecular masses of 75 kDa. When analysed by SDS/polyacrylamide-gel electrophoresis, form A consists of a single polypeptide of molecular mass 78 kDa, whereas form B contained 48 kDa and 32 kDa polypeptide subunits. Neither form A nor form B was taken up from the culture medium into cultured human skin fibroblasts. The two other forms (C and D), with pI values of 5.8 and 5.4 respectively, represented approx. 7% and 3% of the total recovered enzyme activity. The native molecular masses of forms C and D were 94 kDa and approx. 75 kDa respectively. Form C contained three polypeptides with molecular masses of 48, 45 and 32 kDa. N-Acetylglucosamine-6-sulphate sulphatase activity was measured with a radiolabelled disaccharide substrate derived from heparin. The development of this substrate enabled the isolation and characterization of N-acetylglucosamine-6-sulphate sulphatase to proceed efficiently. Forms A, B and C had pH optima of 5.0, Km values of 11.7, 14.2 and 11.1 microM respectively and Vmax. values of 105, 60 and 53 nmol/min per mg of protein respectively. The molecular basis of the multiple forms of this sulphatase is not known. It is postulated that the differences in structure and properties of the four enzyme forms are due to differences in the state of processing of a large subunit.
Department of Chemical Pathology, Adelaide Children's Hospital, Australia.
C
C
Freeman
P R
PR
Clements
J J
JJ
Hopwood
D002853:Chromatography, Liquid; D004187:Disaccharides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D013429:Sulfatases / Q000302:isolation & purification* / Q000378:metabolism
D004187:Disaccharides; D007527:Isoenzymes; C054198:O-((Nalpha)-acetylglucosamine 6-sulfate)-(1-3)-idonic acid; D013429:Sulfatases; C018719:N-acetylglucosamine-6-sulfatase