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Human liver N-acetylglucosamine-6-sulphate sulphatase. Purification and characterization.

ABSTRACT: 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.

SUBMITTER: Freeman C

PROVIDER: S-EPMC1148283 | biostudies-other | 1987 Sep

REPOSITORIES: biostudies-other

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Project description:Kinetic parameters (Km and kcat.) of the two major forms (A and B) and a minor form (C) of human liver N-acetylglucosamine-6-sulphate sulphatase [Freeman, Clements & Hopwood (1987) Biochem. J. 246, 347-354] were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparin, heparan sulphate and keratan sulphate. Enzyme activity is highly specific towards glucosamine 6-sulphate or glucose 6-sulphate residues. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, are hydrolysed with catalytic efficiencies up to 3900 times above that observed for the monosaccharide substrate N-acetylglucosamine 6-sulphate. Forms A and B both desulphate substrates derived from keratan sulphate and heparin. Aglycone structures that influence substrate binding and/or enzyme activity were penultimate-residue 6-carboxy and 2-sulphate ester groups for heparin-derived substrates and penultimate-residue 6-sulphate ester groups for keratan sulphate-derived substrates. The 4-hydroxy group of the N-acetylglucosamine 6-sulphate or the 2-sulphaminoglucosamine 6-sulphate under enzymic attack is involved in the catalytic mechanism. The presence of a 2-amino group in place of a 2-acetamido or a 2-sulphoamino group considerably decreases the catalytic efficiency of the sulphatase, particularly in the absence of a penultimate-aglycone-residue 6-carboxy group. Both forms A and B are exo-enzymes, since activity towards internal sulphate ester bonds was not observed. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure. The presence of aglycone 2-sulphate ester, 6-carboxy group and 6-sulphate ester groups on the glucosamine 6-sulphate residue under attack considerably affects the pH response. Sulphate and phosphate ions are potent inhibitors of enzyme activity.

Project description:Human iduronate-2-sulphatase (EC 3.1.6.13), which is involved in the lysosomal degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate, was purified more than 500,000-fold in 5% yield from liver with a six-step column procedure, which consisted of a concanavalin A-Sepharose-Blue A-agarose coupled step, chromatofocusing, gel filtration on TSK HW 50S-Fractogel, hydrophobic separation on phenyl-Sepharose CL-4B and size separation on TSK G3000SW Ultrapac. Two major forms were identified. Form A and form B, with pI values of 4.5 and less than 4.0 respectively, separated at the chromatofocusing step in approximately equal amounts of recovered enzyme activity. By gel-filtration methods form A had a native molecular mass in the range 42-65 kDa. When analysed by SDS/PAGE, dithioerythritol-reduced and non-reduced form A and form B consistently contained polypeptides of molecular masses 42 kDa and 14 kDa. Iduronate-2-sulphatase was purified from human kidney, placenta and lung, and form A was shown to have similar native molecular mass and subunit components to those observed for liver enzyme. Both forms of liver iduronate-2-sulphatase were active towards a variety of substrates derived from heparin and dermatan sulphate. Kinetic parameters (Km and Kcat) of form A were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparan sulphate, heparin and dermatan sulphate. Substrate with 6-sulphate esters on the aglycone residue adjacent to the iduronic acid 2-sulphate residue being attack were hydrolysed with catalytic efficiencies up to 200 times above that observed for the simplest disaccharide substrate without a 6-sulphated aglycone residue. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure, substrate concentration, buffer type and the presence of other proteins. Sulphate and phosphate ions and a number of substrate and product analogues were potent inhibitor of form A and form B enzyme activities.

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Human liver N-acetylglucosamine-6-sulphate sulphatase. Purification and characterization.

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