Human liver glucuronate 2-sulphatase. Purification, characterization and catalytic properties

C. Freeman, J. J. Hopwood

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Abstract

Human glucuronate 2-sulphatase (GAS), which is involved in the degradation of the glycosaminoglycans heparan sulphate and chondroitin 6-sulphate, was purified almost 2000000-fold to homogeneity in 8% yield from liver with a four-step six-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, a DEAE-Sephacel/octyl-Sepharose coupled step, CM-Sepharose chromatography and gel-permeation chromatography. Although more than 90% of GAS activity had a pI of > 7.5, other forms with pI values of 5.8, 5.3, 4.7 and < 4.0 were also present. The pI > 7.5 form of GAS had a native molecular mass of 63 kDa. SDS/polyacrylamide-gel-electrophoretic analysis resulted in two polypeptide subunits of molecular mass 47 and 19.5 kDa. GAS was active towards disaccharide substrates derived from heparin {O-(β-glucuronic acid 2-sulphate-(1 → 4)-O-(2.5)-anhydro[1-3H]mannitol 6-sulphate (GSMS)} and chondroitin 6-sulphate {O-(β-glucuronic acid 2-sulphate-(1 → 3)-O-(2,5)-anhydro[1-3H]talitol 6-sulphate (GSTS)}. GAS activity towards GSMS and GSTS was at pH optima of 3.2 and 3.0 respectively with apparent K(m) values of 0.3 and 0.6 μM respectively and corresponding V(max) values of 12.8 and 13.7 μmol/min per mg of protein respectively. Sulphate and phosphate ions are potent inhibitors of enzyme activity. Cu2+ ions stimulated, whereas EDTA inhibited enzyme activity. It was concluded that GAS is required together with a series of other exoenzyme activities in the lysosomal degradation of glycosaminoglycans containing glucuronic acid 2-sulphate residues.

Original languageEnglish
Pages (from-to)209-216
Number of pages8
JournalBiochemical Journal
Volume259
Issue number1
DOIs
Publication statusPublished - 1 Jan 1989

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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