Purification, characterization and catalytic properties

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 0 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 900 of GAS activity had a pl of > 7.5, other forms with pl values of 5.8, 5.3, 4.7 and <4.0 were also present. The pl > 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-(3-glucuronic acid 2-sulphate)-(1-+4)-O-(2,5)-anhydro[l-3H]mannitol 6-sulphate (GSMS)} and chondroitin 6-sulphate {O-(/J-glucuronic acid 2-sulphate-(1 -+3)-O-(2,5)-anhydro[l-3H]talitol 6-sulphate (GSTS)}. GAS activity towards GSMS and GSTS was at pH optima of 3.2 and 3.0 respectively with apparent Km values of 0.3 and 0.6 juM respectively and corresponding VKax. values of 12.8 and 13.7 ,umol/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.