Glucuronate-2-sulphatase activity in cultured human skin fibroblast homogenates

Abstract

The optimization of the assay conditions to detect glucuronate-2-sulphatase (GS) activity present in cultured human skin fibroblast homogenates towards a heparin-derived disaccharide substrate O-(beta-D-glucuronic acid 2-sulphate)-(1----4)-D-O-2,5-anhydro[l-3H]mannitol 6-sulphate (GSMS) has shown that a complex relationship exists between pH, buffer composition, ionic strength and the influence of added BSA and salts (NaCl, Na2SO4, CuCl2 and ZnCl2) to achieve maximum sulphatase activity. Whereas albumin stimulated GS activity by more than 2-fold over the pH range 2.7-5.7, CuCl2 stimulated GS activity over the narrow pH range 3.0-4.2, and inhibited GS activity at higher pH. ZnCl2 stimulated GS activity more than 3-fold at pH 3.0 and by more than 10-fold at pH 4.8. NaCl inhibited GS activity at pH 3.0, while activity between pH 4.2 and 4.8 was stimulated by up to 10-fold, resulting in a shift in the observed pH optimum from 3.0 to 4.8 in the presence of 315 mM-NaCl. Skin fibroblast GS activity toward GSMS had apparent Km values of 0.5-1.2 microM at pH 3.0, and 27.0-33.2 microM at pH 4.8. Albumin stimulated GS activity at both low and high pH by an increase in the apparent Vmax. values without significant alteration in the respective Km values. At pH 4.8, NaCl stimulated GS activity as a result of an increase in Vmax. values. These observations raise the possibility that two forms of GS activity are present in skin fibroblast homogenates: a low-Km form that has a pH optimum of 3.0 and is stimulated by BSA and a high-Km form with a pH optimum of 4.8 which is stimulated by NaCl.