Human N-acetylgalactosamine-4-sulphate sulphatase. Purification, monoclonal antibody production and native and subunit M(r) values

Initial purification of N-acetylgalactosamine-4-sulphate sulphatase from human liver homogenates containing approx. 1 mg of enzyme in 26 g of soluble proteins was achieved by a six-column chromatography procedure and yielded approx. 40 μg of a single major protein species. Enzyme thus prepared was used to produce N-acetylgalactosamine-4-sulphate sulphatase-specific monoclonal antibodies. The use of a monoclonal antibody linked to a solid support facilitated the purification of approx. 0.5 mg of N-acetylgalactosamine-4-sulphate sulphatase from a similar liver homogenate. Moreover the enzyme isolated contained a single protein species, shown by SDS/polyacrylamide-gel electrophoresis to have an M(r) of 57,000, which dissociated into subunits of M(r) 43,000 and 13,000 in the presence of reducing agents. Essentially identical enzyme preparations were isolated from homogenates of human kidney and lung and from concentrated human urine. The native protein M(r) of enzyme from human liver and kidney was assessed by gel-permeation chromatography to be 43,000 on Ultrogel AcA and Bio-Gel P-150. The liver N-acetylgalactosamine-4-sulphate sulphatase was shown to have pH optima of approx. 4 and 5.5 with the oligosaccharide substrate (GalNAc4S-GlcA-GalitolNAc4S) and fluorogenic substrate (methylumbelliferyl sulphate) respectively. K(m) values of 60 μM and 4 mM and V(max.) values of 2 and 20 μmol/min per mg were determined with the oligosaccharide and fluorogenic substrates respectively.