The sulphatase of ox liver. XVI. A comparison of the arylsulphatase and cerebroside sulphatase activities of sulphatase A

Abstract

The initial velocities for the hydrolyses of cerebroside sulphate and nitrocatechol sulphate by the sulphatase A of ox liver have been studied under similar conditions in the pH-stat. Cerebroside sulphatase activity requires the presence of sodium taurodeoxycholate and MnCl 2 (or certain other salts) in the reaction mixture. The MnCl 2 lowers the critical micellar concentration of the inhibitory ionic form of the bile salt and allows the formation of mixed micelles of taurodeoxycholate and cerebroside sulphate. The K m varies with taurodeoxycholate concentration and is 0.07 mM cerebroside sulphate a 2 mM bile salt. The reaction is noncompetitively inhibited by K 2SO 4 with a K i of 0.47 mM. It is 50% inhibited by 25 mM hydroxylamine · HCl. The kinetics of the reaction are consistent with a uni-ter mechanism in which the substrate is a mixed micelle of taurodeoxycholate and cerebroside sulphate, and the products are released in the order cerebroside, sulphate and taurodeoxycholate. The arylsulphatase activity is inhibited by ionic taurodeoxycholate. Salts lessen this inhibition by lowering the critical micellar concentration but they also activate the enzyme. At pH 4.5 in the presence of taurodeoxycholate and MnCl 2 the properties of the arylsulphatase are not significantly different from those at the optimum pH of 5.6. The arylsulphatase is competitively inhibited by K 2SO 4 with a K i of 0.15 mM. Explanations of the apparent differences between the arylsulphatase and cerebroside sulphatase activities of sulphatase A are given and it is concluded that both are due to a single site on the protein. It has been shown that certain cations form complexes with 4-nitrocatechol: this reaction is accompanied by the liberation of H + and by an increased absorption at 430 nm.