The sulphatase of ox liver. XXII. Further observations on the cerebroside sulphatase activity of sulphatase A.

Further studies have been made of the cerebroside sulphatase activity of the sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) of ox liver. It is concluded that a cerebroside sulphate-modified form of the enzyme is not produced and that the kinetics of the reaction can be explained by the utilisation of the substrate and accumulation of (SO4)2-. The hypothesis is advanced that this difference between the cerebroside sulphatase and arylsulphatase activities arises from non-polar binding of the cerebroside to the enzyme. Possible reasons for the differences between these results and those of other (Stinshoff, K. and Jatzkewitz, H. (1975) Biochim. Biophys. Acta 377, 126-138) are considered.     

The sulphatase of ox liver. XXIV. The glycosulphatase activity of sulphatase a

The rhodizonic acid method for the determination of SO2-4 has been used to investigate the glycosulphatase activity of the sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) of ox liver. Sulphatase A hydrolyses D-glucopyranose and D-galactopyranose 2-, 3-, 4- and 6-sulphates: glucose sulphates are hydrolysed more rapidly than galactose sulphates and the 3-sulphates more rapidly than the other isomers. 2-Acetamido-2-deoxyglucopyranose 6-sulphate is not hydrolysed, nor is 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranose 1-sulphate. Sulphate is a competitive inhibitor of the glycosulphatase activity. Hydrolysis proceeds through fission of the O-S bond. Evidence is given that the hydrolysis of glucose 3-sulphate is accompanied by the formation of substrate-modified sulphatase A, although this has not been isolated. Sulphatase A has no detectable alkylsulphatase activity.     

The sulphatase of ox liver. XXI: kinetic studies of the substrate-induced inactivation of sulphatase A.

The theoretical basis is given for methods of determining the apparent velocity constant, k*, for the substrate-induced inactivation of sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) and the initial velocity, vo, of the catalytic reaction. The expression is of the same form as the empirical relationships previously used but the significance of the various terms is clearly established. The method has been applied to the characterisation of the inactivation occurring during the hydrolysis of a number of substrates and it has been shown that k* varies with so in a hyperbolic relationship described by k, a velocity constant at infinite substrate concentrations and by K, a constant analogous to the Michaelis constant. Although K varies considerably for different substrates, and is consistently less than the corresponding Km, k is almost constant at 0.23 min-1. It is therefore suggested that the inactivation of the enzyme does not proceed through an enzyme . substrate complex but through the enzyme . SO2-4 complex produced during the catalytic reaction. The effects of several variables on these parameters are described.     

Enzymic studies of sulphatases in tissues of the normal human and in metachromatic leukodystrophy with multiple sulphatase deficiencies: arylsulphatases A, B and C, cerebroside sulphatase, psychosine sulphatase and steroid sulphatases

Abstract— Several sulphatases (arylsulphatases A, B and C, cholesterol sulphatase, dehydroepiandroster-one sulphatase, cerebroside sulphatase and psychosine sulphatase) were deficient in various tissues from two patients with a variant form of metachromatic leukodystrophy. Deficient activities of cerebroside sulphatase and psychosine sulphatase, using physiological substrates, in tissues from metachromatic leukodystrophy with multiple sulphatase deficiencies provided another example that these enzymes may be identical to arylsulphatase A. β-Galactosidase activity was reduced to about 30-50 per cent of normal in brain and liver. Other lysosomal enzyme activities were found to be normal or elevated five to eight times. Arylsulphatase B isolated from the liver of one patient was abnormal, with respect to pi (70) and enzyme kinetics. In mixing experiments with normal enzymes the reduced activities of arylsulphatases A. B and C, cerebroside sulphatase and steroid sulphatases were shown not to be due to the presence of endogenous inhibitors. No arylsulphatase A or B activity in the brain specimen from the patient with multiple sulphatase deficiencies could be detected on isoelectric focussing. In normal brain tissue arylsulphatase A had a pi of 4-6-4-8 while arylsulphatase B had a pi of 7-8 and 8-1. When 4-methylumbelliferyl sulphate was used as a substrate the elution patterns of normal brain and liver arylsulphatase B were more heterogeneous and showed more variation than that when p-nitrocatechol sulphate was used. Arylsulphatase C and steroid sulphatases (cholesterol sulphatase, dehydroepiandrosterone sulphatase and oes-trone sulphatase I were solubilized by the addition of lysolecithin and Triton X-100 and subjected to isoelectric focussing. The pi of cholesterol sulphatase, oestrone sulphatase and arylsulphatase C was 6-8, and the elution patterns of the activities of these enzymes were similar. The pattern of dehydroepiandrosterone sulphatase was more heterogeneous and two major peaks were observed at pi 6 5 and 70. Residual enzyme activities of arylsulphatase C and steroid sulphatases from the brain of the patient with multiple sulphatase activities were not detectable by isoelectric focussing. Simultaneous deficiencies of arylsulphatase C and steroid sulphatases plus isoelectric focussing findings in tissues suggest that these enzymes are closely related in regard to their function. The nature of the genetic defect in metachromatic leukodystrophy with multiple sulphatase deficiencies is discussed.     

Comparison of the cerebroside sulphatase and the arylsulphatase activity of human sulphatase A in the absence of activators.

A cerebroside sulphatase (cerebroside-3-sulphate 3 sulphohydrolase, EC 3.1.6.8) assay based on radio thin-layer chromatography is described. The substrate was labelled by the catalytic addition of tritium to cerebroside sulphate. Using this assay the cerebroside sulphatase activity of sulphatase A (Aryl-sulphate sulphohydrolase, EC 3.1.6.1) from human liver and kidney in the absence of activators was investigated. The pH optimum of this reaction depends on the buffer concentration, being pH 4.5 at 50 mM and 5.3 at 10 mM sodium formate. With the latter concentration the apparent Km for cerebroside sulphate is 0.06 mM; SO2-4 and nitrocatechol sulphate inhibit noncompetitively with a Ki of 4.51 mM for Na2SO4 and 0.43 mM for nitrocatechol sulphate. The cerebroside sulphatase activity of sulphatase A is highly dependent on the ionic strength. The optimum sodium formate concentration is 10 mM, and the cerebroside suophatase activity decreases rapidly with increasing buffer concentration. The same concentration dependence is observed in the inhibitory effect of cerebroside sulphate on the arylsulphatase reaction. The inhibition decreases at increasing buffer concentrations, becoming an activation at 70 mM sodium formate. The progress curve of the cerebroside sulphatase reaction shows a deviation from linearity similar to that of the arylsulphatase reaction. Investigation of the effect of preincubation with cerebroside sulphate on the arylsulphatase activity of the enzyme shows that cerebroside sluphatase activity and inactivation of the enzyme by cerebroside sulphate occur simultaneously. These observations are interpreted as supporting the assumption that cerebroside suophate and arylsulphates are degraded at an identical active site on the same enzyme. Differences in the properties of the cerebroside sulphatase and the arylsulphatase reaction of the enzyme may be attributed to the differences in the physiocochemical state of the two substrates.