3',5'-Cyclic nucleotide phosphodiesterase activity of the sulphatase A of ox liver

The sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) of ox liver hydrolyses adenosine 3',5'-monophosphate (cyclic AMP) to adenosine 5'-phosphate at an optimum pH of approx. 4.3, close that for the hydrolysis of cerebroside sulphate, a physiological substrate for sulphatase A. The Km is 11.6 mM for cyclic AMP. On polyacrylamide gel electrophoresis sulphatase A migrates as a single protein band which coincides with both the arylsulphatase and phosphodiesterase activities, suggesting that these are due to a single protein. Cyclic AMP competitively inhibits the arylsulphatase activity of sulphatase A, showing that both activities are associated with a single active site on the enzyme. sulphatase A also hydrolyses guanosine 3',5'-monophosphate, but not uridine 3',5'-monophosphate nor adenosine 2',3'-monophosphate.     

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. XIX. On the nature of the polymeric forms of sulphatase A present in dilute solutions.

Weight-average elution volumes of sulphatase A (an arylsulphate sulphohydrolase, EC 3.1.6.1) from Sephadex G-200 have been determined as functions of protein concentration, pH, ionic strength and temperature. The results are used to calculate the apparent association equilibrium constants for tetramer formation and the associated standard-state thermodynamic parameters. While the apparent association constant decreased from 10(28) to 10(21) M-3 on increasing the pH from 4.5 to 5.6 at ionic strength 0.1, at any particular pH value studied it was relatively insensitive to temperature variation so that deltaH is close to zero and tetramer formation in solution is associated with a positive entropy change. At pH 5.0, increasing the ionic strength from 0.1 to 2 decreased the association constant by a factor of 100. Methylumbelliferone sulphate has no effect on the association of sulphatase A. The equilibrium results are used to define the degree of association of sulphatase A likely to encountered in experiments designed to elucidate its kinetic properties. In the liver lysosome, the tetramer is probably the dominant species. The monomer and tetramer of sulphatase A have similar, or identical, specific activities with nitrocatechol sulphate and 4-methylumbelliferone sulphate as substrates. With nitrocatechol sulphate, sulphatase A shows Michaelis kinetics under conditions where the monomer is the dominant species and non-Michaelis kinetics where the tetramer is dominant. There is apparently a negative cooperativity between the monomer units in the tetramer. In 2 mM sodium taurodeoxycholate and 0.035 M MnCl2, but not in 0.1 M NaCl, the tetramer shows Michaelis kinetics. This is not due to dissociation of the tetramer. The critical micellar concentration of sodium taurodeoxycholate is about 0.8 mM in both 0.1 M NaCl and 0.035 M McCl2 but the aggregation number is greater in the latter.     

The sulphatase of ox liver. XXV. Sulphatase A as a hysteretic enzyme

The kinetic behaviour of the system native--substrate-modified sulphatase A (arylsulphate sulphohydrolase, EC 3.1.6.1) has been investigated and it has been shown that the progress curve of the complete reaction, including both the inactivation and reactivation stages, can be treated as that of a simple hysteretic system in which the substrate-modified enzyme is activated by a product of the reaction. It has been concluded that the early suggestions that the modification of sulphatase A was accompanied by the exposure of a second ligand-binding site are incorrect. It has been shown that, in the absence of sulphate, the rate of reversion of substrate-modified to native sulphatase A is increased by 4-nitrocatechol but not by the same concentration of 2-nitrophenol. A detailed reaction sequence is proposed. This explains the kinetic behaviour of sulphatase A with nitrocatechol sulphate or 2-nitrophenyl sulphate as substrate and in the presence or absence of sulphate.     

L-ascorbic acid 2-sulphate. A substrate for mammalian arylsulphatases.

Ascorbic acid 2-sulphate has a stability in acid comparable to that of phenyl sulphate and is rather more acid-labile than simple carbohydrate sulphates. At its optimum pH of 4.8 sulphatase A(aryl-sulphate sulphohydrolase EC 3.1.6.1.) hydrolyses ascorbic acid sulphate with a specific activity of 90 mumol/mg per min (150 mumol/mg per min with nitrocatechol sulphate at pH 5.6). At pH 4.8 the kinetics are non-Michaelis. At pH 5.6 Michaelis kinetics are obeyed and Km 12 21 mM ascorbic acid 2-sulphate. K2SO4 is a competitive inhibitor with a Ki of 0.2 and 0.6 mM at pH 4.8 and 5.6, respectively. Sulphatase A is converted into a substrate-modified form during its hydrolysis of ascorbic acid sulphate. Sulphatase B also hydrolyses ascorbic acid 2-sulphate. At pH 4.8 and in the presence of 0.15 M NaCl the specific activity is 0.92 mumol/mg per min (90 mumol/mg per min for nitrocatechol sulphate at pH 5.6). In the absence of NaCl the activity is greatly decreased. Km is 8 mM. K2SO4 is a competitive inhibitor with a Ki of 0.1 mM. Ascorbic acid is not hydrolysed at a detectable rate by the arylsulphatases of the mollusc Dicathais orbita or of Aerobacter aerogenes.?     

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.     

Bile salt activation of cerebroside sulphate sulphohydrolase.

The cholate and taurodeoxycholate activations of cerebroside sulphate sulphohydrolase (cerebroside-3-sulphate 3-sulphohydrolase, EC 3.1.6.8) activity of arylsulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) were compared. Taurodeoxycholate caused a sharp peak of response at a concentration of 1.25 mg/ml (type-I activation). Cholate also showed type-I activation but, in addition, it evoked a second, higher, response plateau at concentrations between 5 and 10 mg/ml (type-II activation). At the pH of the reaction, cholate is converted largely to the sparingly soluble free aicd, so at the high concentrations associated with type-II activation, copious precipitates were formed. It was found that the precipitated material was essential for the type-II activation. Type-I activation appears to involve bile salt interaction with substrate, while type-II activation appears to involve enzyme interaction with solid-phase cholic acid. the putative mutant arylsulphatase A in an unusual form of metachromatic leukodystrophy hydolysed cerebroside sulphate only in the presence of high levels of cholate. The type-II activation may thus be simulating a physiological desulphation reaction.     

The effect of the interdependence of protein and bile salt concentrations on the measurement of cerebroside sulphate sulphatase activity in human leucocyte and fibroblast extracts

The previously observed differences in properties of human leucocyte and fibroblast cerebroside sulphate sulphatase (cerebroside-3-sulphate 3-sulphohydrolase, EC 3.1.6.8) measured in vitro have been found to be due to subtle differences in incubation conditions. Maximum enzyme activity was observed with either crude sodium taurocholate or with pure sodium taurodeoxycholate. The optimum bile salt concentration of the enzyme in leucocyte or fibroblast extracts, but not the pure ox liver enzyme, was critically dependent on protein concentration. At low concentrations of the latter (less than 0.1 mg/ml), maximum activity was observed at taurocholate concentrations less than 0.5 mg/ml; at protein concentrations greater than 0.20 mg/ml substantially more bile acid (more than 1.3 mg/ml) was required to stimulate maximum activity. Addition of Triton X-100 or bovine serum albumin to the incubation mixtures increased the optimum taurocholate concentration. The dependence of the bile salt optimum on protein concentration appears to be related to the binding of the lipid substrate to membranous protein present in the tissue extracts. Release of the bound lipid is effected either by increasing the bile salt concentration or by adding Triton X-100. In the presence of excess bile salt human leucocyte, fibroblast and liver cerebroside sulphate sulphatase activity is stimulated by Triton at low protein concentrations; under identical conditions the pure or crude ox-liver enzyme is substatially inhibited. Our data also show that cerebroside sulphate sulphatase activity measured in extracts from leucocytes and fibroblasts, the tissues normally used to effect a diagnosis of metachromatic leucodystrophy, is the result of a complex interaction of bile salt, protein, Triton X-100 and probably the substrate itself. Any slight alteration in any of those factors, without a corresponding change in any or all of the others, can have a marked effect on the measured enzyme activity, and may lead to errors in the diagnosis of metachromatic leucodystrophy.     

Micelles of cerebroside sulphate

The critical micelle concentration of cerebroside sulphate in water is 0-01 mM: it increases with increasing concentrations of buffer to 0-07 mM in 0-1 M sodium acetate and formate buffers, pH 5-6 and 4-5 respectively. The partial specific volume of the micelles is about 0-94. The behaviour of the micelles in the ultracentrifuge and on Sephadex G-200 shows them to be grossly heterogeneous with respect to size. In 0-1 M buffer s20,w is about 26 S; in water or 0-01 M buffer smaller micelles with an s20,w of about 6 S are also present. In 0-01 M formate, pH 4-5, the smallest species detectable by equilibrium ultracentrifugation had a micellar weight of about 180,000 corresponding to an aggregation number of about 180. Much larger aggregates were also present. It is suggested that the smallest micelles are the substrate for sulphatase A when this is acting as a cerebroside sulphatase in buffers of low ionic strength.     

Lysosomal aryl sulphatase in the liver

Summary Lysosomal aryl sulphatase activity in rat liver is demonstrated by a modification of the existing processes of fixation, incubation and processing. The choice and concentration of the fixative, duration of fixation and thickness of liver slices are found to be important factors in maintaining the levels of enzyme activity. Reliable and reproducible results are obtained by fixing thin liver slices (1 mm) for 18–24 h, in 2% glutaraldehyde buffered to pH 7.4 by 0.1M cacodylate buffer and incubating sections inHopsu et al. (1967) medium using (160 mg) nitrocatechol sulphate as substrate. Aryl sulphatase activity is localised in discrete pericanalicular granules recognised as lysosomes, which stain less intensely than acid phosphatase by the lead method.Supported by a grant from the Nuffield Foundation.     

The activator of cerebroside sulphatase. Lysosomal localization.

1) An activator protein necessary for the enzymic hydrolysis of cerebroside sulphate could be partially purified from unfractionated rat liver. This activator, which is similar to that of human origin, proved to be a heat-stable, non-dialyzable, low molecular weight protein with an isoelectric point of 4.1. Its activity could be destroyed by pronase. 2) For elucidation of the subcellular localization of the activator, rat liver was fractionated by differential centrifugation. The intracellular distribution of the cerebroside sulphatase activator was compared to the distribution patterns of marker enzymes for different cell organelles and found to coincide with the lysosomal arylsulphatase, thus indicating a lysosomal localization. 3) This was confirmed using highly purified secondary, i.e. iron-loaded, lysosomes. After disruption by osmotic shock, these organelles hydrolyzed cerebroside sulphate when incubations were performed under physiological conditions with endogenous as well as exogenous sulphatase A as enzyme. 4) After subfractionation of the disrupted secondary lysosomes into membrane and lysosol fractions by high speed centrifugation, it was found that the activator protein was exclusively associated with the lysosol, whereas the acid hydrolases were distributed differently between the two fractions. 5) The lysosol was further fractionated by semi-preparative electrophoresis on polyacrylamide gels. Two protein fractions were obtained: a high molecular weight fraction, containing the activator-free acid hydrolases, and a low molecular weight fraction, containing the enzyme-free activator of cerebroside sulphatase. 6) The significance of these findings for the hydrolysis of sphingolipids in the lysosomes is discussed.     

Studies of the oestrogen sulphatase and arylsulphatase C activities of rat liver

Detailed studies on the hydrolysis of p-acetylphenyl sulphate and oestrone sulphate by rat liver preparations strongly indicate that arylsulphatase C and oestrogen sulphatase are the same enzyme. Liver is the richest source of both enzymes, which have identical intracellular distributions, being localized mainly in the microsomal fraction. Low oestrogen sulphatase and arylsulphatase C activities were present in foetal liver and these increased at a similar rate after birth. The activities of the enzymes in an ethionine-induced hepatoma were similarly low. Results of heat inactivation, mixed-substrate and competitive-inhibition experiments employing liver microsomal fractions were also consistent with one enzyme being involved. Oestradiol-17beta 3-sulphate was also hydrolysed by microsomal preparations and activity towards both this substrate and oestrone sulphate was inhibited by oestrone and oestradiol-17beta. The physiological significance of this inhibition is discussed.     

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.     

Lysosomal aryl sulphatase in pulmonary alveolar cells

Summary Lysosomal aryl sulphatase has been localised in the lung at the electron microscopic level using a nitrocatechol sulphate barium chloride medium. Variations in fixative concentration and incubation time were found to be important in minimising lysosomal leakage. The distribution of aryl sulphatase in the lung corresponded closely to that of acid phosphatase. Large amounts were found in alveolar macrophages and small quantities in the type II alveolar epithelial cell. In the latter cell the enzyme was found in the lamellar vacuoles thought to represent the site of surfactant production. The significance of this in regard to the function of these organelles is discussed.     

Purification and properties of N-acetylgalactosamine 6-sulphate sulphatase from human placenta

1. N-Acetylgalactosamine 6-sulphate sulphatase was purified about 20000-fold from the soluble extract of human placenta with N-acetylgalactosamine 6-sulphate-glucuronic acid-N-acetyl[1-(3)H]galactosaminitol 6-sulphate as substrate in the activity assay. The enzyme appears to be a glycoprotein with a mol.wt. of about 100000 as determined by gel filtration. On gel electrophoresis in the presence of sodium dodecyl sulphate the major protein band had a mol.wt. of 78000. Variable charge heterogeneity was observed in several enzyme preparations. 2. The purified enzyme released up to one sulphate molecule from the disulphated trisaccharide. It was active towards N-acetylgalactosamine 6-sulphate and exhibited no measurable N-acetylglucosamine 6-sulphate sulphatase or any other known lysosomal sulphatase activity. Hydrolysis of [1-(3)H]galactitol 6-sulphate was achieved by incubation neither with a crude nor with a purified enzyme preparation. Chondroitin 6-sulphate and keratan sulphate, as well as heparin and heparan sulphate, served as competitive inhibitors of the enzyme. 3. Purified N-acetylgalactosamine 6-sulphate sulphatase activity was optimal at pH4.9 and 4.4 when assayed in 0.02m-sodium acetate buffer and at pH4.2 and 5.2 in 0.1m-sodium acetate buffer. A single pH-optimum at pH4.8 was observed for the crude enzyme and for the purified enzyme after mild periodate treatment. The sulphatase activity was inhibited by a variety of anions and cations and activated by thiol-specific and thiol reagents.     

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.     

Purification of steroid sulphohydrolase from human placenta microsomes

A procedure for purification of oestrone sulphate sulphohydrolase from human placenta microsomes was elaborated. The use of Concanavalin-A-Sepharose chromatography made it possible to separate, for the first time, oestrone sulphate sulphohydrolase (Mr 36,000, optimum pH 7.0, Km 5.5 X 10(-5) M, specific activity 1563 nmol X min-1 X mg protein-1) from arylsulphatase C (Mr 45,000, optimum pH 7.6, Km 0.96 X 10(-3) M). The observed third subfraction showed both arylsulphate C and oestrone sulphate sulphohydrolase activity. Sigmoidal kinetics of oestrone sulphate sulphohydrolase after DEAE-cellulose chromatography (Mr 130,000) points to the allosteric character of the enzyme.     

THE PATTERNS OF ARYLSULPHATASES A AND B IN HUMAN NORMAL AND METACHROMATIC LEUCODYSTROPHY TISSUES AND THEIR RELATIONSHIP TO THE CEREBROSIDE SULPHATASE ACTIVITY

Abstract— The arylsulphatase A and B patterns of human tissues and leucocytes have been established by isoelectric focussing. Assay conditions, which enable an evaluation of these patterns as quantitatively as possible, have been studied. The dependences of the enzyme patterns on the origin of the tissues and on the storage conditions have been determined. The arylsulphatase A obtained by isoelectric focussing exhibits cerebroside sulphatase activity in the presence of detergents. A purified preparation of the arylsulphatase B likewise shows a significant, although low, cerebroside sulphatase activity. In cases of the conventional types of metachromatic leucodystrophy the arylsulphatase A activity is missing, while in an atypical form of this disease ('ML Variant' according to A ustin et al . (1965) the arylsulphatase A, B and C activities are deficient. In both forms, however, residual activities of the deficient enzymes could be detected which showed isoelectric points identical to those of the normal enzymes.
The following nomenclature is proposed: 'Variant B' for the conventional type, in which the arylsulphatase B activity is present, and 'Variant O' for the exceptional cases, in which all arylsulphatase activities are deficient. The significance of the cerebroside sulphatase activity of arylsulphatase B for a possible residual turnover of cerebroside sulphates in the conventional type of the disease is discussed.     

Primary alkylsulphatase activities of the detergent-degrading bacterium Pseudomonas C12B. Purification and properties of the P1 enzyme.

The P1 primary alkylsulphatase of Pseudomonas C12B was purified 1500-fold to homogeneity by a combination of streptomycin sulphate precipitation of nucleic acids, (NH4)2SO4 fractionation and chromatography on columns of DEAE-cellulose, Sephacryl S-300 and butyl-agarose. The protein was tetrameric with an Mr of 181000-193000, and exhibited maximum activity at pH 6.1. Primary alkyl sulphates of carbon-chain length C1-C5 or above C14 were not substrates, but the intermediate homologues were shown to be substrates, either by direct assay (C6-C9 and C12) or by gel zymography (C10, C11, C13 and C14). Increasing the chain length from C6 to C12 led to diminishing Km. Values of delta G0' for binding substrates to enzyme were dependent linearly on chain length, indicating high dependence on hydrophobic interactions. Vmax./Km values increased with increasing chain length. Inhibition by alk-2-yl sulphates and alkane-sulphonates was competitive and showed a similar dependence on hydrophobic binding. The P1 enzyme was active towards several aryl sulphates, including o-, m- and p-chlorophenyl sulphates, 2,4-dichlorophenyl sulphate, o-, m- and p-methoxyphenyl sulphates, m- and p-hydroxyphenyl sulphates and p-nitrophenyl sulphate, but excluding bis-(p-nitrophenyl) sulphate and the O-sulphate esters of tyrosine, nitrocatechol and phenol. The arylsulphatase activity was weak compared with alkylsulphatase activity, and it was distinguishable from the de-repressible arylsulphatase activity of Pseudomonas C12B reported previously. Comparison of the P1 enzyme with the inducible P2 alkylsulphatase of this organism, and with the Crag herbicide sulphatase of Pseudomonas putida, showed that, although there are certain similarities between any two of the three enzymes, very few properties are common to all three.     

Purification and properties of arylsulphatase A from chicken brain

1. Chicken brain arylsulphatase A was purified 2000-fold, with overall recovery 14%, by using ammonium sulphate fractionation, ethanol precipitation, Sephadex G-200 gel filtration and DEAE-Sephadex column chromatography. 2. The purified preparation was free from beta-glucuronidase, beta-galactosidase, acid phosphatase, inorganic pyrophosphatase and adenosine 3'-phosphate 5'-sulphatophosphate sulphohydrolase activities. 3. Polyacrylamide-gel electrophoresis indicated that the purified preparation was not homogeneous. 4. Chicken brain arylsulphatase was markedly inhibited by carbonyl reagents in the presence of traces of Cu(2+) in the system. Other metal ions such as Fe(2+) and Zn(2+), were inactive. 5. Ascorbic acid alone had no effect on enzyme activity but enhances the inhibition by Cu(2+). 6. Chicken brain arylsulphatase A resembled arylsulphatase A of other animal species in its kinetic properties such as K(m) value, anomalous time-activity relationship and the inhibitory effect of phosphate, sulphite and sulphate ions. However, its electrophoretic mobility, behaviour under zinc acetate fractionation and stimulation by Ag(+) were similar to arylsulphatase B of other animal species. Thus, this enzyme did not correspond to either arylsulphatase A or arylsulphatase B but properties of both. 7. The purified enzyme preparation can degrade cerebroside 3-sulphate.