The sulphatase of ox liver

366

Biochimica et Biophysica Acta, 704 (1982) 366-373 Elsevier Biomedical Press

BBA31174

THE SULPHATASE OF OX LIVER XXIV. THE GLYCOSULPHATASE ACTIVITY OF SULPHATASE A A.B. ROY and JENNIFER TURNER

Department of Physical Biochemistry, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra City, A.C T. 2601 (Australia) (Received September 9th, 198 I) (Revised manuscript received December 21st, 1981)

Key words: Sulfatase A; Glycosulfatase; (Ox liver)

The rhodizonic acid method for the determination of SO42- 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-2deoxyglucopyranose 6-sulphate is not hydmlysed, nor is 2,3,4,6-tetra-O-acetyl-/3-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 sulphatas~ A, although this has not been isolated. Sulphatase A has no detectable alkylsulphatase activity.

Introduction In 1933 Soda and Egami [1] showed that the arylsulphatase (aryl-sulphate sulphohydrolase, EC 3.1.6.1) and glycosulphatase (sugar-sulphate sulphohydrolase, EC 3.1.6.3) activities in extracts of mollusc tissues were apparently due to distinct enzymes. On the other hand, more recent work [2] has shown that a purified arylsulphatase from the mollusc Charonia lampas still shows glycosulphatase activity. The relationship between these two activities, or enzymes, therefore remains obscure, even in molluscs which are the classical sources of glycosulphatases [1]. A similar situation holds in mammalian tissues. The sulphatase A of ox river has long been known [3] as an arylsulphatase, yet the purified enzyme is also a cerebroside sulphatase [4], hydrolysing the galactose 3-sulphate residues of cerebroside sulphate, and so is a type of 016%4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

glycosulphatase. This enzyme has also been shown to hydrolyse ascorbate 2-sulphate [5] and some monosaccharide sulphates [6]. The purified sulphatases A of pig kidney [7] and rabbit river [8] als0 show glycosulphatase activity, hydrolysing galactose 3-sulphate and glucose 6-sulphate, respectively, as well as cerebroside sulphate. A more detailed study of the glycosulphatase activity of sulphatase A was required to provide information about, first, its specificity, second, the rather striking kinetic differences between the arylsulphatase and cerebroside sulphatase activities of sulphatase A [9,10], and third, the bond sprit during the glycosulphatase reaction. When functioning as an arylsulphatase, sulphatase A attacks the O-S bond of its substrate [11], but the assumption that this will also be the case with its glycosulphatase activity could be unwise because the latter is essentially a type of alkylsulphatase

367 activity and microbial alkylsulphatases attack not the O-S bond but the C-O bond of their substrates [ 12]. This could be a reflection of the very different properties of the C-O bonds in alcohols and phenols rather than of enzyme specificity. The present paper describes an investigation of the glycosulphatase activity of sulphatase A.

Experimental Monosaccharide sulphates. The synthesis and characterisation of the potassium D-glucopyranose and D-galactopyranose sulphates used in this work have already been described [13]. They were analysed for SO42- immediately before use and, if necessary, were treated with the requisite amount of Ba2+ before being used as substrates. Sulphatase A. Homogeneous preparations of this enzyme were obtained from ox liver by minor modifications of the method already published [14]. Sulphate determination. A modification of the methods of Ginsberg and Di Ferrante [15] and of Waheed and Van Etten [16] was used. To 0.25 ml of a solution of SO2- (0-60 nmol) in 0.2 M pyridine-acetic acid buffer was added 1 mi acid BaC12 reagent then, after mixing and standing for 10 rnin, 2 ml rhodizonic acid reagent were added. After 20 min the absorbance of the solution at 520 nm was measured, with water as a blank. A calibration curve was prepared with each set of determinations. The acid BaC12 reagent was 75 /~M BaCl 2 in 80% (v/v) ethanol containing sufficient acetic acid to lower the pH of the pyridine/acetic acid buffer to approximately 4: for buffers of pH 4.5 and 5.6 the reagents contained 0.35 rnl and 0.55 ml, respectively, of acetic acid per 100 mi. When larger or smaller amounts of SO2- were to be determined the concentration of BaC12 in the reagent was adjusted appropriately. The rhodizonic acid reagent was prepared as described by Waheed and Van Etten [16], except that the concentrations of sodium rhodizonate and ascorbic acid were 4 mg and 75 mg, respectively, per 100 ml of 80% (v/v) ethanol. It was kept overnight in the dark before use and discarded when 24 h old. Enzyme assays. Routine assays of arylsulphatase activity with nitrocatechol sulphate as substrate were carried out in a pH-stat (assembly

P H M 2 6 - T T I 1-SBR2-ABU 12; Radiometer, Copenhagen) as previously described [9], as were the determinations of substrate-modified sulphatase A [17]. Some assays of the glycosulphatase activity of sulphatase A were similarly carried out and others were made in a pH-stat assembly with a modified micro-electrode vessel (TTA31; Radiometer, Copenhagen) so that only 1 ml of substrate solution was required. In all cases the ionic strength was kept at 0.1 with KCI, the temperature was 37°C and v0 was computed from progress curves recorded during the first 3 rain of the reaction [9]. In most cases the glycosulphatase activity was followed by measuring the m o u n t of SO42 liberated in 5 min at 37°C in 0.2 M pyridine/acetic acid buffers, pH 4.5 or 5.6. The enzyme concentrations ranged from 1.5 to 200 ptg/ml. The reaction was stopped by heating the solution for 2 rain in a boiling water bath. After cooling, 1 ml of the appropriate acid BaC12 reagent was added. If the protein concentration in the incubation mixture was less than about 25/tg/ml the amount of SO2was determined exactly as described above by adding 2 ml rhodizonic acid reagent. At higher protein concentrations the reaction mixture was centrifuged for 10 min at 2500 rev./min to remove the precipitated protein, 0.5 ml of the clear supernatant was carefully removed and to this was added 0.8 ml rhodizonic acid reagent. Assays and appropriate controls were always carried out in duplicate. Hydrolysis in H~80. One ml of a reaction mixture containing 0.1 mmol potassium glucose 3sulphate, 0.5 mmol sodium acetate/acetic acid buffer, pH 5.6, 0.2 mg sulphatase A and 29 atom% excess H[sO was incubated in a sealed ampoule for 24h at 37°C. Determination of SO2- showed that the hydrolysis of the glucose 3-sulphate was about 97% complete. The liberated SO2- was precipitated as BaSO4 which was then collected and washed as described by Tudball and Thomas [18]. The percentage of BaSO]SO which it contained was measured by infrared spectrometry [18,19]. Chromatography of sugars. Hexoses were separated by the method of Hansen [20] on thin-layer plates of silica gel (Silica gel 60, precoated plates, Merck, Darmstadt) impregnated with NaH2PO 4. The solvent system was propan-2-ol/acetone/

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0.1 M lactic acid (4:4:2, v / v ) and sugars were located by spraying with a diphenylamine reagent [20]. As previously reported [20], a good separation of galactose, glucose and mannose (Rf 0.29, 0.41 and 0.51, respectively) was obtained. Allose was inseparable from glucose under the above conditions.

TABLE I STABILITY OF OX LIVER SULPHATASE A IN 64% ETHANOL To 0.25 ml of a solution of sulphatase A (100 #g/ml) in 0.2 M pyridine-aceticacid buffer, pH 5.6, was added i ml acid BaCI2 reagent (to give a pH of about 4) and the mixture allowed to stand at room temperature. At intervals 25-/~i samples were taken for the spectrophotometricdetermination of sulphatase A activity with nitrocatecholsulphate as substrate.

Results

Determination of sulphate The method described above was found to be satisfactory: 12 replicate assays on a single solution gave a value (mean -+ S.D.) of 29.1 -+ 0.7 nmol SO2 - . There were significant day-to-day variations in the calibration curves, and occasional values were obviously aberrant. There were considerable differences between samples of sodium rhodizonate. Of the three available only one (that used in the present work) gave a solution which, in agreement with the observations of Ginsberg and Di Ferrante [15], required aging. Another sample did not require aging, as found by Waheed and Van Etten [16], while a third gave no colour with Ba 2+ . Carbohydrate sulphates, and at least some aryl sulphates, interfered with the determination of SO2 - by rapidly reacting with the rhodizonlc acid reagent (in the presence or absence of Ba 2+) to give a yellow colour (Xm~ 480 nm) which increased in intensity for about 1 h before slowly fading. The absorbance at 480 nm varied with the sulphate ester and the p H of the reaction mixture, and with 25 #tool ester, corresponding to a substrate concentration of 0.1 M in the assay described above, ranged from 0.1 to 0.6. Only with glucose 2-snlphate was the absorbance greater at p H 4.5 than at pH 5.6, so that the previously described methods [15,16] were modified, as described above, to drop the p H of the reaction mixture to about 4 before the rhodizonic acid reagent was added. This decreased, but did not eliminate, the interference from sulphate esters.

Time (h)

Activity (# tool. rag- I. min- i)

0.03 2 4 6 8 24

88 70 57 47 45 27

fiver. In preliminary experiments it was noted that the apparent specific activity of this enzyme, based on the amount of SO2 - produced, varied with the length of time between the addition of the acid BaC12 reagent (without prior heating to denature the enzyme) and completing the determination of SO2 - . This was traced to the fact that ox fiver sulphatase A is remarkably stable in ethanol, as shown in Table I. Even after 24 h in 64~ ethanol the enzyme activity had dropped only by about 70~. In 86~ ethanol about 60~ inactivation occurred in 5 h. On the other hand, heating the enzyme solution in a boiling water bath gave rapid and complete inactivation, more than 97~ inactivation occuring within 1 rain, and this procedure was therefore adopted in the standard assay described above. The method was quite precise. 20 replicate assays in 0.05 M glucose 3-sulphate at p H 5.6 gave a specific activity of 14.8 -+ 1.4 # m o l . m g - I . rain- i: discarding three apparently low values (12.3, 10.9 and 12.1) improved the precision to give a value of 15.2 -+0.8 # m o l . mg - I . min -m.

Enzyme assay

Uptake of H ÷ on dissolution of monosaccharide sulphates

Waheed and Van Etten [16] reported that the sulphatase A of rabbit liver was completely and rapidly inactivated in 70~ ethanol. Surprisingly, this was not the case with the sulphatase A of ox

When potassium glucose 3- and 6-sulphates or galactose 2- and 6-sulphates were dissolved to give a 0.1 M solution in 0.5 mM sodium acetate, taken to 37°C and the p H adjusted to 5.6, there was a

369 41 []

o- 002

3--

.~_

~ 2

- D.01 ~ .c_

U

o

0

I

I

I

2

Ic~o

3

Time (h)

Fig. 1. The uptake of H + and change in optical rotation when 0.035 M potassium glucose 3-sulphate is dissolved at pH 5.6 and 37°C. The uptake of H + was measured in a pH-stat in 0.065 M KC1/0.5 mM acetate (. ), the change in optical rotation in 0.055 M KCI/0.01 M acetate at 579.1 nm (0).

rapid increase in pH which continued for some time but eventually ceased. More detailed studies were made only with glucose 3- and 6-sulphates and the results with the former are shown in Fig. 1. There was an initial rapid uptake of H + which stopped after about 2 h when some 3.5 /~mol H + had been consumed,

compared with the 350/~mol glucose 3-sulphate in the reaction mixture. The reaction had a tit 2 of about 25 rain. There was a simultaneous, but small, change in the optical rotation of the solution (measured in a 1 dm cell in a Polarimeter 241 MC, Perkin-Elmer, Oberlingen). With glucose 6sulphate the uptake of H + was similar to that shown in Fig. 1, with a tl/2 of about 14 rain, but the change in rotation was smaller, amounting to only +0.014 ° in 3h. Whatever the explanation of these effects, when the hydrolysis of a monosaccharide sulphate was to be followed in the pH-stat the substrate solution was kept at the appropriate pH and temperature until the uptake of H + had ceased.

Glycosulphatase activity The pH optimum for the hydrolysis of 0.1 M potassium glucose 3-sulphate by sulphatase A was about 5.3 when measured by the amount of hydrolysis occurring during 5 rain in 0.2 M pyridine/acetic acid buffers and about 5.6 when measured from v 0 determined in the pH-stat. These values are similar to those for the hydrolysis of, for example, nitroquinol sulphate by snlphatase A [9]. Sulphatase A hydrolysed all the simple Oglucopyranose and D-galactopyranose sulphates tested, and in every case the rate of hydrolysis was

TABLE II KINETIC CONSTANTS FOR THE HYDROLYSIS OF MONOSACCHARIDE SULPHATES BY SULPHATASE A The values of v were measured at a substrate concentration of 0,1 M either by the amount of SO~- liberated during 5 vain or in the pH-stat where the values (in italics) are for v0: the reaction conditions were different in the two cases. The values of K m and V were computed by the method of Wilkinson [21]: the latter, and v, are given in units of p mol. rag-I, rain-I. pH 4.5

pH5.6

V

V

Glucose 2-sulphate Glucose 3-sulphate

1.2 12 8.0

Glucose 4-sulphate Glucose 6-sulphate

0.84 1.5

Galactose 2-sulphate Galaetose 3-sulphate

0.15 2.5

1.2 23

83--- 7 1 1 0 - 18

27

110 + -- 6

1.3 4.0

51 -- 9 ~ ~. 100

2.2

> >"

0.20 3.9 2.6

Galactose 4-sulphate Galactose 6-sulphate

0.15 0.23

K m (mM)

0.21 0.40

V 2.3 50 58 2,0

---0.I ---5 -- 2 ±0.2

100

190- 28 9 0 - 12

0.57 ----0.08

55 + 12

3,9 +---0.4 0.44±0.04 0.79±0.08

1004-14 97---+15

6.0 - 0 . 7

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greater at pH 5.6 than at pH 4.5. Some kinetic parameters arc shown in Table II. Values for Kin and V are given only at pH 5.6 because previous work [5,9] had shown that the kinetics of sulphatase A are non-Michaelis at pH 4.5. The values of v measured by the amount of SO2- produced during 5 rain were not sufficiently accurate to show whether or not the double-reciprocal plots were non-linear at the latter pH with monosaccharide sulphates as substrates, as they are with aryl sulphates, but with glucose 3-sulphate, where v0 was measured in the pH-stat, the non-linear double-reciprocal plots were obvious at pH 4.5. Nevertheless, from the plots of v against substrate concentration it appeared that in all cases the enzyme was saturated by lower concentrations of substrate at pH 4.5 than at pH 5.6. Neither 2-acetamido-2-deoxyglucopyranose 6sulphate nor 2,3,4,6 - tetra- O - acetyl- fl- D glucopyranose 1-sulphate were hydrolysed at a detectable rate by sulphatase A at either pH 4.5 or 5.6. With the former compound the rate of any hydrolysis must have been at least an order of magnitude less than the lower values in Table II, but the situation with the 1-sulphate is less certain because the high rate of non-enzymic hydrolysis of this ester made it difficult to detect any slight enzyme activity. The hydrolysis of glucose 3-sulphate by sulphatase A was competitively inhibited by SO2- : the value of K i, from measurements of v0 in the pH-stat, was 1.4 raM, similar to that previously found for the hydrolysis of nitrocatechol sulphate [9]. Glucose had no inhibitory effect at a concentration of 0.1 M: it likewise had no effect on the arylsulphatase activity of sulphatase A.

Sugars produced by glucosulphatase action Glucose 2-sulphate and glucose 4-sulphate (0.12 M) in 0.4 M pyridine/acetic acid buffer, pH 5.6, were hydrolysed for 18h at 37°C with sulphatase A (40 #g/ml) and 5-#1 samples of the hydrolysates applied, along with appropriate controls and standards, to phosphate-impregnated silica-gel plates, as described above. In both cases the only sugar produced was glucose. If the glycosulphatase reaction had involved a stereospecific fission of the C-O bond with inversion of configuration, as in the alkylsulphatase reaction [12],

then the products would have been mannose and galactose, respectively, and these would have been readily detected in the chromatographic system used [20], as was confirmed by the addition of these sugars to samples of the hydrolysates prior to chromatography.

Hydrolysis m H 180 The SO2- formed during the hydrolysis of glucose 3-sulphate in 295g H~sO was isolated as BaSO4, as described above. Infrared spectrophotometry showed a peak at 961 cm -t due to BaSO~SO and measurements of peak heights showed that this was present to the extent of about 35% of the total BaSO4, clearly demonstrating fission of the O-S bond during the hydrolysis of glucose 3-sulphate by sulphatase A.

Formation of substrate-modified sulphatase A As shown in Fig. 2, the observed rate of hydrolysis of glucose 3-sulphate by sulphatase A at pH 5.6 decreased more rapidly than predicted from the observed values of K m and of the K i for SO2 - . This suggested that a substrate-modified form of sulphatase A was formed during the glycosulpha-

I

I

20

I

I

40

60 Time (rain) Fig. 2. Progress curves from pH-stat recordings of the hydrolysis of 0.05 M glucose 3-sulphate at pH 5.6, 37°C, with the ionic strength adjusted to 0.1 with KCI or BaC12 as indicated. Enzyme concentration, 10 ~g/ml. The shaded area includes computed progress curves for normal enzyme reactions competitively inhibited by one of the reaction products ( S O ~ - ) with K m 0.114-0.011 M glucose 3-sulphate and K i 1.4+-0.14 mM SO42-. The dotted line shows a computed curve for a reaction in which the enzyme was undergoing a substrate-induced modification, as with nitrocatechol sulphate [9], using values of 0.165 mmol.1 - I and 0.16 rain - l for vo and k*, respectively.

371

tase reaction, as it is during the arylsulphatase reaction [17]. On the other hand, as is shown in Fig. 2, even in the presence of Ba2+ to remove the SO~- produced during the reaction, the velocity did not fall as rapidly as predicted from the values of v0 and k*, the apparent velocity constant for the substrate-induced modification computed from the early stages of the reaction [9]. The fact that the activity falls more rapidly in the presence of Ba2+ , that is, in the absence of SO~-, shows that the accumulation of the latter is not responsible for the drop in activity. The high residual activity in the presence of Ba2+ was not due to the accumulation of the other reaction product, glucose, because the addition of 2 mM glucose to such a reaction mixture, 60 min after the start of the reaction, did not give any increase in activity. Comparison of the observed progress curve in Fig. 2 with the theoretical one, allowing for the inhibition by SO~-, showed that after 60 min the velocity had fallen by about 36~: this suggested that the enzyme isolated from such a reactioir mixture should contain about 36~ of substratemodified sulphatase A. Nevertheless, experiments carried out under a variety of conditions all failed to detect any substrate-modified enzyme in the sulphatase A isolated from such reaction mixtures by the chromatographic techniques used previously [17]. For example, in the experiment shown in Fig. 2 (in KC1), 102 pg enzyme were present and the expected recovery after chromatography of the reaction mixture was about 75 pg: the enzyme recovered amounted to 63 pg and contained no detectable substrate-modified sulphatase A.

Possible alkylsulphatase activity When tested under the same general conditions as used for the determination of glycosulphatase activity, but with the incubation time increased to 15 rain, sulphatase A (0.2 mg/ml) did not hydrolyse at a detectable rate, at either pH 4.5 or 5.6, potassium ethyl sulphate (0.1 M) potassium octan1-yl sulphate (20 mM), potassium DL-octan-2-yl sulphate (8 mM) or potassium nonan-5-yl sulphate

(2o mM). Discussion

The method of sulphate determination, based on those previously described [15,16], was satisfac-

tory but the day-to-day variation in the calibration curves was inconvenient. The source of this variation was not identified but a contributing factor must be the changes which occur during the aging of rhodizonate solutions, which presumably involves the complex oxidation-reduction reactions of this compound [22]. A minor disadvantage is the differences between samples of sodium rhodizonate which means that a thorough check of the method is necessary with each batch of reagent. Further problems were caused by the yellow colour which developed when at least some sulphate esters reacted with the rhodizonic acid reagent, but this could be minimized by keeping the pH of the reaction mixture about 4. Despite these limitations the method provided a useful assay for the glycosulphatase activity of sulphatase A and the only important disadvantage was that it could not be used to obtain progress curves for the first few minutes of the reaction because the inability to rapidly denature the enzyme made difficult the precise control of incubation time. For this reason, the pH-stat was used to obtain accurate kinetic data for the more rapidly hydrolysed substrates. The latter method, in its standard form with a reaction volume of 10 ml, is not useful for routine assays with pure monosaccharide sulphates which can only be prepared by tedious methods [13] and must be used in concentrations of at least 0.1 M (Table II). The rise in pH which occurs when at least some monosaccharide sulphates are dissolved in dilute buffer at pH 5.6 does not appear to have been noted previously. In the case of potassium glucose 3- and 6-sulphates, the only esters examined in some detail, the uptake of H + was accompanied by a small increase in the optical rotation. It should be noted that the mutarotation of glucose is associated with a pH change because t-D-glucose is a stronger acid than a-D-glucose, the apparent pK values at 25°C being 12.20 and 12.49, respectively [23]. However, although the pK values of glucose sulphates are likely to be less than those of glucose, it is unlikely that they would be sufficiently small to account for an uptake of H + of the magnitude shown in Fig. 1. The explanation of the uptake of H + on the dissolution of at least some monosaccharide sulphates must therefore remain in doubt. If it is associated with mutarota-

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tion then its study must await the separation of the different anomeric forms. All the simple glucose and galactose sulphates tested were hydrolysed by sulphatase A, although at considerably different rates and with different values of K m, as summarized in Table II. Quite apart from the problems introduced by using values of v obtained from the amount of SO~liberated during 5 min where reactions are not of zero order, the values of K m and V are in many cases of limited significance because the highest substrate concentration used, 0.1 M, was obviously less than Km. In both the glucose and galactose series the 3-sulphates were hydrolysed considerably more rapidly than the other sulphates, both at pH 4.5 and 5.6. It was somewhat surprising that the glucose sulphates were aU hydrolysed considerably more rapidly than the corresponding galactose sulphates because, as far as is known, the physiological substrates for sulphatase A are sulpholipids containing galactose 3-sulphate residues. Although glucocerebrosides are well known [24], there is no evidence for the existence of the corresponding sulphates and glucocerebrosides are apparently not substrates for cerebroside sulphotransferase [25]. This wide specificity of sulphatase A in vitro suggests that in vivo the specificity may be governed not only by the structure of the carbohydrate sulphate but also by the adjacent part of the molecule, a. situation which certainly pertains with other sulphatases [26]. In all cases the hydrolysis of the monosaccharide sulphates was more rapid at pH 5.6 than at pH 4.5 and certainly with glucose 3-sulphate, the only ester investigated in detail, the pH optimum (from measurements of %) was 5.6, the same as that for the arylsulphatase activity [9]: this is in sharp contrast to the cerebroside sulphatase activity, which has an optimum pH of about 4.5 [4], or to the hydrolysis of ascorbate 2-sulphate, which has an optimum pH of about 4.7 [5]. Sulphate is a competitive inhibitor (Ki, 1.4 mM) of the glycosulphatase activity of sulphatase A, as it is of the arylsulphatase activity, again in contrast to the cerebroside sulphatase activity, where the inhibition by SO~- is noncompetitive. Like the arylsulphatase activity, the glycosulphatase activity of sulphatase A is accompanied by the formation of a substrate-modified enzyme,

as is shown in Fig. 2. The inactivation does not go as nearly to completion as it does with the arylsulphatase activity, and the modified enzyme has not been isolated. The reason for both these affects may be the relative values of K m and g i for SO~-. For glucose 3-sulphate the ratio of K m / / K i is about 100, while for nitrocatechol sulphate, the substrate normally used for the preparation of substrate-modified enzyme [17], it is about 1. As the formation of the substrate-modified enzyme involves complex equilibria between the enzyme, substrate and products, [17] the magnitude of the ratio of K m / / K i is likely to be important. However, the data in Fig. 2 clearly show that the rate of hydrolysis of glucose 3-sulphate by sulphatase A falls much more rapidly than can be explained by the disapperance of substrate or the accumulation of SO~-. Further, the fact that the decrease in rate is even greater in the presence of Ba2+ is consistent with formation of a substrate-modified form of sulphatase A which is activated by SO42-. The glycosulphatase and arylsulphatase activities are also similar in that it is the O-S bond of the substrate which is split so that the sugar produced through the glycosulphatase activity of sulphatase A is that present in the substrate. It seems reasonable to assume that it will also be the O-S bond which is split when sulphatase A is functioning as a cerebroside sulphatase, but the relative insolubility of cerebroside sulphate, its low rate of hydrolysis and the complex reaction mixture required to detect cerebroside sulphatase activity [4] combine to make experimental proof difficult. Its action in attacking the O-S bond of its substrates dearly distinguishes sulphatase A from the alkylsulphatases which attack the C-O bond of their substrates [12,27,28] and indeed, as pointed out above, sulphatase A shows no alkylsulphatase activity, although carbohydrate sulphates can be regarded as substituted alkyl sulphates: so also can steroid sulphates, and it may be pertinent that their enzymic hydrolysis also involves fission of the O-S bond [29]. This obviously throws some doubts on the validity of the present classification of the sulphatases [30], doubts which are reinforced when it is recalled that other 'arylsulphatases' - such as the sulphatases B of ox liver [31] or of hen oviduct [32], or the arylsulphatase of Charonia lampas [2] - also show some glyco-

373

sulphatase activity. On the other hand, not all arylsulphatases have been shown to be glycosulphatases and there are apparently glycosulphatases which are devoid of arylsulphatase activity [33]. Acknowledgement We are indebted to Professor K.S. Dodgson for the gift of several alkyl sulphates. References 1 Soda, T. and Egami, F. (1933) Bull. Chem. Soc. Japan 8, 148-160 2 Hatanaka, H., Ogawa, Y. and Egami, F. (1976) Biochem. J. 159, 445-448 3 Roy, A.B. (1953) Biochem. J. 55, 653-651 4 Jerry, A. and Roy, A.B. (1973) Biochim. Biophys. Acta 293, 178-190 5 Roy, A.B. (1975) Biochim. Biophys. Acta 377, 356-363 6 Roy, A.B. (1980) Ciba Syrup. (New Series) 72, 177-190 7 Mehl, E. and Jatzkewitz, H. (1968) Biochim. Biophys. Acta 151,619-627 8 Waheed, A. and Van Etten, R.L. (1980) Biochim. Biophys. Acta 614, 92-101 9 Roy, A.B. (1978) Biochim. Biophys. Acta 526, 489-506 l0 Roy, A.B. (1979) Biochim. Biophys. Acta 568, 103-1 l0 II Spencer, B. (1958) Biochem. J. 69, 155-159 12 Bartholomew, B,, Dodgson, K.S., Matcham, G.W.J., Shaw, D.J. and White, G.F. (1977) Biochem. J. 165, 575-580 13 Archbald, P.J.,Fenn, M.D. and Roy, A.B. (1981) Carbohydr. Res. 93, 177-190

14 Nichoi, L.W. and Roy, A.B. (1964) J. Biochem., Tokyo 55, 643 -651 15 Ginsberg, L.C. and Di Ferrante, N. (1977) Biochem. Med. 17, 80-86 16 Waheed, A. and Van Etten, R.L. (1978) Anal. Biochem. 89, 550-560 17 Nicholls, R.G. and Roy, A.B. (1971) Biochim. Biophys. Acta 242, 141-151 18 Tudball,N. and Thomas, P. (I972) Biochem. J. 126, 187-191 19 Spencer, B. (1959) Biochem. J. 73, 442-447 20 Hansen, S.A. (1975) J. Chromatog. 107, 224-226 21 Wilkinson, G.N. (1961) Biochem. J. 80, 324-332 22 Sehwarzenbach, G. and Suter, H. (1941) Helv. Chim. Acta 24, 617-638 23 Los, J.M. and Simpson, L.B. (1954) Rec. Trav. Chim. Pays-Bas 73, 941-958 24 Martensson, E. (1967) Prog. Chem. Fats Lipids I0, 365-407 25 Farrell, D.F. and McKhann, G.M. (1971) J. Biol. Chem. 246, 4694-4702 26 Hopwood, J.J.and EIIiott,H. (1981) Clin. Claim. Acta I12, 55-66 27 Cloves, J.M., Dodgson, K.S., Games, D.E., Shaw, D.J. and White, G.F. (1977) Biochem. J, 167, 843-846 28 Shaw, D.J.,Dodgson, K.S. and White, G.F. (1980) Biochem. J. 187, 181-196 29 Logan, G.G. and Warren, J.C. (1969) Biochem. J. 114, 707 30 Enzyme Nomenclature (1979) pp. 254-257, Academic Press, New York 31 Farooqui, A.A. and Roy, A.B. (1976) Biochim. Biophys. Acta 452, 431-439 32 Tsuji, M., Nakanishi, Y., Habuchi, H., Ishihara, K. and Suzuki, S. (1980) Biochim. Biophys. Acta 612, 373-383 33 Hatanaka, H., Ogawa, Y. and Egami, F. (1976) J. Biochem., Tokyo 79, 27-34