Comparative studies of rodent anionic arylsulfatases

Comp. Biochem. Physiol. Vol. 82B, No. 1, pp. 55-61, 1985 Printed in Great Britain

0305-0491/85 $3.00+0.00 © 1985PergamonPress Ltd

COMPARATIVE STUDIES OF RODENT ANIONIC ARYLSULFATASES D. BRUCE THOMPSON,* WILLIAM L. DANIEL* and JANET H. GLASER'~ *Department of Genetics and Development, University of Illinois, Urbana, IL 61801, USA; and tDepartment of Biochemistry, University of Illinois, Urbana, IL 61801, USA (Received 6 February 1985) A~tract--1. Approximately 25 and 40~, respectively, of murine (Mus musculus) and rat (Rattus norvegicus) hepatic arylsulfatase (EC 3.1.6.l) activity eluted from DEAE-ion exchange resins under high salt conditions. 2. This high salt fraction contained arylsulfatase A and an enzyme which was immunologically similar to arylsulfatase B. 3. The latter enzyme was thermostable, resistant to inhibition by silver, completely inhibited by phosphate, displayed linear kinetics, and had a higher pH optimum than arylsulfatase A. 4. Anionic arylsulfatase B also hydrolyzed chondroitin-4-SO4 heptasaccharide. 5. Sephacryl S-300 gel filtration resolved anionic arylsulfatase B into 55 and 115 kd fractions. 6. Rodent arylsulfatase A activity was grossly underestimated when 4-methyl-umbelliferylsulfate was employed as substrate.

INTRODUCTION

detected in murine (Harrison et al., 1982) and human brain (Stevens et al., 1977) which have weak affinities for DEAE-cellulose and isoelectric points intermediate with respect to arylsulfatase A and cationic arylsulfatase B. The murine anionic isozymes were converted to the major cationic form by neuraminidase treatment (Harrison eta/., 1982). This charge heterogeneity suggests that more highly anionic species of arylsulfatase B may exist which coelute from ion exchange resins with arylsulfatase A. This report describes the isolation and characterization of anionic arylsulfatases from rodent liver and compares their substrate ranges.

Arylsulfatases (arylsulfate sulfohydrolase, EC 3.1.6.1) occur in a wide variety of organisms, but have been most extensively studied in mammals. These enzymes catalyze hydrolysis of arylsulfate esters ( R - O - S O ; + H20~-~ROH + H + + SO~-), and are primarily localized in the lysosomal (arylsulfatases A and B) or microsomal (arylsulfatase C) subcellular fractions (Roy, 1960; Rowden, 1967). Mammalian arylsulfatase A appears to function as a cerebroside sulfatase, whereas arylsulfatase B hydrolyzes N-acetylgalactosamine-4-sulfate ester bonds occurring in chondroitin-4-sulfate and dermatan sulfate in glycosaminoglycans of connective tissue (Mehl and Jatzkewitz, 1968; Gorham and Cantz, 1978). Arylsulfatase C may function as a steroid sulfatase in vivo (Burns, 1983). Although arylsulfatases A and B both hydrolyze p-nitrocatechol sulfate (pNCS), they differ with respect to a number of other properties. Arylsulfatase A has an acidic isoelectric point, is strongly inhibited by silver ion, and has a dimeric structure at neutral pH (Farooqui, 1980; Bleszynski and Leznicki, 1967). Arylsulfatase B has a basic isoelectric point, is resistant to silver inhibition, and is usually monomeric at neutral pH. Furthermore, arylsulfatase A is more thermolabile than arylsulfatase B (Lenzi and Daniel, 1981). Both arylsulfatase A and arylsulfatase B occur in multiple forms in certain tissues. Human arylsulfatase A can be separated into multiple isozymes by narrow range isoelectric focusing (Stevens et al., 1976). Part of this heterogeneity was due to differential sialylation. Anionic forms of arylsulfatase B have been

MATERIALS AND

METHODS

Materials SWR/J inbred mice (Mus musculus) were purchased from the Jackson Laboratory (Bar Harbor, ME), and Sprague-Dawley rats (Rattus norvegicus) were obtained from Holtzman (Madison, WI). Beef liver was supplied by local sources, pNCS and 4-methylumbelliferyl sulfate (4MUS) were obtained from Sigma Chemical Co. (St Louis, MO), and 4MUS was repurified by the method of Rinderknecht et al. (1970). Chondroitin-4-sulfate heptasaccharide was prepared and tritiated as previously described (Glaser and Conrad, 1979). Rabbit anti-murine kidney arylsulfatase B was prepared (Daniel et al., 1980) and purified by ammonium sulfate fractionation and DEAE-cellulose chromatography. Immobilized Clostridium perfringens neuraminidase Type VI was purchased from Sigma; and DEAESephacel, Sepharose S-300, and molecular weight standards were obtained from Pharmacia (Uppsala, Sweden). All other chemicals used for these experiments were reagent grade. Isolation of arylsulfatases ,4 and B All procedures were conducted at 4°C unless otherwise specified. Aqueous homogenates were prepared, sonicated, and centrifuged as previously reported, and arylsulfatases A and B were separated by DEAE-cellulose chromatography

*Abbreviations: C4S, chondroitin-4-SO4 heptasaccharide; 4MUS, 4-methylumbelliferyl sulfate; pNCS, p-nitrocatechol sulfate. 55

56

D. BRUCETHOMPSON et al.

using a 1.5 × 4 c m column (Daniel et al., 1982). Arylsulfatase activity was sequentially eluted with 8 ml of 20 mM Tris-acetate (pH 7.4), 7ml of 100mM Tris-acetate (pH 7.4), and 7 ml of 200 mM Tris-acetate, 0.4 M NaC1 (pH 7.4). The major portion of arylsulfatase B elutes with the void fraction, and arylsulfatase A elutes with the high salt fraction. The high salt eluate was saved for further analysis. Enzyme assays Arylsulfatase A activity was estimated using 10mM pNCS in 0.5 M sodium acetate-acetic acid buffer (pH 5.1) (Lenzi and Daniel, 1981). Arylsulfatase B activity was measured using either 10mM p N C S in acetate buffer (pH 5.9) (Daniel et aL, 1980) or 5 mM 4MUS in the same buffer. For the latter assay, 50/~1 of appropriately diluted enzyme and 100#1 substrate were incubated at 45°C for 10min. The reaction was stopped with 4ml of 0.08M glycine-sodium carbonate (pH 10), and the fluorescence of the released 4-methylumbelliferone was measured with a Turner 111 fluorometer using 7~50 primary and 4-8 secondary filters. Chondroitin-4-sulfate sulfatase activity was determined using 3H-labelled chondroitin-4-sulfate heptasaccharide as substrate. The reaction mixture containing 10/~1 0.5M acetate buffer (pH4.0), 10#1 substrate ( ~ 80,000 cpm, 10.5 × 106cpm//~mol), 10#1 of appropriately diluted enzyme, and 70 td H20 were incubated at 37°C for 1 hr; and the reaction was terminated by heating at 100°C for 10min. The reaction mixture was spotted on Whatman 3 paper, chromatographed for 46hr in nbutanol:glacial acetic acid: 1 M NH4OH (2:3:2.5), cut into strips, and counted using standard methods (Glaser and Conrad, 1979). Partial purification o f arylsulfatases A and B Ten percent (w/v) homogenates were prepared in 10 mM Tris-HCl buffer (pH 7.5) containing 0.05~o v/v Triton X100, sonicated 30 sec, and centrifuged at 20,000 g for 20 min. The supernatant was adjusted to 60~ saturation with solid (NH4)2SO4 and allowed to stand at 4°C overnight. In some experiments, arylsulfatase A was destroyed by heating prior to ammonium sulfate fractionation. The 20,000g supernatant was adjusted to pH 5.9 with 1 M acetic acid, heated at 50°C for 30 min, and centrifuged at 20,000g for 20 min. The supernatant was adjusted to pH 7 by dropwise addition of 2 M Tris prior to addition of (NH4)2SO4. The protein suspension was pelleted by centrifugation at 3000g for 15 min, and the pellet was resuspended in a solution containing 0.1 M Tris-HCl (pH 7.5), 0.15 M NaC1, 0.1~ (w/v) NaN 3, and 0.05~o (v/v) Triton X-100. The suspension was clarified by centrifugation at 20,000g for 20min, and the supernatant was dialyzed against 20 mM Tris-acetic acid (pH 7.4) overnight. The retentate was concentrated to 20 ml by pressure filtration and applied to a 25 x 1 cm column of DEAE-Sephacel pre-equilibrated with the 20 mM Tris-acetate buffer. The column was washed with 100 ml of the same buffer, and developed with a linear gradient formed from 100ml of 20mM Tris-acetate buffer and 100ml of 0.2M Tris-acetate buffer (pH7.4) containing 0.4 M NaCI. Two ml fractions were collected and monitored for protein (Lowry et al., 1951) and arylsulfatase activity using the pNCS assay. The bound fraction eluting at 0.2MNaC1 and 0.1 MTris was dialyzed against 20mM Tris-HC1 (pH 7.2) containing 0.1 M NaC1, concentrated to 5 ml, and applied to a 50 x 2.5 cm column of Sephacryl S-300. The arylsulfatases were eluted with 20 mM Tris-HC1, 0.1 M NaCI (pH 7.2), dialyzed against 20mM Tris-acetate (pH 7.4), and used for enzyme characterization. Enzyme characterization Immunotitration of arylsulfatase activities with rabbit anti-mouse arylsulfatase B IgG was performed as previously described (Daniel et al., 1982). Residual arylsulfatase activ-

ities in the supernatant following immunoprecipitation were measured using pNCS or 4MUS as substrates, pH optima of the pNCS- and 4MUS-sulfatase activities were estimated using 0.5 M sodium acetate-acetic acid buffers covering a pH 4.8 to 5.9 range. Linearity of these activities was followed at optimal pH and at 5 rain intervals. The effect of silver ion upon arylsulfatase activities was assessed by incubating enzyme with 0.2 mM AgNO 3 at 37°C for 15 min prior to enzyme assay (Lenzi and Daniel, 1981). Inhibition of arylsulfatase activity by other cations and anions was studied by incorporating the respective inhibitors in the reaction mixture at the specified concentrations. Thermal stabilities of arylsulfatases A and B were measured in 0.5 M sodium acetate--acetic acid buffer (pH 5.9) at 65°C (Daniel et al., 1982). Apparent molecular weights of DEAESephacel arylsulfatase fractions were estimated by Sephacryl S-300 filtration using 0.02 M Tris-HCl, 0.1 M NaC1 buffer (pH 7.2) employing rabbit muscle aldolase (158 kd), bovine serum albumin (67 kd), and ovalbumin (43 kd) as standards. Neuraminidase treatment Enzyme recovered in the 0.2MNaC1 fraction from DEAE-Sephacel was dialyzed overnight against 0.05 M sodium acetate--acetic acid buffer, pH 5. The retentate was concentrated to 10 ml by pressure filtration and split into 5 ml aliquots containing about 1 #mol/hr 4MUS-sulfatase activity. Five units of neuraminidase immobilized on beaded agarose were added to one aliquot, and both aliquots were incubated at 37°C for 16 hr with agitation according to the procedure of Farooqui and Srivastava (1979). The immobilized neuraminidase was pelleted by centrifugation, and the treated and untreated supernatants were dialyzed against 20 mM Tris-acetate (pH 7.4) at 4°C overnight. The retentates were rechromatographed on DEAE-Sephacel as described above, and arylsulfatase activities were located using 4MUS as substrate. RESULTS D E A E - c e l l u l o s e chromatography Batch elution o f hepatic arylsulfatases from DEAE-cellulose is presented in Table 1. The majority o f the rodent activity eluted with fraction I regardless o f whether p N C S or 4 M U S was used as substrate, although a larger p r o p o r t i o n o f 4MUS-sulfatase than p N C S - s u l f a t a s e activity occurred in this fraction. By contrast, the distribution o f bovine p N C S - and 4MUS-sulfatase activities between fractions I and III were strikingly different. M o r e than 80~o o f bovine p N C S - s u l f a t a s e activity occurred in fraction III, while 80~o o f the 4MUS-sulfatase activity was recov-

Table 1. Batch elution of hepatic arylsulfatasesfrom DEAE-cellulose Fraction II II1 (% of recoveredactivity) I

Substrate pNCS Mouse Rat Bovine 4MUS Mouse Rat Bovine

70 _ 3 60_+2 16 + 1

2 _+l <1 < 1

26 +_3 40_+2 82 -+ 2

88 + 2 84_+ 1 80-+2

2+ 1 <1 2-+1

9+ 2 16+2 18-+2

Activitiesare expressedas the mean and SE of three experiments. Fractions I (0.02 M Tris-aeetate); fraction II (0.1MTris-acetate); fraction I|I (0.2 M Tris-acetate; 0.4 M NaCl).

Rodent anionic arylsulfatase ered in fraction I. One gram of murine liver yielded 185+ 10/~mol/hr of pNCS-sulfatase activity and 17.4 + 2.0/~mol/hr of 4MUS-sulfatase activity. Corresponding yields for 1 g of rat and 1 g of bovine liver were 181 +20/tmol/hr (pNCS) and 12.3+l.5/lmol/hr (4MUS) and 56+3/~mol/hr (pNCS) and 15.3 + 1.5/~mol/hr (4MUS), respectively. Recoveries of activity from DEAEcellulose were comparable for bovine, rat, and murine liver, approximating 8570 regardless of substrate used to assay enzyme activity. Properties ofpNCS- and 4MUS-sulfatase activities present in fraction I from murine liver (Table 2) were similar and consistent with previously reported characteristics of arylsulfatase B (Daniel and Caplan, 1980). However, the pNCS- and 4MUS-sulfatase activities occurring in fraction III exhibited quite different properties. The pH profile for pNCSsulfatase activity in fraction III displayed a pH optimum at 5.1 with a shoulder at 5.9. pNCS sulfatase activity in fraction 1II was strongly inhibited by silver ion and was non-linear after 5 min. Approximately 70~ (by extrapolation) of fraction III pNCSsulfatase activity possessed a half-denaturation time of 5 min at 65°C, while the remainder decayed more slowly (q/2=60min). By contrast, fraction III 4MUS-sulfatase activity displayed a pH optimum of 5.9, and no shoulder was observed at pH 5.1. Furthermore, fraction III 4MUS-sulfatase activity was relatively thermostable at 65°C, was resistant to silver, and displayed linear kinetics for more than 30 min. p NCS- and 4M US-sulfatase activities in both fractions I and III were completely inhibited by phosphate, indicating that arylsulfatase C was not appreciably contributing to hydrolysis of 4MUS under conditions of the assay employed. Properties of rat p N C S - and 4MUS-sulfatase activities eluting Table 2. Properties of crude murine arylsulfatases eluting with fractions I and III from DEAE-cellulose Fraction I 4MUS

Fraction III 4MUS

pNCS pH optimum Kinetics q/265°C (rain) 70 Residual activity: 0.2 mM A g N O 3 0.2 M Na2HPO 4

pNCS

5.9 Linear 60 _+ 3

5.9 Linear 60 + 4

5.1 Non-linear Biphasic

5.9 Linear 60 + 4

100+2 0

100+4 0

16_+4 0

86_+4 0

57

from DEAE-cellulose in fractions I and III closely resembled those of the corresponding murine activities (data not shown). Isolation and characterization of anionic arylsulfatase B Murine and rat anionic arylsulfatase B isozymes were isolated using the protocol presented in Table 3. Arylsulfatase A was inactivated by heating at 50°C and by inhibition of any residual arylsulfatase A activity by inclusion of 0.2 mM AgNO 3 in the pNCS reaction mixture, and therefore only arylsulfatase B was measured during purification. Recoveries of arylsulfatase activity for rat and mouse were comparable, regardless of whether pNCS-Ag ÷ or 4MUS was used to estimate enzyme activity. Three fractions of arylsulfatase activity were recovered from DEAESephaceh I (0.02 M Tris; 0 NaCI), II (0.025 M Tris; 0.04M NaC1), and III (0.09M Tris; 0.19MNaC1). Approximately 917o of murine arylsulfatase B activity recovered from DEAE-Sephacel occurred in fraction I, and about 8~ of the recovered activity was present in fraction III. The ratios o f p N C S - A g ÷- to 4MUS-sulfatase activity in peaks I and III were comparable. The rat enzyme was similarly partitioned among the three DEAE-Sephacel fractions; however, a larger proportion of rat arylsulfatase activity was recovered in fraction III compared to murine enzyme activity irrespective of the substrate employed for assay. The distributions of the 4MUS-sulfatase activities of rat and mouse following the DEAE-Sephacel chromatography step closely approximated those of the crude 4MUS-sulfatase activities from the two species (Table 1). By contrast, a much larger proportion of p NCS-sulfatase activity than 4MUS-sulfatase activity was recovered in fraction III when assays were performed under conditions permissive to both arylsulfatase A and B (Table 1). The properties of the rat and murine arylsulfatases occurring in fraction III (Table 3) are presented in Table 4. The pH optimum, linearity of reaction, thermostability, molecular weight, and resistance to silver inhibition are consistent with properties reported for arylsulfatase B from these two species. Furthermore, both the rat and murine enzyme were quantitatively precipitated by an IgG preparation known to precipitate arylsulfatase B but not arylsulfatase A (Harrison et al., 1982).

Table 3. Isolation of rodent hepatic anionic arylsulfatase B pNCS #mol/hr S.A.

4MUS /lmol/hr S.A.

Ratio of S.A. pNCS/4MUS

Supernatant

Mouse Rat

2152 1890

0.7 0.6

203 190

0.06 0.06

11.7 10.0

Heat (50°C, 30 min)

Mouse Rat

1644 1474

1.3 1.2

156 141

0.16 0.11

8.1 10.9

(NH4)2SO4

Mouse Rat

1315 1135

3.5 3.0

137 113

0.40 0.30

8.8 10.0

DEAE-Sephacel Mouse

I I1 1II I II III

1060 5 94 882 6 179

10.4 0.8 1.5 11.0 2.0 1.8

95 2 7 76 1 18

0.9 0.33 0.11 0.9 0.33 0.18

11.6 2.4 13.6 12.2 6.1 10.0

Rat

Enzyme was extracted from 12 g of liver. S.A. = specific activity (#mol/mg protein/hr). The p N C S assay for steps 1 4 included 0.2 mM AgNO 3.

58

D. BRUCETHOMPSONet

Table 4. Properties of rodent anionic arylsulfatase B Mouse Rat pNCS 4 M U S pNCS 4MUS pH optimum 5.9 5.9 5.9 5.9 Kinetics Linear Linear Linear Linear tl, 2 65C (min) 60 60 60 60 Molecular weight (kd) 55 _+2 55 _+2 58 _+3 58 _+3 c'~,Residual activity: 0.2mM AgNO3 100_+2 100_+4 100-+3 100-+1 0.2 M Na2HPO4 0 0 0 0 anti-Aryl B 3 -+2 5 _+2 4 +_3 5+ 1 Anti-Aryl B: rabbit anti-murine renal arylsulfatase B IgG. Molecular weight was estimated by gel filtration through Sephacryl S-300. Table 5. Copurificationof murine hepatic 4MUS- and C4S-sulfatase activities 4MUS C4S 4MUS (#mol/mg/hr) (/~mol/mg/hr) " C4S Crude supernatant 0.06 0.0020 29.6 (NH~)2SO4 0.40 0.0234 17.1 DEAE-Sephacel (III) 0.11 0.0043 25.5 Sephacryl S-300 0.12 0.0047 25.5

Arylsulfatase B hydrolyzes sulfate ester bonds of chondroitin-4-sulfate oligosaccharides. If the anionic 4MUS-sulfatase activity isolated by the protocol described above is contributed by arylsulfatase B, then this enzyme fraction should also hydrolyze the sulfate ester bonds of chondroitin-4-SO4 heptasaccharide (C4S). F o u r of the fractions listed in Table 3 were tested for both 4 M U S - and C4S-sulfatase activities (Table 5). Both activities copurified, supporting the contention that anionic 4MUS-sulfatase activity may be contributed by an isozyme of arylsulfatase B. Extensive post-translational modification of lysosomal hydrolases occurs during their maturation, producing a variety of differently charged isozymes. If hepatic anionic and cationic arylsulfatase B differ with respect to their sialic acid content, treatment of the anionic form with neuraminidase should convert the anionic isozyme to a form resembling the cationic isozyme. Murine and rat anionic arylsulfatase B (fraction III from DEAE-Sephacel) were incubated with or without immobilized neuraminidase at 37°C for 16 hr (see Materials and Methods). Recoveries of 4MUS-sulfatase activity in the neuraminidase-treated

al.

specimen averaged 70 + 6 ~ of the activity present in the specimen lacking neuraminidase. The neuraminidase-treated or untreated samples were applied to DEAE-Sephacel, and the columns were developed with 0.02 M Tris-acetate followed by a linear salt-buffer gradient as described above. All of the recovered 4MUS-sulfatase activity eluted with fraction III (0.09 M Tris; 0.19 M NaCI) regardless of the method of pretreatment. Recoveries from the column averaged 80 + 5~o of the applied activity. These trends suggest that increased sialylation is not responsible for retention of anionic arylsulfatase B by DEAE-Sephacel. Comparison of the characteristics of p N C S - and 4MUS-sulfatase activities present in the "high salt" fraction obtained from DEAE-cellulose chromatography (Tables 1 and 2) suggest that a least two enzymes contribute to hydrolysis of p N C S and that one enzyme is largely responsible for hydrolysis of 4 M U S . Twenty grams of murine or rat liver were homogenized, sonicated, and centrifuged as described (see Materials and Methods), and the supernatant was divided into two aliquots. One was treated as outlined in Table 3. The heat treatment was omitted for the second aliquot, and the p N C S assay was performed at p H 5.5 in the absence of silver ion. These alterations minimized loss of arylsulfatase A and permitted better monitoring of both arylsulfatase A (pH o p t i m u m = 5 . 1 ) and arylsulfatase B (pH optimum = 5.9) during partial purification. The resuits of this experiment are presented in Table 6. Yields of 4MUS-sulfatase activity from the split unheated and heated samples were comparable. Furthermore, the distribution of 4MUS-sulfatase activity between fractions I and III following D E A E Sephacel chromatography were not altered by omission of the heat treatment. Similar trends were observed for both rat and murine 4MUS-sulfatase activity. By contrast, both overall yield of p N C S sulfatase activity and the proportion of activity occurring in fraction III were increased when the heating step was omitted during partial purification of murine or rat arylsulfatases. This is reflected in the increase in ratio of p N C S to 4 M U S activity. Murine and rat anionic arylsulfatases from fraction III were sized by gel filtration through Sephacryl S-300, using bovine serum albumin, aldolase, and ovalbumin as standards. Murine p N C S and

Table 6. DEAE-Sephacelchromatography of heated and unheated rodent arylsulfatases pNCS 4MUS Ratio of S.A. #mol/hr S . A . ,umol/hr S.A. pNCS/4MUS Heated I Mouse 804 9.1 90 1.0 9.1 Rat 915 8.0 80 0.7 11.4 III Mouse 63 1.6 6 0.2 8.0 Rat 141 2.0 11 0.3 6.7 ~o I . A . Mouse 51 58 Rat 48 61 Unheated 1 Mouse 781 5.0 93 0.6 8.3 Rat 814 5.2 79 0.5 10.4 Ill Mouse 314 2.8 11 0.1 28.0 Rat 557 3.5 19 0.1 35.0 ~o I . A . Mouse 68 60 Rat 65 66 ~o I.A. = % of one-half of crude supernatant activity recovered from DEAE-Sephacel(see text). Fraction II contained <2% of the recovered activity and has been omitted from the table.

Rodent anionic arylsulfatase

i:

59

+I

A_

"64

E ='3

03

~2

•

,

15

I °°'° 0.5-

.

20

25 Fraction

-

.

30 Number

.

35

,

40

'I

~

o.4m 0.2

2

15

20

25 30 Fraction Number

35

4"0

Fig. I. Sephacryl S-300 fractionation of murine hepatic arylsulfatase activities from DEAE-Sephacel fraction III. (A) Unheated aliquot; (B) heated aliquot. Fraction 1 is the first fraction following elution of the void volume. Fraction volume = 2 ml. BSA = bovine serum albumin (67 kd); OVAL = ovalbumin (43 kd); ALD = rabbit muscle aldolase (158 kd).

4MUS-sulfatase activities from the unheated preparation were each resolved into two fractions having apparent molecular weights approximating 115,000 and 55,000 daltons, respectively (Fig. IA). The major portion of murine pNCS-sulfatase activity was

100. ~ ~\ \

~ ~

.. ,

o--o pNCS 115kd = ;4MUS 115kd .--~4Mus

ArylB {B

-

t~ ~ 4O

2C

associated with the l l 5 k d peak, while the 4MUS-sulfatase activity was predominantly found in the 55 kd peak. The pNCS-sulfatase activity of the 115 kd peak was largely denatured by heating at 50°C; however, the 55 kd p N C S activity was essentially unaltered by this treatment (Fig. 1B). The distribution of 4MUS-sulfatase activity between the 55 kd and 115 kd peaks was unaffected by heating, Similar trends were observed for rat anionic arylsulfatases. Murine arylsulfatases from the 115 and 55 kd peaks were incubated with increasing quantities of rabbit anti-murine arylsulfatase B (Fig. 2). About 95~o of the 4MUS-sulfatase activity associated with the 55 kd peak and approximately 70Y/oof the 115 kd 4MUS-sulfatase activity were precipitated by antiarylsulfatase B. By contrast, the l l 5 k d p N C S sulfatase activity was only slightly affected by this antiserum, supporting the contention that much of the 115 kd pNCS-sulfatase activity was contributed by arylsulfatase A. DISCUSSION

g I0 /J[ onti-Aryl B

1"5

Fig. 2, Immunopreeipitation of murine anionic arylsulfatases separated by Sephacryl S-300 gel filtration. 115 and 55kd=115,000 and 55,000 dalton isozymes, respectively. Aryl B = purified arylsulfatase B from murine brain.

The data presented in this report demonstrate the existence of at least two anionic arylsulfatase B isozymes (115 and 55 kd) in rodent liver that coelute with arylsulfatase A from DEAE-ion exchange resins. Collectively these isozymes may account for as much as 10 and 20% of the murine and rat arylsulfatase B activity, respectively, present in crude homogenates.

60

D. BRUCETHOMPSONet al.

Anionic arylsulfatase B is structurally related to the cationic isozyme, as demonstrated by its crossreactivity with antiserum directed against the cationic enzyme. This and several other features distinguish anionic arylsulfatase B from arylsulfatase A. The major anionic arylsulfatase B isozyme (55 kd) has a higher pH optimum and smaller molecular weight than arylsulfatase A. Furthermore, anionic arylsulfatase B exhibits linear kinetics, is thermostable, hydrolyzes C4S, and is resistant to inhibition by silver; whereas, arylsulfatase A exhibits non-linear kinetics, is thermolabile, does not hydrolyze C4S, and is strongly inhibited by silver ion. Anionic arylsulfatase B was completely inhibited by phosphate, a property which differentiates it from arylsulfatase C. Neuraminidase treatment did not convert anionic arylsulfatase B to the cationic isozyme, suggesting that it does not contain more sialic acid than the cationic isozyme. However, other forms of posttranslational modification, such as phosphorylation, could be responsible for the higher electronegativity of anionic arylsulfatase B. Anionic arylsulfatase B isozymes have been detected in brain (Harrison et al., 1982; Stevens et al., 1977), skin fibroblasts (Stevens, 1974), and peripheral leukocytes (Uehara et al., 1983); however, these enzymes were clearly separable from arylsulfatase A by DEAE-cellulose or DEAE-Sepharose chromatography. The anionic isozyme from human leukocytes and human brain is enriched in mannose-6-PO4 (Uehara et al., 1983; Kureha and Eto, 1983). We have observed small quantities of a comparable arylsulfatase B isozyme in rodent liver which accounts for < 2~o of total arylsulfatase B activity (fraction II, Tables 1 and 3). This enzyme is distinct from the more negative form we have described in this report. Several investigators have described assays for arylsulfatases A and B employing 4MUS as substrate (Stevens et al., 1977; Chang et al., 1981; Gravel et al., 1982). Both enzymes are typically assayed in combination with or without an inhibitor of arylsulfatase A, and arylsulfatase A activity is obtained by difference. This assay assumes that 4MUS-sulfatase activity adequately estimates arylsulfatase A and B activity, and that all of the hydrolysis of 4MUS is catalyzed by these two enzymes. Two earlier reports (Delvin et al., 1976; Hultberg, 1977) raised questions relevant to the suitability of 4MUS as a substrate for arylsulfatases A and B, both with respect to its ability to accurately detect inherited deficiencies of these enzymes and with respect to interference by arylsulfatase C which hydrolyzes 4MUS at alkaline pH. These problems appear to have been corrected by appropriate selection of the method of extraction (Chang et al., 1981) and by inclusion of Pb 2+ in the assay to sequester interfering anions (Chang et al., 1981; Gravel et al., 1982). The relatively small proportion of arylsulfatase B compared to arylsulfatase A in human fibroblasts would tend to minimize problems created by differential hydrolysis of 4MUS by these two enzymes. Our data indicate that 4MUS-sulfatase activity grossly underestimates rodent arylsulfatase A activity and predominantly reflects the arylsulfatase B content of rodent liver. This discovery greatly simplifies assay of rodent arylsulfatase B activity in crude preparations. Fur-

thermore, arylsulfatase C does not appreciably interfere with estimation of arylsulfatase B activity by measuring 4MUS-sulfatase activity under the conditions specified. 4MUS is also a poor substrate for bovine arylsulfatase A. The ratio of arylsulfatase A to arylsulfatase B is reversed in bovine and human liver compared to rodent liver. The elution profile of bovine p NCS-sulfatase activity from DEAE-cellulose (Table 1) may be explained by the excess of arylsulfatase A occurring in bovine hepatic cells; however, the elution profile of bovine 4MUS-sulfatase activity paralleled that for rodent liver, supporting the contention that bovine arylsulfatase A activity is seriously underestimated when 4MUS is used as substrate. Comparison of Figs 1 and 2 suggests that a small proportion of rodent anionic arylsulfatase B may be dimeric. About one-quarter of murine anionic 4MUS-sulfatase activity coeluted from Sephacryl S300 with proteins approximating 115 kd in size. The apparent molecular weight of this protein(s) is close to that expected for an arylsulfatase B dimer. Antimurine arylsulfatase B antiserum precipitated 70-75% of the 115 kd 4MUS-sulfatase activity, indicating that a majority of this activity was contributed by arylsulfatase B. Arylsulfatase A may be responsible for the remainder of the 4MUS-sulfatase activity in this fraction. Feline arylsulfatase B is a homodimer (McGovern et al., 1982), providing precedent for a dimeric isozyme of arylsulfatase B. Summarizing, about 10-20% of rodent hepatic arylsulfatase B coelutes from DEAE-cellulose or DEAE-Sephacel with arylsulfatase A. This highly anionic arylsulfatase B fraction occurs in monomeric and dimeric forms. Rodent arylsulfatase A activity is underestimated when 4MUS is used as substrate. Acknowledgement--We would like to thank Lorie Hatfield

for assistance with the preparation of this manuscript. REFERENCES

Bleszynski W. and Leznicki A. (1967) Activators and inhibitors of soluble arylsulphatases from ox brain. Enzymologia 33, 373-389. Burns G. (1983) On the identity of arylsulphatase C and steroid sulphatase. Hum. Genet. 65, 189. Chang P. L., Rosa N. E. and Davidson R. G. (1981) Differential assay of arylsulfatase A and B activities: a sensitive method for cultured human cells. Analyt. Biochem. 117, 382-389. Daniel W. L. and Caplan M. S. (1980) Comparative biochemistry of murine ary[sulfatase B. Biochem. Genet. 18, 625-642. Daniel W. L., Abedin K. and Langelan R. E. (1980) Murine arylsulfatase B: evidence favoring control of liver and kidney activity by two regulatory elements. J. Hered. 71, 161-167. Daniel W. L., Harrison B. W. and Nelson K. (1982) Genetic regulation of murine hepatic arylsulfatase B activity during development. J. Hered. 73, 24-28. Delvin E. E., Pottier A. and Glorieux F. H. (1976) Comparative activity of arylsulphatases A and B on two synthetic substrates. Biochem. J. 157, 353-356. Farooqui A. A. (1980) Sulfatases, sulfate esters and their metabolic disorders. Clin. Chim. Acta 100, 285-299. Farooqui A. A. and Srivastava P. N. (1979) Isolation, characterization and the role of rabbit testicular arylsulfatase A in fertilization. Biochem. J. 181, 331-337.

Rodent anionic arylsulfatase Glaser J. H. and Conrad H. E. (1979) Chick embryo liver fl-glucuronidase: comparison of activity on natural and artificial substrates. J. biol. Chem. 254, 6588-6597, Gorham S. D. and Cantz M. (1978) Arylsulphatase B, an exosulphatase for chondrotin 4-sulphate tetrasaccharide. Hoppe-Seyler's Z. Physiol. Chem. 359, 1811-1814. Gravel R. A., Leung A., Tsui F. and Kolodny E. H. (1982) A micromethod for the detection of arylsulfatases A and B in cultured fibroblasts and amniocytes. Analyt. Biochem. 119, 360-364. Harrison B. W., Daniel W. L. and Abbas K. J. (1982) Comparative studies of murine anionic and cationic arylsulfatase B. Experientia 38, 73-75. Hultberg B. (1977) Comparative activity of sulphatases in human liver on two synthetic substrates. Clin. Chim. Acta 80, 423-429. Kureha Y. and Eto Y. (1983) A comparative study of brain arylsulfatases B~ and B2: the differences between the two forms in rates of uptake by multiple sulfatase deficient (MSD) disorder fibroblasts. J. Biochem. 94, 1489-1492. Lenzi V. D. and Daniel W. L. (1981) Partial purification and characterization of murine arylsulfatase A. IRCS Med. Sci. 9, 751-752. Lowry O, H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. McGovern M. M., Vine D. T., Haskins M. E. and Desnick

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R. J. (1982) Purification and properties of feline and human arylsulfatase B isozymes. J. biol. Chem. 257, 12605-12610. Mehl E. and Jatzkewitz H. (1968) Cerebroside 3-sulfate as a physiological substrate of arylsulfatase A. Biochim. biophys. Acta 151, 619-627. Rinderknecht H., Geokas M. C., Carmack C. and Haverback B. J. (1970) The determination of arylsulfatases in biological fluids. Clin. Chim. Acta 29, 481-491. Rowden G. (1967) Lysosomal aryl sulphatase in mouse kidney and liver. Nature, Lond. 215, 1283-1284. Roy A. B. (1960) The sulphatase of ox liver. 7. The intracellular distribution of sulphatases A and B. Biochem. J. 77, 380-386. Stevens R. L. (1974) Minor anionic arylsulfatase in cultured human fibroblasts. Bioehim. biophys. Acta 370, 249-256. Stevens R. L., Fluharty A. L., Killgrove A. R. and Kihara H. (1976) Microheterogeneity of arylsulfatase A from human tissues. Biochim. biophys. Acta 445, 661-671. Stevens R. L., Fluharty A. L., Killgrove A. R. and Kihara H. (1977) Arylsulfatases of human tissue: studies on a form of arylsulfatase B found predominantly in brain. Biochim. biophys. Acta 481, 549-560. Uehara Y., Gasa S., Makita A., Sakurada K. and Miyazaki T. (1983) Lysosomal arylsulfatases of human leukocytes: increment of phosphorylated B variants in chronic myelogenous leukemia. Cancer Res. 43, 5618-5622.