Comp. Biochem. PhysioL Vol. 98B, No. 2/3, pp. 313-322, 1991

0305-0491/91 $3.00 + 0.00 © 1991 Pergamon Press pie

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COMPARATIVE BIOCHEMISTRY OF M A M M A L I A N ARYLSULFATASE C A N D STEROID SULFATASE BERTHIE M. RUOFF and WILLIAML. DANIEL Department of Cell and Structural Biology, University of Illinois, 506 Morrill Hall, 505 S. Goodwin Ave., Urbana, IL 61801, USA (Tel: 217-333-8172) (Received 20 July 1990) Abstract--1. Hepatic arylsulfatase C (ASC) and steroid sulfatase (SS) from six of eleven mammals (rat, dog, baboon, cow, goat, and sheep) eoeluted from DEAE-Sephacel as a single anionic species. A minor cationic peak of ASC and SS activity was also recovered from solubilized microsomes derived from the domestic cat. Characterization of the cationic activities indicated they were most likely contributed by a protein structurally related to the anionic isozyme. Properties of ASC and SS activities occurring in these seven species were most consistent with the presence of both activities in the same enzyme. 2. Guinea-pig liver SS activity was partitioned between an alkylsulfatase (hydrolyzing dehydroepiandrosterone sulfate (DHEAS)) and an arylsulfatase (hydrolyzing both estrone sulfate (E1S) and 4-methylumbelliferyl sulfate (4MUS) at a common active site). These enzymes were physically separable by ion-exchange chromatography and possessed distinct immunological and chemical properties. 3. Porcine, squirrel, and human livers possessed a major isozyme of ASC that lacked both E1S- and DHEAS-sulfatase activities. The human hepatic ASC was separable from SS by electrophoresis and was partially resolved from SS by DEAE-Sephacel chromatography. The ASC isozyme lacking SS activity was heat-labile in all three species.

INTRODUCTION There has been considerable controversy regarding whether mammalian arylsulfatase C (ASC; EC 3.1.6.1) and steroid sulfatase (SS; EC 3.1.6.2) activities reside in the same enzyme. Furthermore, there is also disagreement concerning the ability of the same SS to hydrolyze both the arylsulfate, estrone sulfate (E1S), and the alkylsulfate, dehydroepandrosterone sulfate (DHEAS). Human placental ASC-, EIS°, and DHEAS-sulfatase activities all appear to reside in the same enzyme (Vaccaro et al., 1987), and its structural gene, STS, has been mapped to the short arm of the X-chromosome adjacent to the pseudoautosomal region (Curry et al., 1984). A second human ASC isozyme lacks appreciable E1S- and DHEAS-sulfatase activities (Chang et al., 1986) and is the major isozyme occurring in liver and certain other tissues (Munroe and Chang, 1987). The gene encoding the ASC enzyme lacking SS activity has been mapped to the short arm of the X chromosome near STS (Chang et al., 1990). Human male ceils contain a pseudogene corresponding to STS that has been mapped to the long arm of the Y-chromosome (Yen et al., 1988). Data addressing the relationships between ASC and SS activities in other mammals is fragmentary. Sheep brain ASC, EIS-sulfatase, and DHEASsulfatase activities appear to be contributed by three different enzymes (Balasubramanian, 1976; Mathew and Balasubramanian, 1982). Sheep brain ASC and Abbreviations used--ASC, arylsulfatase C; DHEAS, dehydroepiandrosterone sulfate; EIS, estrone sulfate; 4MUS, 4-methylumbelliferyl sulfate; SS, steroid sulfatase.

E1S-sulfatase required a dialyzable activator that was not essential for the respective hepatic activities (Lakshmi and Balasubramanian, 1981a). Monkey brain appears to possess an arylsulfatase (ASC and E1S-sulfatase activities) and an alkylsulfatase (DHEAS-sulfatase activity) (Lakshmi and Balasubramanian, 1981 b). Rat and murine ASC, E 1S-sulfatase, and DHEAS-sulfatase activities appear to be provided by a single enzyme (Kawano et al., 1989; Nelson et al., 1983; Keinanen et al., 1983); however, the rat enzyme may possess separate active sites for arylsulfatase and alkylsulfatase activities (Kawano et al., 1989). The initial uncertainty regarding the location of the murine steroid sulfatase gene, Sts, has been resolved by the discovery that the murine X- and Y-chromosomes carry functional Sts loci (Keitges et al., 1987). The literature cited above suggests that extensive variation of ASC and SS occurs among mammals, ranging from one enzyme with one active site exhibiting ASC, E1S-sulfatase, and DHEAS-sulfatase activities to separate enzymes for each activity. Furthermore, different tissues of the same species may display heterogeneity of ASC and SS activities. Different purification protocols were utilized to isolate the respective enzymes, and methods employed for their characterization also varied considerably from one study to another, making interspecific comparisons difficult. The purposes of the present study were: (1) to isolate liver ASC activity from representative mammals using a common purification protocol, (2) to compare the properties of these ASC enzymes and their SS activities, and (3) to search for an ASC isozyme resembling the human liver isozyme that lacks SS activity.

313

314

BERTHIE M. R u o ~ and WmLIAM L. DANIEL MATERIALS AND METHODS

Materials Fresh cat (Felis catus ), dog (Canis familiaris ), rat (Rattus norvegicus), and guinea-pig (Cavia porcellus) livers were obtained from animal caretakers for the School of Life Sciences of the University of Illinois. Fresh goat (Capra hircus), cow (Bos taurus), sheep (Ovis aries), and pig (Sus scrofa) livers were collected from the Meat Science Laboratory, University of Illinois. Dr Raymond Lee provided fresh squirrel (Sciurus carolinensis) liver. Frozen baboon (Papio hamadryas) liver was supplied by Dr John VandeBerg (Southwest Foundation for Biomedical Research, San Antonio, TX), and frozen human (Homo sapiens) liver and placenta were obtained from Dr Patricia Chang (McMaster University Medical Center, Hamilton, Ontario, Canada). DEAE-Sephacel, Concanavalin A-Sepharose, Protein A Sepharose, Sephacryl S-500, Phenyl-Sepharose, Pharmalyte ampholytes, and unlabeled substrates (4MUS, EIS, and DHEAS) were purchased from Sigma Chemical Co. (St Louis, MO). [7-3H(N)]-dehydroepiandrosterone sulfate, ammonium salt, and [6,7:H(N)]-estrone sulfate, ammonium salt, were obtained from New England Nuclear (Boston, MA). 4MUS was repurified before use (Rinderknecht et al., 1970). All other chemicals were reagent grade.

Enzyme assays ASC activity was assayed using 5 mM 4MUS in 0.2 M phosphate buffer, pH 8.6, containing 1% Triton X-100 (Nelson and Daniel, 1979). DHEAS-sulfatase and EISsulfatase activities were estimated by a method modified from Ropers et al. (1981). The labeled product was extracted directly into the scintillation cocktail. Zero time blanks were employed for all assays.

buffer containing 0.3% Triton X-100. Fractions containing 10% or more of peak activity were pooled, concentrated to 5-10 mg/ml protein, and dialyzed against 0.02 M Tris-HC1, pH 8.0. Purified ASC was stored in 1 ml aliquots at -20°C until needed for antibody generation.

Antibody production Two ml of ASC was emulsified in 2 ml of Freund's complete adjuvant and injected subcutaneously into New Zealand White rabbits. Booster injections were administered at 2, 3, and 4 weeks after the initial injection, using antigen emulsified in Freund's incomplete adjuvant. Animals were bled, and the IgG fraction was isolated from serum by the method of Goding (1976). IgG was stored at -20°C until USe.

Enzyme extraction for comparative characterization Microsomes were isolated as described in the preceding section. The microsomes were resuspended in 0.02 M TrisHCI, 0.1% NAN3, pH 8.0 at a concentration of 8 mg/ml and diluted with an equal volume of 0.6% Triton X-100 in the same buffer. The suspension was stirred for 2 hr at 4°C to solubilize ASC and centrifuged at 100,000g for 60min. Human hepatic ASC required 2% Triton X-100 for solubilization, and the solubilization procedure was modified accordingly. The solubilized ASC was further purified by DEAE-Sephacel chromatography as described above, and the fractions were monitored for ASC, ElS-sulfatase, and DHEAS-sulfatase activities. Fractions containing 10% or more of the peak activity were pooled, dialyzed against 0.02 M Tris-HCl, 0.1% NAN3, pH 8.0, and concentrated under nitrogen. The concentrates were stored in 1 ml aliquots at - 20°C.

Enzyme extraction for Cellogel electrophoresis ASC purification for antibody generation ASC was purified from fresh dog, cow, and rat livers. Homogenates of 20% w/v were prepared in 0.25 M sucrose in 0.01 M Tris-HC1, pH 8.0, containing 0.1% NAN3. The homogenates were centrifuged at 10,000g for 15 min, and the supernatants were centrifuged at 100,000g for 60 min. The microsomal pellet was washed in 0.02 M Tris-HCl, 0.1% NAN3, pH 8.0, resuspended in wash buffer, and frozen at - 2 0 ° C until continuation of the purification. The microsomes were thawed at 4°C, diluted with an equal volume of 0.02 M Tris-HCl, 0.3 M NaC1, 0.1% NaN 3, pH 8.0 buffer, and resuspended by homogenization. The homogenate was sonicated with a Biosonik sonicator at high-maximum setting for 30 sec, and the sonicate was applied to a 2.6 x 35 cm column of Phenyl-Sepharose that had been equilibrated with 0.02 M Tris-HC1, 0.15 M NaC1, 0.1% NaN 3, pH 8.0 buffer. The column was washed with equilibration buffer, and ASC was eluted with 1% Triton X-100 in equilibration buffer. Fractions containing at least 10% of the peak activity were pooled and dialyzed against 0.02 M Tris-HCl, 0.1% NaN 3, pH 8.0 buffer. The retentate was applied to a 2.5 x 42cm column of DEAE-Sephacel equilibrated in 0.02 M Tris-HC1, 0.1% NaN 3, 0.3% Triton X-100, pH 8.0 buffer, and the column was washed with equilibration buffer. ASC was eluted with a linear gradient of 0-0.3 M NaCI in equilibration buffer. Fractions containing 10% or more of the peak activity were combined and dialyzed against 0.02 M Tris-HC1, pH 8.0, containing 0.1 M NaC1, 1 mM MnC12, 1 mM CaCI, 0.1% NAN3, and 0.3% Triton X-100 (start buffer). A 2.5 x 4.5 cm column of Concanavalin A-Sepharose was charged with the retentate, and washed with start buffer. ASC activity was eluted with 0.5 M l-O-methyl-~-D-glucopyranoside in start buffer. Fractions containing enzyme activity were pooled, concentrated to 5-10 ml by pressure filtration under N 2, and dialyzed against 0.02 M Tris-HCl, 0.2 M NaC1, 0.1% NAN3, pH 8.0 buffer. The retentate was loaded onto a 2.5 x 38cm column of Sephacryl S-500, and ASC activity was eluted with dialyzing

Human liver ASC and placental ASC display fast or slow mobility, respectively, when extracted with Miranol H2M, but exhibit similar mobilities when extracted with Triton X-100 (Chang et al., 1986). Therefore, a mini-extraction was performed to isolate ASC for Cellogel electrophoresis. Approximately, 4 g of tissue were minced in 0.9% NaC1 and centrifuged at 10,000g for 10 min. The pellet was washed three times with 0.25 M sucrose in 0.04 M Tris-HC1, 0.1% NAN3, pH 8.0, and then homogenized in this sucrose-Tris buffer. The homogenate was centrifuged at 100,000g for 60 min, and the pellet was homogenized in 2% Miranol H2M in 0.04 M Tris-HC1, 0.1% NaN 3, pH 8.0. The homogenate was sonicated with a Biosonik sonicator, and the sonicate was centrifuged at 100,000g for 60 min. The supernatant was used for electrophoresis.

Enzyme characterization Protein concentrations were estimated by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Suspensions were clarified by centrifugation following incubation to remove interfering detergent. ASC from the DEAE-Sephacel fraction was diluted to approximately 704MU fluorescence units (Turner fluorometer) with 0.1 M Tris-HC1, 0.15 M NaCI, 0.1% NaN 3, 0.3% Triton X-100, pH 7.5 fiBS) and mixed with 0-200/~1 of anti-ASC IgG and diluted to 400/11 with TBS. The mixture was incubated at 37°C for 1 hr and at 4°C for 16 hr. A volume of 200#1 of packed Protein A-Sepharose was added and incubated at room temperature for 15 min, vortexing at 5-min intervals. The mixture was centrifuged for 5 min, and the supernatant was assayed for residual 4MUS-, E1S-, and DHEAS-sulfatase activities. Isoelectric focusing of 4MUS-, E1S-, and DHEASsulfatase activities was performed in 5% polyacrylamide gels containing 0.1% Triton X-100 and pH 6.5-9 or pH 5-8 ampholytes. 4MUS-sulfatase activity was located by incubation with a filter paper overlay containing the assay mixture for 30 min at 37°C, flooding the gel with glycine-carbonate,

Mammalian arylsulfatase C and steroid sulfatase pH 10, and visualized under u.v. light. Parallel lanes were sliced at 1-cm intervals. The slices were incubated in 1 ml 0.025 M KCI, 0.1% NAN3, 0.1% Triton X-100, and incubated overnight at 4°C. The extracts were tested for pH and for E1S- and DHEAS-sulfatase activities. Thermal stabilities were estimated by incubating 0.5 ml aliquots of enzyme (diluted to appoximately 70 4MU fluorescence units with 0.02M Tris-HCl, 0.1% NaN 3, 0.1% Triton X-100, pH 8.0) at 50°C for 0-60 min. The aliquots were cooled on ice for I0 min and centrifuged at 1500g for 5 min. The supernatants were assayed for residual 4MUS-, EIS-, and DHEAS-sulfatase activities. Inhibition of 4MUS-sulfatase activity by E IS and DHEAS was monitored at 2.5 and 1.25 mM concentrations of 4MUS and 0-500/~M concentrations of E1S or DHEAS. 4MUS assays were performed at pH 7.1 for human ASC and pH 8.5 for the ASC from other species. Effects of other inhibitors upon 4MUS-, E1S-, and DHEAS-sulfatase activities were studied by preincubation of 5 #1 of inhibitor solution or assay buffer and 45/zl of enzyme for 15 min at 37°C, and activities were compared in the presence and absence of the inhibitor. Phosphate was eliminated from the 4MUS substrate mixture for phosphate inhibition studies. The pH optima for 4MUS-, E 1S-, and DHEAS-sulfatases were determined over the range pH 6.5-9.0. Cellogel electrophoresis was performed as described by Chang et al. (1986). RESULTS

ASC (4MUS) and SS (DHEAS and E1S) activities present in crude liver homogenates derived from eleven mammalian species are listed in Table 1. More than 100-fold variation of both ASC and SS activities was observed among the liver homogenates. Rat, cow, and sheep liver homogenates possessed high activities with all three substrates, while human, guinea-pig, and cat homogenates exhibited uniformly low activities. By contrast, squirrel liver homogenate contained high 4MUS- and E1S-sulfatase activity and low DHEAS-sulfatase activity. ASC from rodent (rat), carnivore (dog), and artiodactyl (bovine) liver was partially purified using the sequence: phenyl-Sepharose hydrophobic chromatography, DEAE-Sephacel ion-exchange chromatography, Concanavalin A-Sepharose affinity chromatography, and Sephacryl S-500 gel filtration. ASC behavior during purification was monitored by measuring its 4MUS-sulfatase activity. Hepatic ASC from all three species was retained by phenylSepharose, DEAE-Sepharose, and Concanavalin A-Sepharose, suggesting that ASC is a hydrophobic anionic glycoprotein. Final yields were 42%, 29%, and 4% for rat, dog, and bovine ASC, respectively (Table 2). The relatively poor yield for bovine ASC appeared to be the result of low recoveries from phenyl-Sepharose and Concanavalin A-Sepharose compared to those for rat and dog ASC. Properties of the partially purified enzymes resembled those of

315

Table 1. Rates of hydrolysis of 4MUS, DHEAS, and EIS by mammalian liver homogenates* Species

4MUS

DHEAS

E 1S

Rat Squirrel Sheep Cow Goat Pig Baboon Dog Human Guinea-pig Cat

19680 8800 7280 4320 1900 1520 1280 864 432 226 129

6.70 0.37 2.06 2.85 0.54 0.75 1.36 1.21 0.31 0.10 0.06

14.07 17.97 12.81 20.65 12.55 5.93 8.32 9.45 1.52 2.49 0.19

*Activities expressed as nmol/g wet wt/hr.

their crude counterparts and will be described in a later section of this paper. The partially purified ASC from each species was used to raise polyclonal antibodies for comparative purposes. Hepatic 4MUS-, E1S-, and DHEAS-sulfatase activities from four mammals (rat, dog, cow, and goat) eluted from DEAE-Sephacel as a single peak at salt concentrations ranging from 0.15 to 0.25 M. The elution profile for the rat enzyme is shown in Fig. 1. The elution profiles of the remaining three species were comparable. Cat, baboon, and sheep hepatic 4MUS-, EIS-, and DHEAS-sulfatase activities coeluted in both minor cationic and major anionic fractions. The cationic fraction contained 10-25% of the total 4MUS-, E1S-, and DHEAS-sulfatase activities, respectively, recovered from the column. The cat elution pattern is illustrated in Fig. 1. Similar elution profiles were observed for the baboon and sheep liver activities. Neither arylsulfatase A nor arylsulfatase B contributed significantly to the 4MUS-sulfatase activity in either fraction, since 0.2 M phosphate was incorporated in the assay mixture, completely inhibiting these arylsulfatases. Properties of rat anionic 4MUS-, E1S-, and DHEAS-sulfatase activities and the corresponding cationic and anionic sulfatase activities from the cat are given in Table 3. Characteristics of the rat activities were generally representative of those from all seven mammalian species. 4MUS- and E1S-sulfatase activities had pH optima near 9, while that for DHEASsulfatase activity approximated 6.5. The cat was exceptional, with a pH optimum for E1S-sulfatase approximating that of DHEAS-sulfatase. All three sulfatase activities were resistant to denaturation at 50°C, with more than 80-90% of the respective activities remaining after 60 min at this temperature. Similar isoelectric points were observed for 4MUS-, E1S-, and DHEAS-sulfatase activities from each of the seven species, approximating 6.0-6.5 in five of the seven species. Bovine hepatic ASC and SS focused in two bands (pI = 4.5 and 7.0), both possessing activity

Table 2. Partial purification of rat, dog, and bovine ASC* Step Homogenate Microsomes Phenyl-Sepharose DEAE-Sephacel ConA-Sepharose Sephacryl S-500

S.A.

Rat yield

150 1048 611 4216 37,333 65,116

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54 92 228 171 770 1417

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Bovine Yield 100 56 17 14 4 4

Fold 1X 2X 4X 3X 14X 26X

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activities. Fraction volume was 4 ml. toward all three substrates. A similar pattern was also observed for goat SS and ASC (pI = 6.0 and 7.2). Comparisons of the respective properties of the cationic and anionic ASC and SS activities from cat liver strongly suggest that the cationic activities may reside in a protein that is derived from or otherwise closely related to the anionic protein. Similar relationships were also observed for the cationic and anionic isozymes of ASC and SS from baboon and sheep liver.

Anionic DHEAS-, EIS-, and 4MUS-sulfatase activities from bovine, rat, and dog liver were treated with rabbit polyclonal anti-ASC IgG induced by the respective partially purified ASC enzymes (Fig. 2). Anti-ASC from each species precipitated all three hepatic activities. This result is compatible with the presence of catalytic sites for the three substrates within the same protein or in very similar proteins that co-purified during the preparation of antigen for antibody production. Baboon hepatic E1S-, DHEAS-,

Table 3. Properties of rat and cat hepatic ASC and SS

4MUS 9.0 6.4

pH Optimum pI % Residual activity: 20mM NaCN 42_+2 2mM Na2HPO4 101-+7 25raM Na2SO4 127_+11 0.2 mM AgNO3 48_+2 T5o (50°C) (min) * *Approximately 90-100% of these

Rat anioni EIS 9.0 --

Cat DHEAS 6.5 6.4

Cationic 4MUS EIS 9.0 6.5-7.0 ---

DHEAS 6.5 --

4MUS 9.0 6.0

83+1 50+1 32+1 24+2 71+_1 87_+2 91_+2 97-+2 64-t-5 83+_2 87_+1 90-1-1 1064-1 65+2 103-+4 7 6 _ + 5 107_+1 80_+2 52_+4 96_+7 * * * --activities remained after heating for 60 min at 50°C.

60+2 97+_.2 96-+1 91_+1 *

Anionic EIS 6.5 6.0

DHEAS 6.5 6.0

26+1 58-+2 7 6 - 1 - 1 91-+1 75-+3 100-t-5 64_+3 101 -+ 1 ---

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Fig. 2. Immunoprecipitation of rat ASC and SS by anti-rat ASC IgG (A), bovine ASC and SS by anti-bovine ASC IgG (B), dog ASC and SS by anti-dog ASC IgG (C), and baboon ASC and SS by anti-bovine ASC IgG (D). Hepatic anionic enzyme was incubated with the indicated IgG preparation, mixed with Protein A-Sepharose, and centrifuged as described in Materials and Methods. Residual supernatant activity is displayed in the figure. EIS, DHEAS, and 4MUS represent the respective hydrolytic activities remaining in the supernatants. and 4MUS-sulfatase activities were coprecipitated by anti-bovine ASC (Fig. 2), indicating substantial sharing of immunological determinants between the baboon and bovine enzymes. Furthermore, this result supports the occurrence of hydrolytic sites for the three substrates in the same or very similar proteins. A Dixon plot of inhibition of baboon ASC by EIS and DHEAS is illustrated in Fig. 3 and supports noncompetitive inhibition of 4MUS-sulfatase activity by these sulfated steroids. Similar plots were observed for rat ASC, bovine ASC, and dog ASC. By contrast, hydrolysis of 4MUS by human placental ASC, an enzyme known to function as a steroid sulfatase, was competitively inhibited by E1S and DHEAS (Fig. 3). Anionic fractions of guinea-pig, porcine, and human hepatic 4MUS-, E1S-, and DHEAS-sulfatases eluted in two or more peaks from DEAE-Sephacel, suggesting that these activities were associated with more than one enzyme (Fig. 4). Two peaks of guinea-pig 4MUS-sulfatase activity, eluting with approximately 0.07M and 0.15 M NaCI, were observed. Both of these fractions coincided with peaks of E1S-sulfatase activity; however, DHEAS-sulfatase activity eluted as a distinct peak at about 0.11 M NaC1. Considerable overlap among the three fractions occurred, making it impossible to resolve isozymes in neighboring peaks. Fractions eluting under low-salt conditions (0.07 M NaCI) or high-salt conditions (0.15 M), respectively, were combined in separate pools for characterization. DHEAS-sulfatase activity was apportioned between both pools. Properties of the E1S- and 4MUSsulfatase activities occurring in the low- and high-salt

pools are compared in Table 4. The data suggest that the E1S- and 4MUS-sulfatase activities in both fractions are associated with the same or very similar proteins. The low-salt pool was utilized for further characterization of the guinea-pig isozymes. Thermal denaturation of guinea-pig 4MUS-, E1 S-, and DHEAS-sulfatase activities are shown in Fig. 5A. DHEAS-sulfatase activity was more therrnolabile than either 4MUS- or E1S-sulfatase activity, supporting the contention that DHEAS is hydrolyzed by an enzyme distinct from that responsible for hydrolysis of 4MUS and DHEAS. Anti-bovine ASC IgG more effectively precipitated guinea-pig DHEAS-sulfatase activity than either 4MUS- or E1S-sulfatase activities (Fig. 6A), further supporting the occurrence of two different sulfatase isozymes in guinea-pig liver. Guinea-pig 4MUS-sulfatase activity was not inhibited by 500/x M DHEAS (Table 4), but was competitively inhibited by EIS (Fig. 7A). Summarizing, these experiments are Table4. Characteristicsof low-and high-saltfractionsof guinea-pig hepatic E1S- and 4MUS sulfataseactivities Low-salt High-salt 4MUS E1S 4MUS EIS pH Optimum 8.0-9.0 6.5 9.0 6.0 pI 7.2 7.4 % Residualactivity: 20 mM NaCN 26 -+2 13 + 1 22+2 25_.+I 2mM Na~HPO4 101 _+3 70_+2 90+__4 7 6 + 3 25mM Na2SO4 97_+2 61 _+3 93_+4 71_+4 0.2 mM AgNO3 72 _+2 48 _+1 75_+1 45_+1 500#M DHEAS 100 _+5 -Ts0 (50°C) (min) 53 _+2 56 _+3 55_+3 54+3

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(Fig. 5B). Anti-bovine hepatic ASC IgG precipitated all three activities; however, both E1S- and DHEASsulfatase activities were more effectively precipitated by this antibody preparation than was 4MUS-sulfatase activity (Fig. 6B). These trends suggest that two ASC isozymes may be present in porcine liver, only one of which is a SS. Both ASC and SS activities were very low in human liver extracts compared to those of many other species. DHEAS- and EIS-sulfatase activities co-eluted in a minor low-salt (0.07 M NaCI) and major high-salt (0.21 M NaC1) peak. Elution of 4MUS-suifatase activity peaked at 0.28 M NaCI (Fig. 4C). Considerable overlap between the major E1S/DHEAS-sulfatase peak and the 4MUS-sulfatase peak prevented resolution of the respective activities, so both'peaks were pooled for characterization. Human 4MUS-sulfatase activity was not inhibited by 500/tM DHEAS nor E1S and was more thermolabile at 50°C than both E1S-

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319

affectedsquirrelA S C and SS activities.4MUS-, EIS-, and DHEAS-sulfatase activitiesappeared to be precipitated to the same extent, although anti-rat ASC IgG precipitatedlessthan 25% of the squirrelsulfatase activities(Fig. 6E). Both EIS and D H E A S inhibited squirrelliver4MUS-sulfatase activityby a non-competitivemechanism (inhibitionby EIS ispresented in Fig. 7B). Hepatic A S C of six mammals was extracted with Miranol H 2 M and subjectedto cellogclelectrophoresis to search for a fast variant comparable to that of human fiver A S C reported by Chang et aL (1986). Human placental A S C was used as a standard for comparison. Hepatic ASC from baboon, goat, mouse, rat,and squirrelpossessed relativemobiliticsequal to or less than that of human placental A S C (Fig. 8, Table 6). A single band corresponding to the fast variant was observed in crude liverextracts.However, partialpurificationof human hepatic arylsulfatascC using a protocol similar to that employed for purification of the rat, bovine, and dog enzymes revealed two bands of 4MUS-sulfatasc activity, one corresponding to the fast variant and a faint band with a mobility identical to that of the placental isozymc (Fig. 8).

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.

60

MIN. S0°C Fig. 5. Thermal denaturation of guinea-pig hepatic ASC and SS (low-salt fraction) (A), porcine ASC and SS (B), human hepatic ASC and SS (C), human placental ASC and SS (D), and squirrel hepatic ASC and SS (E).

320

BERTHIEM. RUOFFand WILLIAML. DANIEL

c

40

~" 80

D

o E1S • DHEAS

1,0

A 4MUS

100

100

pi igG

Fig. 6. Immunoprecipitation of guinea-pig hepatic ASC and SS (low-salt fraction) (A), porcine ASC and SS (B), human hepatic ASC (C), human placental ASC and SS (D), and squirrel ASC and SS (E). Anti-rat hepatic ASC IgG was used to precipitate the squirrel hepatic activities, while anti-bovine hepatic ASC IgG was used for precipitation of the activities from the other species. DISCUSSION

Several ASC models have been proposed for the hydrolysis of synthetic arylsulfates and steroid sulfates, ranging from one enzyme with one catalytic site to separate enzymes for each substrate. The human placental ASC possesses essentially all of the SS activity occurring in that organ (Burns, 1983) and behaves according to the expectations for one active site catalyzing the hydrolysis of synthetic arylsulfates, E1S, and DHEAS (Vaccaro et al., 1987). Therefore, this enzyme was used as a standard for comparison with hepatic ASC from eleven mammals derived from four orders. Antibodies used for comparative purposes were raised against ASC that was purified under conditions which optimized recovery of phosphateresistant 4MUS-sulfatase activity. This protocol was followed to detect ASC isozymes with variable affinities for E 1S and/or DHEAS. Relatively crude preparations of hepatic ASC extracted from microsomes and subjected to DEAE-Sephacel chromatography were then examined for evidence of enzyme heterogeneity. The most common ASC pattern, occurring in seven of the eleven mammalian species, involved a single

anionic fraction of ASC activity which also contained both SS activities. 4MUS-sulfatase (an ASC substrate) and E1S- and DHEAS-sulfatase activities were stable at 50°C. No evidence for a thermolabile ASC was observed among any of the seven mammalian preparations. Furthermore, anti-ASC IgG precipitated all three substrate activities from dog, bovine, rat, and baboon in the Protein A-Sepharose system. 4MUSsulfatase activity from baboon, rat, dog, and bovine fractions were inhibited by both EIS and DHEAS, with the respective Dixon plots supporting noncompetitive inhibition. These trends are most consistent with a single enzyme catalyzing hydrolysis of all three substrates, but two or more sites of the enzyme appear to be involved with substrate-inhibitor binding. Iwamori et al. (1976) reported non-competitive inhibition of rat hepatic E1S-sulfatase by DHEAS, and Kawano et al. (1989) also observed non-competitive inhibition for rat liver ASC by both E1S and DHEAS. In the latter case, the rat enzyme had been purified to homogeneity, indicating that the multiple sites resided in the same enzyme protein. Guinea-pig hepatic DHEAS-sulfatase activity was separated from both E1S- and 4MUS-sulfatase activities by DEAE-Sephacel chromatography.

Table 5. Biochemical properties o f porcine, h u m a n , and squirrel liver arylsulfatases 4MUS pH Optimum pl % Residual activity: 2mM NaCN 2 mM Na2HPO 4 25 m M Na2SO 4 0.2mM AgNO 3 500/~M D H E A S 500#M EIS T~ (50°C) (min)

8.5-9

95+3 82+_6 52+_1 6+_1 100+_4 95+_5 5

Pig EIS 6.5 All 6.0, 7.3 36+_1 79+_2 78+_2 8+_1 --*

DHEAS

4MUS

Human E1S

6.5

6.5-7 6.9

81+_2 81+_3 98+_2 22+_2 --*

12+_1 78+_2 58+_1 24+1 100_+4 100_+3