Biochimica et Biophysica Acta, 704 (1982) 353-360 353 Elsevier Biomedical Press

BBA31185

EVIDENCE FOR HETEROGENEITY OF HEPATIC BILE SALT SULFOTRANSFERASES IN FEMALE HAMSTERS AND RATS

STEPHEN BARNES a., and JERRY G. SPENNEY b

Department of Biochemistry and Comprehensive Cancer Center and b Division of Gastroenterology, University o/Alabama and Veterans Administration Medical Center, Birmingham, AL 35294 (U.S.A.)

(Received November 26th, 1981)

Key words: Sulfotransferase heterogeneity; Bile salt; (RodenO

Gel filtration and anion-exchange chromatography have been used to investigate whether 3'-phosphoadenylyl- sulfate:bile salt sulfotransferase activity from female rat and hamster liver is heterogeneous. Using these techniques at least three different enzyme activities were demonstrated with two different bile salt substrates. In both animals, but particularly the rat, there was a marked difference in the substrate specificity between each of the peaks of enzyme activity. The reducing agent, 2-mercaptoethanol, enhanced the proportion of the highest molecular weight (130000) form of the enzyme from rat liver detected with glycochenodeoxycholate as substrate. This effect was duplicated by alkylation of sulfhydryl groups with iodoacetamide and is interpreted as being due to intermolecular association caused by disruption of intramolecular disulfide bonds.

Introduction

Hepatic Y-phosphoadenylylsulfate:bile salt sul- fotransferases ** are soluble enzymes found in the rat [1], guinea pig [2], hamster [3] and man [41. Gel filtration chromatography of the partially purified rat bile salt sulfotransferase activity using Sep- hadex G-100 produced a single peak of activity with an apparent molecular weight of 130000 [1]. The partially purified human liver bile salt sulfotransferase was also a single activity, but had a molecular weight of 65000 [5], a value similar to

* To whom correspondence should be addressed at: R123 Volker Hall, Department of Pharmacology, University of Alabama in Birmingham, Birmingham, AL 35294, U.S.A.

** This name is utilized in the text to describe the group of sultotransferases which catalyze the formation of bile salt sulfate esters. One such enzyme, 3-phosphoadenylyl- sulfonate:taurolithocholate sulfotransferase (EC 2.8.2.14) has been previously described (see Ref. 1).

0167-4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

that reported for certain steroid and phenol sulfotransferases found in liver homogenates [6-9]. In contrast to the apparent homogeneity of bile salt sulfotransferase activities in human [4] and rat liver [1], steroid and phenol sulfotranserases have been reported to be heterogeneous [8,9]. Similar heterogeneity should be anticipated for bile salt sulfotransferase enzymes from hamster liver, since bile salt sulfotransferase activity measured with glycochenodeoxycholate (trivial name of the glycine conjugate of 3a,7a-dihydroxy-5/3-cholan- 24-oate) has a marked sex difference in affinity (Kin), rather than Vm~ x [3], implying that sex- specific bile salt sulfotransferase enzymes or differ- ing amounts of multiple enzymes may be present.

The present investigation was therefore under- taken to determine whether the bile salt sulfo- transferase activity from hamster liver was a ho- mogeneous enzyme activity, and to compare the bile salt sulfotransferase activities of rat and ham- ster liver. We report that bile salt sulfotransferase

354

from both hamster and rat liver exists in several forms as demonstrated by ion-exchange chro- matography and gel filtration chromatography.

This work has been published in part in ab- stract form in Gastroenterology 79, 1300 (1980).

Materials

Female adult golden Syrian hamsters weighing 100-120 g and female Wistar rats weighing 200- 250g were purchased from Engle Laboratory Animals Inc. (Farmsburg, IN), and Southern Animal Farms (Prattville, AL), respectively. [1- 3H]Glycochenodeoxycholate (2 Ci/mmol) and 3'- phosphoadenosine-5'-phospho[ 35 S]sulfate (1.5 Ci/mmol) were purchased from New England Nuclear Corp. (Boston, MA). The latter labeled compound was obtained in an ethanol/water mix- ture. This was freeze-dried to remove the ethanol, and redissolved in water. It was stored frozen and periodically checked for purity by paper electro- phoresis in 100 mM pyridine acetate buffer (pH 5.3). Non-radioactive bile salts, steroids, sodium pyruvate, reduced nicotinamide adenine dinucleo- tide and reduced glutathione were purchased from Sigma Chemical Co. (St. Louis, MO). DEAE-cel- lulose (DE-52) was obtained from Whatman (Clif- ton, N J) and Sephacryl S-200 from Pharmacia Fine Chemicals (Piscataway, N J). Y-Phosphoa- denosine-5'-phosphosulfate (PAdoPS) was pur- chased from PL-Biochemicals (Milwaukee, WS).

Methods

Preparation of hepatic cytosol. Non-fasted animals which had been fed ad libitum on a chow diet were killed between 09.00 and 10.00h under ethyl carbamate anethesia (0.15 g/100 g body weight, given intraperitoneally). The livers were quickly excised and placed in ice-cold 100 mM sodium phosphate/5 mM MgSO 4 buffer (pH 7.0). The livers were blotted dry, weighed and then minced in 1 ml ice-cold sodium phosphate/MgSO 4 buffer per g wet weight liver; the mince was ho- mogenized using a motorized loose-fitting Teflon- glass homogenizer. In some experiments 2mM 2-mercaptoethanol was added to the homogeniza- tion medium. The soluble 105000 x g supernatant fraction was isolated by differential centrifugation

[3] and stored frozen at -20°C. When stored at -20°C bile salt sulfotransferase activity of the homogenate is preserved for at least 9 months.

Alkylation of proteins in the soluble super- natant fraction which contained 80 mg/ml was carried out by reduction with 10 mM dithiothreitol for 1 h at room temperature, followed by the addi- tion of iodoacetamide to a final concentration of 40 mM and reaction for a further 1 h at room temperature. The samples were then dialyzed over- night at 4°C against the sodium phosphate/MgSO 4 buffer.

DEAE-cellulose chromatography. DEAE-cellu- lose (DE52) was equilibrated with 10 mM sodium phosphate/5 mM MgSO 4 buffer (pH 7.0) and packed to form a 1 X 20 cm column. The soluble supernatant fraction was dialyzed at 4°C against two changes of 4 liter of the same buffer and 3 ml, containing 80 mg/ml protein, were applied to the column at 0.7 ml/min. Unbound proteins were eluted with 100 ml of the starting buffer. The bound proteins were eluted with 500 ml of a linear 0-500 mM NaCI gradient in the starting buffer; 5-ml fractions were collected. The gradient was quantitated by conductivity measurements. Pro- tein content of the eluate was determined by mea- surement of absorbance at 280 nm. Bile salt sulfotransferase activity was measured as previ- ously described [3]. Fractions were stored at -20°C after collection. Protein content of indi- vidual fractions was determined by the method of Lowry et al. [10] using bovine serum albumin as standard.

Gel filtration chromatography. Sephacryl S-200 was suspended, washed and equilibrated with 100 mM sodium phosphate/5 mM MgSO 4 buffer (pH 7.0) and poured to form a 1.6 X 90 cm column. Soluble supernatant fractions were equilibrated with this buffer by dialysis and were centrifuged at 100000 rev./min (148000 X g at ravg ) for 20 min in Beckman Airfuge immediately prior to being ap- plied to the column. A sample volume of 0.5 ml was applied to the column, and 1-ml fractions were collected at 0.33 ml/min. The column was calibrated for molecular weight determinations using aldolase, transferrin, bovine serum albumin, ovalbumin, trypsin and chymotrypsinogen (Sigma Chemical Co., St Louis, MO). Additionally, since the hepatic soluble supernatant fractions contain

many enzymes, it was possible to use some of these activities as internal calibration standards. To this end, lactate dehydrogenase and glutathione S-transferase activities were measured to de- termine their profiles in the column eluant frac- tions. In some experiments 2 to 50 mM 2- mercaptoethanol was included in the elution buffer.

Enzyme assays. Radioactive tracer assays were used to measure bile salt sulfotransferase activity [3]. Aliquots of the column fractions (100-150 #1) were incubated in a total volume of 200 #1, with final concentrations of 25 #M glycochenodeo- xycholate, 180 #M PAdoPS, 100 mM phosphate buffer, pH 7.0, and 5 mM MgSO 4. [1-3H]Glyco - chenodeoxycholate was used as the tracer, 0.15 #Ci per assay tube. Reactions were carried out by incubation at 37°C for 1 h. where necessary, in- cubations were extended for 2 h to enhance the sensitivity of the assay. The reaction rate was linear with time, as noted previously [3]. The reac- tion was terminated with 0.8 ml methanol and the precipitated protein was removed by centrifuga- tion. The supernatants were evaporated to dryness and the residue redissolved in 50 #1 aqueous methanol (1:1, v/v); 20-#1 aliquots were applied to individual lanes of a 20 X 20 cm silica gel G TLC plate (Rediplate, Fisher Chemical Co., Norcross, GA) along with standards of glyco- chenodeoxycholate and its mono- and disulfates. After development and localization of standards the glycochenodeoxycholate monosulfate zone was scraped off and the radioactivity on the silica was measured by addition of 0.5 ml methanol and 5 ml scintillation fluid (NE-260, Nuclear Enterprises, Palo Alto, CA) and counting in a Packard model 2240 liquid scintillation spectrometer. Conversion of counts per min to disintegrations per min was carried using a channels ratio method.

In some experiments, PAdoP[35S]S (0.2 #Ci added to each assay tube) was used to quantitate sulfation of glycolithocholate (trivial name for the glycine conjugate of 3a-hydroxy-5fl-cholan-24- oate). The assay procedure was as described for [1-3H]glycochenodeoxycholate, except that sam- ples were boiled to terminate the reaction rather than using methanol. After removal of precipitated protein by centrifugation, 20-#1 aliquots of the supernatant were directly applied to the TLC plate. Unreacted PAdop[35S]S remained at the origin in

355

the solvents used for development. Bile salt sulfotransferase activity was calculated

from the fraction of the tracer (either [3H]glyco- chenodeoxycholate or PAdoP[35S]S) converted to the bile salt sulfate. For assays with [3H]glyco- chenodeoxycholate, correction was made for the apparent activity in the absence of added PAdoPS. In those assays with PAdop[35S]S, correction was made for apparent activity in the absence of added bile salt. Alternatively, deletion of the tissue ex- tract gave similar background values, indicating that endogenous substances were not undergoing sulfation.

Lactate dehydrogenase was measured by the change in absorbance at 340 nm due to the oxida- tion of NADH with pyruvate as substrate [11]. Glutathione S-transferase activities were measured using 1-chloro-2,4-dinitrobenzene and 1,2-dichlo- ro-4-nitrobenzene as substrates, by measuring ab- sorbance changes at 340 and 345 nm, respectively [12]. A Gilford 2400 spectrophotometer equipped with a kinetic changer and recorder was used for these measurements.

Data smoothing. A simple smoothing routine was used to minimize the random error arising from the measurement of enzyme activity to de- scribe the elution profile. The value of the n th data point was replaced by the following:

n = ( n - 2 + 2 ( n - 1 ) + 5 n + 2 ( n + 1) + n + 2)/11

This eliminated most of the small-scale variation resulting from measurement error, without altering the major features of data. In order to use this method at least 20 data points, which had to be evenly distributed accross the data set, were neces- sary. It was applied to data in Figs. 3 and 5, but not in Figs. 2 and 4. In the latter cases linear interpolation was used.

Results

Preliminary experiments demonstrated that bile salt sulfotransferase activity from rat and hamster liver soluble supernatant fraction was stable for at least 4 days at 4°C and was not affected by varying the ionic strength by addition of 0 to 0.5 M NaC1.

The reducing agent 2-mercaptoethanol pro-

356

duced a slight inhibition of bile salt sulfotransferase activity from both rats and hamsters, maximum inhibition (25%) occurring at 100 mM. In con- centrations used in the isolation and chromato- graphic steps (2-5 mM), inhibition did not exceed 7%. These findings indicated that little loss of activity would be expected during collection peri- ods associated with the chromatographic proce- dures.

DEAE-ceilulose chromatography Bile salt sulfotransferase activity in hamster liver

soluble supernatant fractions (equivalent to 5 g liver) was completely absorbed on the 20 X 1 cm DEAE-cellulose column. Elution with a NaCI gradient (0-0.5 M) produced two peaks of activity with both glycochenodeoxycholate and glycolitho- cholate; the first activity eluted at 100 mM NaC1 and the other eluted at 180 mM NaC1 (Fig. 1A). The ratio of activities for glycochenodeoxycholate to glycolithocholate (measured at 100 #M) was 1.00 for for the 100 mM NaC1 peak and 1.73 for the 180 mM peak. Specific activities of each peak were increased 5-8-fold relative to the soluble supernatant fraction.

Similar fractionation of rat liver soluble super- natant fraction also produced two peaks of bile salt sulfotransferase activity with glycochenodeo- xycholate and giycolithocholate, one eluting at 60 mM NaCI and the other at 100.mM NaC1 (Fig. I B). The ratio of activities for glycochenodeo- xycholate and glycolithocholate was 0.96 for the 100 mM NaC1 peak and 0.41 for the 180 mM NaC1 peak. Purification relative to the soluble supernatant was only 3-4-fold because the bile salt sulfotransferase activity eluted near the pro- tein peak at the beginning of the NaCI gradient.

Gei fiitration chromatography Lactate dehydrogenase activity determined on

the eluted fractions gave a single peak with an apparent molecular weight of 135000, in good agreement with the accepted molecular weight of 136700 [14]. The glutathione S-transferase activity had an apparent molecular weight of 38000 in hamsters and 32000 in rats; similar values were obtained for these enzymes in the presence and absence of 2-mercaptoethanol in the elution buffer.

Fractionation of soluble supernatant fractions

of hamster liver homogenates on Sephacryl S-200 produced three peaks of bile salt sulfotransferase activity, corresponding to apparent molecular weights of 45000, 60000 and 200000 (Fig. 2). The 200000 molecular weight protein peak was active with both bile salt substrates. The 60000 molecu- lar weight peak was detected with glycolitho- cholate as substrate and the 45000 molecular weight peak with glycochenodeoxycholate as sub- strate. The relative proportion of bile salt sulfotransferase activity in each peak showed some interanimal variation. In certain animals a large proportion of the bile salt sulfotransferase activity appeared as the 200000 molecular weight peak, whereas in other animals the 45000 molecular weight fraction predominated. Prolonged storage had no effect on the chromatographic profile for individual samples (data not shown).

Similarly, rat liver cytosol yielded three peaks of activity, corresponding to molecular weights of 130000, 64000 and 32000 (Fig. 3). The predomi- nant bile salt sulfotransferase activity with glyco- chenodeoxycholate as substrate (75% of total) was eluted as the 32000 molecular weight peak, whereas with glycolithocholate as substrate the 64000 molecular weight peak predominated. Both results were inconsistent with the work of Chen et al. [1], who found that the 130000 molecular weight peak was the major activity. However, these workers and others routinely add 2-mercaptoethanol to the homogenization and elution buffers to preserve enzyme activity, which may be a factor influencing molecular weight.

Effect of 2-mercaptoethanol In contrast to hamster bile salt sulfotransferase

activity, which was not significantly altered by high concentrations of 2-mercaptocthanol, the elu- tion profile of rat liver bile salt sulfotransferase activity with both bile salts as substrate was sensi- tive to the inclusion of 2 mM 2-mercaptoethanol in the elution buffer (Fig. 4). The 130000 molecular weight peak represented 70% of the total activity when 2-mercaptoethanol was present with either substrate. Raising the 2-mercaptoethanol con- centration to 50 mM did not further increase the proportion of the 130000 molecular weight peak. The other bile salt sulfotransferas¢ activity had the same molecular weight as the major peak seen in

357

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Fig. I. (left-hand figure) Fractionation of hepatic bile salt sulfotransferase (BAST) activity on DEAE-cellulose. Bile salt sulfotransferase activity from hamster (A) and rat (B) livers after loading the supematant fractions and washing with 10 mM sodium phosphate/5 mM MgSO4 buffer (pH 7.0) was eluted with a 0-0.5 M NaCI gradient. Protein (,42so, m, • . . . . . ) and ionic strength (s (mS), ) are shown. Bile salt sulfotransferase activity is shown for glycochenodeoxycholate (0) and glycolithocholate (O) as substrates. Bile salt sulfotransferase attivity was normalized by setting the largest enzyme activity equal to 100 and adjusting all the other activities accordingly.

Fig. 2. (upper right) Fractionation of hamster hepatic bile salt sulfotransferase (BAST) activity by gel filtration. Soluble supernatant fractions (0.5 ml) were separated by gel filtration on a 90× 1.6 cm Sephacryl S-200 column, and eluted with 100 mM sodium phosphate/5 mM MgSO4 (pH 7.0). Bile salt sulfotransferase activity is shown for glycochenodeoxycholate (0) and glycolithocholate (O) as substrates. The first peak of bile salt sulfotransferase activity for both substrates has a molecular weight of 200000. The other peaks correspond to molecular weights of 45000 (for glycochenodeoxycholate) and 60000 (for glycolithocholate).

Fig. 3. (lower fight) Fractionation of rat hepatic bile salt sulfotransferase (BAST) activity by gel filtration. Soluble supernatant fractions (0.5 mi) were separated on a Sephacryl S-200 colunm as described in Fig. 2. Bile salt sulfotransferase activity is shown with glycochenodeoxycholate ( ) as glycolithocholate ( . . . . . . ) as substrates.

358

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Fig. 4. Effect of 2-mercaptoethanol on molecular weight of rat hepatic bile salt sulfotransferase (BAST) activity. Soluble su- pernatant fractions (0.5 ml) were separated as described in Fig. 2. 2-Mercaptoethanol was added to the sample and the buffer to give a final concentration of 2mM. Bile salt sulfotransferase activity is shown with glycochenodeoxycholate (@) and glycolithocholate (O) as substrates. The first peak of bile salt sulfotransferase activity has a molecular weight of 130000. The other two peaks correspond to molecular weights of 32000 (for glycochenodeoxycholate) and 64000 (for glyco- lithocholate).

the absence of 2-mercaptoethanol, namely the 64000 molecular weight peak with glycolitho- cholate as substrate and the 32000 molecular weight peak with glycochenodeoxycholate as sub- strate.

Since 2-mercaptoethanol usually reduces dis- ulfide bridges to their sulfhydryl forms and thereby promotes dissociation of protein subunits, it was surprising that 2-mercaptoethanol apparently caused an increase in molecular weight.

Effect of alkylation by iodoacetamide The effect of alkylation, of the proteins in the

rat liver soluble supernatant fraction with di- thiothreitol and iodoacetamide was studied using gel filtration on Sephacryl S-200. A control sample was treated with 10 mM dithiothreitol, but not iodoacetamide, and was dialyzed to remove di- thiothreitol. When it was chromatographed on Sephacryl S-200 in the presence and absence of 2-mercaptoethanol, two peaks of bile salt sul- fotransferase activity were detected with glyco- chenodeoxycholate as substrate, corresponding to molecular weights of 32000 and 130000 (Fig. 5A). In the presence of 2-mercaptoethanol approxi- mately equal amounts of each of the 32000 and 130000 molecular weight forms were detected, whereas in the absence of 2-mercaptoethanol the

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Fig. 5. Effect of 2-mercaptoethanol and iodoacetamide on the gel filtration of rat liver bile salt sulfotransferase (BAST). A. A soluble supernatant fraction of rat liver, isolated in the absence of 2-mercaptoethanol, was chromatographed in the presence ( . . . . . . ) and absence ( ) of 2 mM 2-mercaptoethanol on Sephacryl S-200. Glycochenodeoxycholate was the bile salt substrate. B. The same soluble supernatant fraction of rat liver used in A was reduced with dithiothreitol and alkylated with iodoacetamide. After dialysis it was separated in the presence ( . . . . . . ) and absence ( ) of 2 mM 2-mercaptoethanol on Sephacryl S-200.

32000 molecular weight form predominated. When alkylation of the same sample was car-

ried out with dithiothreitol and iodoacetamide (see Methods), the resulting elution profile from Sep- hacryl S-200 in the absence of 2-mercaptoethanol was similar to that obtained by adding 2- mercaptoethanol to the non-alkylated sample (Fig. 5B). The inclusion of 2 mM 2-mercaptoethanol in the elution buffer in the chromatography of the alkylated sample had only a minor effect on the elution profile of bile salt sulfotransferases (Fig. 5B).

Discussion

This study has demonstrated that the enzyme(s) from hamster and rat liver which catalyze the transfer of sulfate from PAdoPS to bile salts can be separated into at least three forms. The evi- dence for multiple enzymes was based on separa- tion by charge at pH 7.0 using DEAE-cellulose ion-exchange chromatography and by gel filtra- tion.

In contrast, previous investigators demonstra- ted only a single enzyme activity [1] using gel filtration to fractionate partially purified bile salt

sulfotransferase activity from rat liver. It is possi- ble that their method used to partially purify rat liver bile salt sulfotransferase activity eliminated the 32000 and 64000 molecular weight forms of the enzyme. Alternatively, assay conditions may not have been sufficiently sensitive to detect the 32000 and 64000 molecular weight forms. In addi- tion, the reducing agent 2-mercaptoethanol was used in the previous study [1] and we have shown that this increases the proportion of the 130000 molecular weight peak when both glycochenodeo- xycholate and giycolithocholate were the sub- strates.

In the presence of 2-mercaptoethanol, rat liver bile salt sulfotransferase forms a polymeric unit, possibly a tetramer. Since alkylation of sulfhydryl groups with iodoacetamide produced enzyme elu- tion patterns similar to that obtained with 2- mercaptoethanol, reduction of intramolecular dis- ulfide bonds may expose additional hydrophobic regions and promote association to a tetrameric form. Reduction of intramolecular disulfide bridges could also cause unfolding of a molecule if it is a tightly wrapped ball and hence increase its effec- tive hydrodynamic radius, the parameter which determines its apparent molecular weight when analyzed by gel filtration. In this case, a 60% increase in effective diameter would be necessary to explain the observed data.

A similar association has been reported for other sulfotransferases; estrone sulfotransferase is converted from a monomeric form of molecular weight 65000 to a trimer or tetramer under the influence of 2-mercaptoethanol [16], and andros- tenolone sulfotransferase associates in the presence of cysteine [17]. Purified rat liver hydroxysteroid sulfotransferase II, with a molecular weight of 290000, forms aggregates which are inactive [18] when it is isolated in the presence of 2-mer- captoethanol.

Since the site of sulfation is different for gly- colithocholate (3a-position) [1] and glycocheno- deoxycholate (7a-position) [3], unique bile salt sulfotransferase activities were suspected for gly- colithocholate and glycochenodeoxycholate. None- theless, the peak activities detected with each sub- strate corresponded to the same molecular weights as observed by gel filtration and the same residual charge by ion-exchange chromatography. How-

359

ever, there were distinct differences in substrate specificity between the 32000 and 64000 molecu- lax weights peaks. This could be explained by changing substrate specificity between the pro- posed monomeric, dimeric and tetrameric forms. However, it is also conceivable that separate 32000 molecular weight subunits occur which associate to a differing extent. This would explain the observed results if PAdoPS caused dissociation to the monomer form in the reaction mixture.

Three bile salt sulfotransferase activities of molecular weights 45000, 60000 and 200000 were separated by gel filtration from hamster liver. The 200000 molecular weight peak was observed in variable amounts; in some animals it was more than 50% of the total bile salt sulfotransferase activity. These activities may be either oligomeric, or separate enzymes. Unlike rat liver bile salt sulfotransferase activity, 2-mercaptoethanol had only a small effect on the profile of hamster liver bile salt sulfotransferase activities separated by gel filtration.

Previous attempts to fractionate rat liver bile salt sulfotransferase activity on DEAE-Sephadex yielded only one peak [1]. Our findings have been confirmed in a recent abstract by Kane and his colleagues [15], who separated two bile salt sulfotransferase activities from female rat liver soluble supernatants using DEAE-Sephadex ion- exchange chromatography.

DEAE-cellulose ion-exchange chromatography of hamster liver soluble supernatant also produced two peaks of bile salt sulfotransferase activity; however, the broad nature of the peaks, unlike that observed for rat liver bile salt sulfotransferase activites, suggested that additional enzymes may be present. In both hamsters and rats, the bile salt sulfotransferase peak eluting at 100 mM NaC1 sulfated the monohydroxy bile salt, glycolitho- cholate, more efficiently than the other peak.

Future studies on bile salt sulfotransferase ac- tivity from rat and hamster liver should therefore take into account affects on the component enzymes as well as the overall enzyme activity.

Acknowledgements

This study was supported in part by Federal grant No. CA-13148 from the National Cancer

360

Institute and grant No. AM-25511 from the Na- tional Institut0s of Health, by a Faculty Research Grant from the Graduate School University of Alabama in Birmingham, and by the Veteran's Administration Medical Research Service. S.B. was a Fellow of the National Library of Medicine, Program Grant number 5TI 5LM07015 during part of these studies. The technical assistance of Mr Rodney Waldrop and Ms Diana Howell is appre- ciated. Dr. Awni Sarrif, formerly of the Depart- ment of Pharmacology, provided the substrates for measurement of glutathione S-transferase activity. Ms. Sharon Garrison kindly typed the manuscript.

References

1 Chen, L.-J., Bolt, R.J. and Admirand, W.H. (1977) Biochim. Biophys. Acta 480, 219-227

2 Chen, L.J., Thaler, M.M., Bolt, R.J. and Golbus, M.S. (1978) Life Sci. 22, 1817-1820

3 Barnes, S., Burhol, P.G., Zander, R., Haggstrom, G. and Hirschowitz, B.I. (1979) J. Lip. Res. 20, 952-959

4 Loof, L. and Wengle, B. (1978) Biochim. Biophys. Acta 530, 451-460

5 L66f, L. and Hjert~n, S. (1980) Biochim. Biophys. Acta 617, 192- 204

6 Adams, J.B. and Poulos, A. (1967) Biochim. Biophys. Acta 146, 493-508

7 Ryan, R.A. and Carroll, J. (1976) Biochim. Biophys. Acta 429, 391-401

8 Singer, S.S., Gebhart, J. and Hess, E. (1978) Can. J. Bio- chem. 56, 1028-1135

9 Sekura, R.D. and Jakoby, W.B. (1979) J. Biol. Chem. 254, 5658-5663

10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193,265-275

11 Bergmeyer, H.U. and Brent, E. (1974) in Methods of En- zymatic Analysis (Bergmeyer, H.U., ed.), p. 574, Academic Press, New York

12 Habig, W.H., Pabst, M.J. and Jakoby, W.B. (1974) J. Biol. Chem. 249, 7130-7139

13 Hofstee, B.H.J. (1959) Nature 184, 1296-1298 14 Huston, J.S., Fish, W.W., Mann, K.G. and Tanford, C,

(1972) Biochemistry I1, 1609 15 Kane, R.E., Chen, L.-L and Thaler, M.M. (1981) Gastroen-

terology 80, 1337 16 Adams, J.B. and Edwards, A,M. (1968) Biochim. Biophys,

Acta 167, 122-140 17 Adams, J.B. and Chulavatnatol, M. (1967) Biochim. Bio-

phys. Acta 146, 509-521 18 Marcus, C.J., Sekura, R.D. and Jakoby, W.B. (1980) Anal.

Biochem. 107, 296-304