THE JOURNAL OF BIOLOGICAL CHEMISTRY

Amine N-Sulfotransferase*

Vol. 262, No. 21, Issue of July 25, pp. 10039-10043,1987 Printed in U.S.A.

(Received for publication, January 16, 1987)

Sengoda G. Ramaswamy and William B. Jakoby From the Laboratow of Biochemistry and Metabolism, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes bf Health, Bethesda, Maryland 20892

A highly purified amine N-sulfotransferase has been isolated from guinea pig liver that catalyzes sulfuryl group transfer from 3’-phosphoadenosine 5’-phospho- sulfate to one of a large number of either primary or secondary amines forming the appropriate sulfamate and adenosine 3’,5’-bisphosphate. Amines as differ- ent as aniline, 2-naphthylamine, ocytlamine, 1,2,3,4- tetrahydroquinoline and 1,2,3,4-tetrahydroisquino- line, desmethylimipramine, and cyclohexylamine serve as acceptors; the product of the last of these substrates is the sugar-substitute cyclamate. Amine N- sulfotransferase activity is dependent on the presence of an unprotonated amino group. The purified enzyme preparation also has 0-sulfotransferase activities, sug- gesting that transfer to oxygen could represent an intrinsic function of the N-sulfotransferase.

The conjugation of amino groups with sulfate was suggested as a natural process when 2-naphthylsulfamate was isolated from the urine of animals to which 2-napthylamine had been administered (1). Soon thereafter, preparations from mam- malian liver (2, 3) and from sheep intestinal mucosa (4) were shown to catalyze N-sulfation of 2-naphthylamine in the presence of a sulfate “activating” system. These early results have been confirmed by the recent isolation, in amounts adequate for characterization, of 2-naphthylsulfamate as a product of the reaction catalyzed by preparations from guinea pig liver (5). The scope of the reaction remained undefined although arylamines such as aniline (2, 6, 7), alkylamines such as tridecylamine (6), and secondary amines such as desmethylimipramine (6, 7) were found to serve as acceptors for sulfuryl group transfer with liver preparations.

Guinea pig liver, a tissue found by Roy (2, 3) to be active in naphthylsulfamate formation, has been used to characterize the responsible enzyme with the view that sulfamate forma- tion could participate in the process of detoxication, i.e. in metabolizing foreign compounds so as to render them phar- macologically inactive or more readily excretable. Enzymes active in detoxication are characterized by their affinity for a broad spectrum of lipophilic substrates ( t i ) , a criterion that appears to be fully met by the amine N-sulfotransferase that catalyzes Reaction 1 and is described here. In addition to primary aryl, aliphatic, and alicyclic amines, the enzyme is active with secondary amines (Reaction 2); in each instance

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3”phosphoadenosine 5’-phosphosulfate (PAPS)’ serves as the sulfuryl group donor.

RNH2 + PAPS + RNHSO3H + PAP (1)

RNR’H + PAPS + RNR’SO3H + PAP (2)

MATERIALS AND METHODS

Livers from adult guinea pigs were obtained from Pel-Freez Bio- logicals and stored at -70°C until use. The several amines tested were purchased at the highest grades available from either Sigma or Aldrich; the former also supplied adenosine 3’,5’-bisphosphate (PAP) and adenosine 5’-phosphosulfate. Adenosine 3’,5’-bisphosphate-aga- rose was obtained from P-L Biochemicals. PAPS from Sigma had been used for enzyme assay but was found to contain an inhibitor of the reaction, probably PAP, that was hydrolyzed by bisphosphate nucleotidase (9). PAPS from P-L Biochemicals was of greater purity and was satisfactory. [3SS]PAPS, a product of Du Pont-New England Nuclear, had a specific activity of about 3.1 Ci/mmol.

Electrophoresis Gel electrophoresis was performed in cylindrical nondenaturing

gels according to Davis (10) and with sodium dodecyl sulfate vertical slab gels according to Laemmli (11). For both electrophoresis and gel filtration the following proteins were used as representative globular proteins: pancreatic ribonuclease, soybean trypsin inhibitor, carbonic anhydrase, ovalbumin, and bovine serum albumin (all from Phar- macia).

Sulfotransferase Assays The standard enzyme assay represents a modification of a method

that was described to exploit the differential partition into chloroform of the ion pair formed between many organic sulfate esters or sulfa- mates with methylene blue (12, 13). For assay of N-sulfation, the enzyme was incubated in a total volume of 400 pl containing 200 mM potassium bicine at pH 8.0, 2 mM 2-naphthylamine-HC1 and 1 mM PAPS. After 1 h at 37 “C, the reaction was stopped by addition of 0.5 ml of methylene blue reagent (250 mg of methylene blue, 50 g of anhydrous sodium sulfate, and 10 ml of concentrated sulfuric acid per liter). Chloroform, 2 ml, was added and the mixture agitated with a vortex mixer. After brief centrifugation at 2000 X g, the chloroform layer was transferred to a tube containing 50-100 mg of anhydrous sodium sulfate. Absorbance was determined with a 1-cm light path at 651 nm; an A,, of 6.0 is equivalent to the formation of 1 pmol of 2- naphthylsulfamate. The reaction is a linear function of time and of protein concentration when 10-130 nmol of naphthylsulfamate are formed. Because of the greater solubility of methylene blue in chlo- roform at high ionic strength of the aqueous phase, a control reaction mixture from which PAPS is omitted must be used to compensate for the different conditions encountered in each fraction when eluting columns with a salt gradient.

Since the ion-pair method is relatively insensitive and is very much less effective with other products of the amine N-sulfotransferase- catalyzed reaction, a procedure was adopted by which [3sS]PAPS served as sulfuryl group donor. The reactive sulfamate was subse- quently separated by thin layer chromatography. The enzyme, in a

’ The abbreviations used are: PAPS, 3’-phosphoadenosine 5’-phos- phosulfate (or the synonym, adenosine 3”phosphate 5’-phosphosul- fate); PAP, adenosine 3’,5’-bisphosphate; HPLC, high performance liquid chromatography.

10039

10040 Amine N-Sulfotransferase

total volume of 50 pl, was incubated in 0.2 M potassium bicine at pH 8.0, 2 mM amine, and 1 mM [%]PAPS (0.5 pCi) for 30 min at 37°C. After the reaction was terminated by addition of 20 p1 of 2 M acetic acid, aliquots of 5 pl were applied to a 2 X 10-cm strip of an Eastman cellulose chromatogram sheet (No. 13255), dried, and subjected to ascending chromatography at room temperature for about 20 min, i.e. the period required for the solvent front to move about 8 cm. The solvent system, 1-propano1:ammonia:water (63:1), is generally appli- cable for separating radioactive sulfamates from interfering radioac- tivity (14). After drying, a section of the chromatogram, from the solvent front to 2 cm below it, was cut and added to 10 ml of Aquasol in a scintillation vial and radioactivity was measured. Although the sensitivity of the method can be tailored to need by changing the specific activity of PAPS, the conditions presented were adequate in allowing measurement of between 0.1 and 2 nmol of sulfamate. When tested with 2-naphthylamine, the assay was linear with time and protein concentration over the suggested range of product; the data were within 10% of the value expected when the ion-pair and the chromatographic procedures were compared. The formation of sul- famates was observed qualitatively by autoradiography (Kodak XAR5 film) of the TLC plates.

One unit of activity is defined as that amount of enzyme catalyzing the formation of 1 nmol of sulfamate/min. Specific activity is in terms of units of activity/mg of protein. Protein was determined by the method of Bradford (15) with crystalline bovine serum albumin (Pentex) as standard.

Phenol sulfotransferase activity was determined as described with 2-naphthol (16); alcohol sulfotransferase activity was determined chromatographically under the conditions described for 1-butanol and dehydroepiandrosterone (17), and activity with phenolic steroids was determined with estrone (18).

Isolation and Analysis of Product 2-Naphthylamine, 60 pmol, was incubated in a total volume of 3

pmol PAPS in 0.2 M bicine at pH 8.0 and 37 "C. After 3.5 h, 30 ml of ml with 2.4 units of enzyme (after Step 2 of the purification) and 5

95% ethanol were added and the mixture was cooled on ice and centrifuged. The supernatant liquid was evaporated at 40 "C under nitrogen and the residue dissolved in 1 ml of 50 mM triethylamine carbonate at pH 7.6. The preparation was charged onto a column (0.5 X 4 cm) of DEAE-cellulose (Whatman, DE52), equilibrated with the same buffer, and eluted as described by Sekura and Jakoby (16) with triethylamine carbonate. The column was monitored by absorbance at 277 nm and that peak corresponding to 2-naphthylsulfamate was pooled and subjected to repeated flash evaporation at about 40 "C in order to eliminate triethylamine. The residue was dissolved in 2 ml of water. Portions of 250 p1 were subjected to HPLC using a Zorbax CN column (4.6 X 250 mm, Du Pont) that was eluted with a linear gradient of 0-100% methanol in 12.5 mM formic acid over 30 min. The material eluting as a sharp peak at 10.3 min, at which authentic 2-naphthylsulfamate was found, was retained.

Evaporation under nitrogen of the eluate from several 250-p1 samples that had been combined allowed analysis by mass spectrom- etry. Negative ion fast atom bombardment-mass spectra were ob- tained on a V6 Analytical 7070E mass spectrometer (Altrincham, Chesire, Great Britain) equipped with a V6 fast atom bombardment ion source operated at an accelerating voltage of -6 kV. Glycerol was used as the sample matrix and ionization was effected by a beam of xenon atoms derived by charge exchange neutralization of a 1.2-mA beam of xenon ions accelerated through 8.4 kV. Spectra were acquired at a scan speed of 10 s/decade over the mass range 100-600 by using a V6 Analytical 2035 data system; the background due to the glycerol matrix was automatically subtracted.

Purification of Amine Sulfotransferase The enzyme was purified from guinea pig livers which had been

stored for as long as 6 weeks at -70°C without significant loss in enzyme activity. All preparative methods were conducted at 0-4 "C.

Step I: Extract-Approximately 250 g of liver were homogenized with a Waring blender at maximum speed for 1 min in 600 ml of buffer A (20 mM potassium bicine at pH 7.5 containing 1 mM EDTA, 5 mM P-mercaptoethanol, 20% sucrose (w/v), and 0.02% sodium azide). The homogenate was filtered through cheesecloth and centri- fuged at 10,000 X g for 20 min. The supernatant fluid was also filtered through cheesecloth and then centrifuged at 100,000 X g for 60 min. The residue was discarded.

Step 2: DEAE-Cellulose-The extract (440 ml) was applied directly

TABLE I Summary of purification of amine N-sulfotransferase

Step Total Amine Specific protein activity activity

1. Extract 440 12,400 7,870 0.63 2. DEAE-cellulose 245 3,250 956 0.29 3. Salting out 52 1,640 1,140 0.70 4. PAP-agarose-1 365 1,106 1,000 9.7 5. Chromatofocusing 30 12 186 16 6. PAP-agarose-2 31 3.6 136 37 7. Gel filtration 17 1.9 94 50

to a column (4 X 40 cm) of DEAE-cellulose (Whatman, DE52) which had been equilibrated with buffer A. The column was washed with about 200 ml of the buffer and eluted with a linear KC1 gradient of 0-0.4 M in 1.6 liters of buffer A (Fig. L4). Active fractions of 13 ml each (fractions 45-65) were pooled.

Step 3: Salt Fractionation-To the pooled eluate (245 ml) were added 57.3 g of ammonium sulfate. The suspension was stirred for 45 min and centrifuged at 10,000 X g for 30 min. The precipitate was dissolved in about 40 ml of buffer A and dialyzed against three changes of 4 liters each of buffer A for a total of 36 h. The volume after dialysis was 52 ml.

Step 4: PAP-Agarose-1-The salt fraction was charged onto a column of PAP-agarose (2.5 X 12 cm) previously equilibrated with buffer A. The column was washed with about 200 ml of the same buffer and eluted with a 0-0.3 M gradient of KC1 in 600 ml of the buffer. After passage of all 600 ml, an additional 25% of enzyme activity was recovered by allowing 100 ml of 0.3 M KC1 to flow through the column. The active fractions, 13 ml each, usually eluted at a position corresponding to a conductivity of 3.4-9.5 mmho (Fig. 1B), were pooled (365 ml), concentrated by Amicon filtration using a YM-10 membrane, and dialyzed against buffer A (three changes of 2 liters each over 18 h).

Step 5: Chromatofocusing-The dialyzed enzyme solution was ap- plied to a chromatofocusing column (1.6 X 30 cm, Pharmacia PBE94) which had been equilibrated with 25 mM Tris-HC1 at pH 8.3 contain- ing 1 mM EDTA, 5 mM 2-mercaptoethanol, and 20% sucrose (w/v). The column was eluted with Polybuffer 74 (Pharmacia) which had been diluted 1:4 with water, followed by adjustment of the pH to 5.0 with 1 M acetic acid. The active fractions, 3 ml each between pH 6.6 and 6.9 (Fig. lC), were pooled (fractions 57-65).

Step 6: PAP-Agarose-2-The preparation was applied to a column (0.8 X 10 cm) of PAP-agarose that had been equilibrated with buffer A. After washing with 50 ml of the same buffer, enzyme was eluted with a gradient of 100 ml of the buffer ranging from 0 to 50 p M in PAPS. The active fractions (fractions 16-41, Fig. lD), about 30 ml, were concentrated to 4 ml with an Amicon YM-10 membrane.

Step 7: Sephadex G-100-The concentrated fraction was applied to a column of Sephadex G-100 (1.6 X 80 cm) that had been washed with buffer A and was eluted with the same buffer (Fig. 1E). The active fractions (fractions 14-19) were pooled (20 ml).

At this stage, the enzyme should be concentrated to at least 0.5 mg of protein/ml, at which point it retains its activity for a t least 3 months at 4 "C in buffer A. The purification is summarized in Table I.

RESULTS

The elution pattern from DEAE-cellulose of amine N- sulfotransferase activity consisted of a broad plateau of activ- ity beginning at a salt concentration yielding a conductivity of about 1 mmho, Enzyme activity was completely lost within 12 h unless stabilized with 20% (w/v) sucrose for the fraction which may be considered as the initial "peak" (tubes 45-65 in Fig. lA) and by 20% glycerol for eluates that appeared there- after. Only sucrose, and not glycerol, was effective for the enzyme in the initial peak which was retained and purified further. It will be apparent from Fig. 2 that a number of sulfotransferases are present in guinea pig liver and that each of these activities is spread over the entire elution pattern from DEAE-cellulose.

FIG. 1. Elution patterns of steps in the purification of the amine N- sulfotransferase from columns of DEAE-cellulose ( A ) , PAP-agarose- 1 (Step 4) ( B ) , chromatofocusing (C), PAP-agarose-2 (Step 6) (D) , and gel filtration on Sephadex G- 100 ( E ) . Dotted line represents con- ductance for A and B, and pH for C. The l i n e without points denotes Am. Sulfo- transferase activity is presented for 2- naphthylamine (0) and 2-naphthol (W). The specific fractions used for further processing are indicated by the solid bars.

. f 2

1

0 40 80 l o o 120

Eluant Fraction FIG. 2. Elution pattern of guinea pig liver from a column

(2.5 X 16 cm) of DEAE-cellulose (DE52) in which sulfotrans- ferase activities are shown for 2-naphthlamine (O), 2- naphthol (I), dehydroepiandrosterone (O), and estrone @). Liver, 20 g, was treated as outlined in Step I, charged onto the column, and eluted in a 400-ml salt gradient as in Step 2. Fractions of 3 ml were collected and assayed by the appropriate [%]PAPS method using thin layer chromatography.

Purification was directed at the removal of contaminating sulfotransferases acting on phenols (16, 19) and on alcoholic (17, 20) or phenolic steroids (18). Nevertheless, all of these 0-sulfotransferase activities were present in the final prepa- ration. A (2')3'-bisphosphate nucleotidase (EC 3.2.1.1), an enzyme hydrolyzing both PAPS and PAP at the 3"phosphate and thereby interfering with the assay (9), was removed by Step 4 of the purification procedure. Further attempts at purification in order to reduce phenol and alcohol sulfotrans- ferase activity using HPLC with either DEAE-silica or gel filtration columns were unsuccessful and resulted in large losses in activity. Similar lack of success was encountered with hydrophobic (butyl-, octyl-, and benzyl-agarose) and affinity (ATP- and ADP-agarose) columns.

Upon electrophoresis in nondenaturing polyacrylamide gel, the purified enzyme displays a broad, diffuse band and a minor additional band of protein. This observation is readily reproducible. At higher protein concentrations, the minor band comprises about 5% of stained material (Fig. 3). The broad major band encompasses sulfotransferase activity with amine, phenol, and hydroxysteroid, each of which has its peak activity in the same slice (Fig. 3). Not shown in the figure is activity with estrone which, in a separate experiment and gel, also has its activity in a broad band with the peak coincident

9 15 I

Slice FIG. 3. Sulfotransferase activity eluted after trituration

with 0.2 ml of buffer A of polyacrylamide gel slices of ap- proximately 2 mm each along the length of a gel cylinder. Staining of the gels representing 10 and 80 pg of protein are shown at the top. Activity is shown with 2-naphthylamine (O), dehydroe- piandrosterone (01, and 2-naphtha1 (W). The activities are presented to show their position on the gel but the specific activities are not to be taken quantitatively since the assays were under different condi- tions and for different periods of time.

with that for sulfation of 2-naphthylamine. Electrophoresis in sodium dodecyl sulfate gels revealed two

protein bands of about equal density with M , estimated a t 33,000 and 34,000 in comparison with globular protein standards; no other bands staining with Coomassie Blue were visible. Gel filtration with Sephadex G-75 allowed an estimate of M , of 60,000 based on the assumption that the enzyme is a globular protein of the same shape and partial specific volume as the protein standards used. The PI of the enzyme was found to be 7.0 by the method of Wrigley (21) using Ampholines (Pharmacia) in the range of pH 3-10.

Substrates for N-Sulfation-A wide variety of amines served as substrates despite large differences in carbon skeleton (Table 11). Included were a number of aryl primary amines (aniline, 4-chloroaniline, and 2-naphthylamine) and an aryl secondary amine (1,2,3,4-tetrahydroisoquinoline). Octyla- mine was a substrate as was the secondary amine, des- methylimipramine. Cyclohexylamine was also a substrate, the

10042 Amine N-Sulfotransferase

TABLE I1 Amine sulfotransferase substrates

Assays were conducted by incubation of enzyme, 2 mM of acceptor amine, 1 mM PAPS, and either 0.1 M potassium bicine at pH 7.2 or 0.2 M glycine at pH 10 for 60 min at 37 'C.

Substrate Specific activity

pH 7.2 pH 10.0 nmol min" mg"

Aniline 3.1 0.8 4-Chloroaniline 15.7 9.2 Cyclohexylamine 0.1 0.7 Desmethylimipramine 0 6.3 2-Naphthylamine 29.0 6.3 n-Octylamine 0.2 4.5 4-Phenyltetrahydropyridine 1.4 23.0 1,2,3,4-Tetrahydroisoquinoline 0.6 31.6 1,2,3,4-Tetrahydroquinoline 15.6 6.5

A I

0

6 8 10

pH FIG. 4. Amine N-sulfotransferase activity as a function of

pH and sulfuryl group acceptors. Upper p a n e l , 2-Naphthylamine (0), 1,2,3,4-tetrahydroisoquinoline (A), tetrahydroquinoline (01, and octylamine (0). Lower panel, 4-chloroaniline (U), aniline (01, and desmethylimiprimine (0). The following buffers were used in the indicated pH ranges: succinate (pH 5.0-6.5), cacodylate (pH 6.5-7.51, bicine (pH 7.5-9.0), and glycine (pH 9.0-10.7).

product of which is the sugar-substitute cyclamate. The data in Table I1 were obtained at two pH values because of the pattern observed for the pH optima of the enzyme. It will be evident from Fig. 4 that the arylamines, both primary and secondary, were poor substrates at pH values below 6.0, con- sistent with their relatively acidic pK, (22). In contrast, the two non-arylamines tested had their optimal pH at 9 or above, in keeping with their pK values in the alkaline range (22). The data in Table I1 confirm these relationships for several additional substrates.

The following amines also served as acceptors when tested qualitatively with [35S]PAPS under otherwise standard assay conditions followed by TLC and autoradiography: 4-amino- acetophenone; l-amino-4-nitronaphthalene, 4-tert-butylani- line, N-methylaniline, 1-naphthylamine, tridecylamine, and toluidine. Pyridine and its 4-amino, 4-chloro, and 4-phenyl derivatives were inactive as were diaminoanthroquinone, 6- phenethylamine, and saccharin.

Kinetic data were difficult to obtain, particularly for the less active substrates, because of a tendency toward substrate inhibition at the high concentrations of amine that were

necessary. For the more active acceptor amines, under oth- erwise standard assay conditions of pH 8.8 and 1 mM PAPS, the following values for apparent K , (and Vm,, in terms of nmol/min/mg of protein given in parentheses) were calcu- lated 2-naphthylamine, 3.4 M (59); 1,2,3,4-tetrahydroiso- quinoline, 14 mM (250); and 1,2,3,4-tetrahydroquinoline, 38 mM (740). With 2 mM 2-naphthylamine and otherwise stan- dard assay conditions, a K, of 30 p~ was calculated for PAPS.

Substrates for 0-Sulfation-Under standard assay condi- tions for phenol and alcohol sulfotransferase, respectively, the following compounds were found to serve as substrates when tested at a concentration of 2 mM in comparison to 2-naph- thylamine which is taken as 100: 2-naphthol, 10; norepineph- rine, 5; pentachlorophenol, 3; tyrosine methyl ester, 13; de- hydroepiandosterone, 27; and estrone, 17. I t should be noted that pentachlorophenol, which serves as an effective substrate here, is usually regarded as a potent inhibitor of phenol sulfotransferases (23, 24) and, with the phenol sulfotransfer- ase from rat liver, has a Ki of 0.2 pM (24).

Effect of Magnesium-Although the enzyme could be stim- ulated optimally by approximately 90% in the presence of 20 mM MgC12, the use of M$+ was avoided in the early fraction- ation steps because of the presence of significant (2')3',5'- bisphosphatase (9), an enzyme that requires Mg2+ for activity. In order to avoid phosphatase action, a chelating agent was used in most of the buffers with which the enzyme was in contact.

Identification of Product-2-Naphthylsulfamate was iden- tified by its yield of an ionic species of mass 222 after fast atom bombardment in accord with the same species observed with an authentic sample of the sulfamate. Both standard and isolated product displayed maximum absorbance at 268 nM. Attempts at hydrolyzing the product with a number of sulfa- tases that are effective with 0-sulfates, from a variety of sources (Helix pomatia, limpet, Aerobacter aerogenes, and abalone, all from Sigma), were unsuccessful.

Reuersibility-Attempts to demonstrate reversibility of amine N-sulfotransferase were not successful. With 1 mM 2- naphthylsulfamate, 1 mM PAP, and 6 units of enzyme/ml, no evidence was obtained for the formation of naphthylamine. Reaction products were examined by HPLC with a "NOVA- P A K Cls column (3.9 X 150 mm, Waters) and a gradient of 0-100% methanol in PIC reagent B7 (Waters); in this system, 2-naphthylamine has a retention time of 19.5 min.

Sources of Enzyme-Extracts prepared from a number of freshly obtained organs, homogenized, and centrifuged as for Step 1 of the purification procedure allowed detection of N- sulfotransferase when activity was greater than 10 pmol/min/ mg of protein. For liver from guinea pig, rat, and rabbit these values were 290, 180, and 60, pmol min-' mg", respectively. As a lower limit, based on the specific activity of cell-free preparations, a mature guinea pig liver of about 10 g would be able to convert 0.3 pmollmin 2-naphthylamine to its sul- famate and about half that of 4-chloroaniline or 1,2,3,4- tetrahydroquinoline. Also active were intestinal mucosa from the rat (210 pmol/min/mg) and rabbit (40 pmol/min/mg) but not from guinea pig, and kidney from the rat (50 pmol/min/ mg) but not kidney from either guinea pig or rabbit. Activity in lung, if any, was below 10 pmol/min/mg for each of the three animals as it was from brain, heart and spleen of the rat.

DISCUSSION

The amine N-sulfotransferase from guinea pig liver, al- though highly purified, does not appear to be homogeneous: native polyacrylamide gels show an additional, slower migrat-

Amine N-Sulfotransferme 10043

ing band of protein when high concentrations of enzyme are applied (Fig. 3). The major band contains the sulfotransferase activity that is spread over a broad area, suggesting, as one possibility, a rapidly dissociating-associating protein, an ob- servation consonant with this enzyme's requirement for high concentrations of sucrose for stability (25, 26). The possible lack of homogeneity is reinforced by the observation that amine N-sulfotransferase, phenol 0-sulfotransferase, alcohol 0-sulfotransferase and estrone 0-sulfotransferase activities are all present at the highest level of purity that we were able to achieve.

The results, therefore, may be explained by contamination of a distinct amine N-sulfotransferase by three O-sulfotrans- ferases. All four activities seem to be present as isoenzymes in extracts of guinea pig liver (Fig. 2). However, only a small portion of the total phenol sulfotransferase activity was pres- ent in the early eluates from DEAE-cellulose that were used for purification of the amine sulfotransferase (Fig. 2); eluates from the higher salt concentrations were avoided because they contained the major portion of phenol sulfotransferase. That each of the four sulfotransferases was present in a single protein band would be in accord with the known size of about 60,000 daltons for most of the highly purified phenol sulfo- transferases (16,19) and for an adrenal estrone sulfotransfer- ase (27) but not for the known alcohol sulfotransferases, which are at least twice that size (17, 20, 28).

Alternatively, the data could be viewed as suggesting that all four sulfotransferase activities are intrinsic to a single enzyme. The unusually large spread of concomitant enzyme activity for each of the four sulfotransferases (Fig. 3) and for protein over nearly a centimeter of gel, with all four sulfo- transferase activities peaking at the same gel slice, provides an argument in favor of a single catalytic species. It should be more than coincidence that only the four sulfotransferases, and not any of the marker proteins, failed to yield sharp bands.

For the enzymes of detoxication, such a broad range of reactions residing in one protein is not unknown (29). Among the sulfotransferases, one group of rat phenol sulfotransfer- ases can use either phenols or organic hydroxylamines as substrate (19), and all of the alcohol sulfotransferases (17,20, 28) are active on the vast range of primary and secondary alcohols in compounds as disparate in size as ethanol and hydroxysteroids. A sulfotransferase from the human adrenal (EC 2.8.2.15) is known to act not only with hydroxysteroids but also utilizes phenolic steroids as sulfuryl group acceptors (30). With the glutathione transferases, another example of enzymes participating in detoxication, compounds with elec- trophilic carbon, nitrogen, sulfur, and oxygen serve as sub- strate for nucleophilic attack by the thiolate anion of GSH (31). Thus, there is adequate precedence for a variety of functional groups being conjugated by the catalytic generalists that participate in detoxication. However, despite efforts at separation of the several sulfotransferase activities, the small yield and low initial concentration of amine N-sulfotransfer- ase activities make this enzyme difficult in terms of preparing protein for establishing homogeneity.

It is clear that amines of a variety of carbon skeleton serve as substrates for sulfation, including the interesting situation in which cyclamate, a compound prohibited from use as a sugar substitute, is formed by Reaction 1 from cyclohexyla-

mine. For each acceptor molecule there is the clear depend- ence of the sulfuryl group transfer reaction on the presence of the unprotonated form of the amine as shown by the excellent correlation of activity with pK, of the substrate (Fig. 4). Thus, the amine N-sulfotransferase fits the criteria for membership in the group of enzymes involved in detoxication by reason of the broad range of its substrate spectrum that includes, as a minimum, both primary and secondary amines.

Acknowledgment-It is with pleasure that we thank Dr. James A. Kelley of the National Cancer Institute for devising and conducting all of the operations involving the use of the mass spectrometer.

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