- Email: [email protected]
Thiol S-methyltransferase from rat liver
ARCHIVES OF BIOCHEMISTRYAND BIOPHYSICS Vol. 196, No. 2, September, pp. 631-637, 1979
Thiol S-Methyltransferase RICHARD A. WEISIGER’
AND
from Rat Liver WILLIAM
B. JAKOBY
Section on Enzymes and Cellular Biochemistry, Laboratory of Biochemistry and Metabolism, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014 Received March 12, 1979; revised April 12, 1979 The thiol S-methyltransferase from rat liver has been solubilized and prepared in homogeneous form. The enzyme exists in a monomer of M, 28,000although enzyme activity is highly unstable with a half-life of 4 days under the best conditions of storage. The reaction requires S-adenosylmethionine as methyl donor but, as is the case with many enzymes active in detoxification, a large variety of lipophilic compounds can serve as acceptors. Acceptor activity is limited to thiols. The naturally occurring hydrophilic thiols, glutathione and cysteine, act neither as substrates nor as inhibitors. The course of the reaction is biphasic with an initial rapid formation of product that is followed by a slower linear rate. The suggestion is offered that this behavior reflects the slow dissociation of an enzyme-product complex composed of enzyme and S-adenosyl-homocysteine.
The S-adenosylmethionine-mediated methylation of thiols was observed by Bremer and Greenberg in the microsomal fractions of liver, kidney, and lung of the rat as well as in the livers of other mammals (1). The enzyme, thiol methyltransferase (EC 2.1.1.9>, displayed little specificity for the thiol substrate: Mercaptoethanol, mercaptoacetate, methylmercaptan, and 2,3-dimercaptopropanel, among others, were all active as substrates. Although the assay used (1) was unsuitable for testing compounds such as cysteine and GSH as substrates, these two physiological thiols were not inhibitory in the methylation of mercaptoethanol. Subsequent work with particulate systems has implicated thiol S-methyltransferase in the metabolism of several drugs including diethyldithiocarbamate (Antabuse) (2-4), Z-thiouracil (5, 6), 6-thiopurine (5), and 6-propyl-2-thiouracil (6). The enzyme has been solubilized with Triton X-100 from rat liver microsomes (7) and a subsequent 20-fold purification has been achieved (8). 1 Present address: Department of Medicine, University of California School of Medicine, San Francisco, Calif.
S-Adenosylmethionine
+ RSH +
S-adenosylhomocysteine + RSCH,.
[l]
This report presents the details of solubilization, purification to homogeneity, and partial characterization of the thiol methyltransferase from rat liver. MATERIALS Frozen rat livers were obtained from ARS SpragueDawley rats and stored at -70°C for as long as 3 months prior to use. Hydroxyapatite for chromatography was prepared (9) for us by David Rogerson of this Institute. S-Adenosyl-L&&hyl-3H]methionine (9 to 15 Wmmol) was obtained from Amersham and diluted, when necessary, in 10 mM sulfuric acid. Mercaptans and their derivatives were obtained from the following sources: N-acetyl+cysteine, S-adenosyl-L-homocysteine, L-cysteine, L-cysteine methyl ester, 2,3-dimercaptopropanol, diethyldithiocarbamate glutathione, pmercaptoethanol, 2-mercap topropionic acid, S-methyl+cysteine, S-methyl-glutathione, and thioglycolic acid from Sigma Chemical; dithiothreitol from Schwarz Mann; 2-mercaptoethylamine and 2-amino-Bmercaptopurine from Calbiochem; 2-benzimidazolethiol, 2-mercaptoacetanalide, and 2methylthiobenzothiazole &om Eastman: benzylmethyl sulfide, 2-mercaptobenzothiazole, 4-nitrothiophenol, and thioanisole from Aldrich. 6-Propyl-2-thiouracil and
631
0003-9861/79/100631-07$02.00/O Copyright 0 1979by AcademicPress, Inc. All rights of reproductionin any form reserved.
632
WEISIGER AND JAKOBY
1-methylimidazole-2-thiol (Tappazole) were gifts from Jan Wolff of this Institute. METHODS Radioactivity was measured with a Beckman LB-8100 scintilation spectrometer using 0.75% 2,5-diphenyloxazole and 0.015% 1,4-bis-2-(5-phenyloxazolyl)benzene in toluene as the fluor. Electrophoresis was performed in sodium dodecyl sulfate gels as described by Weber and Osborne (10). Thin layer chromatography of the methylation products of thiols was carried out with Eastman silica coated gel plates (No. 13181) and compared with the authentic derivatives: S-methyl-2-mercaptobenzothiazole (developed in 95% ethanol, R, = 0.54); S-methyl6-thiopurine (developed in 95% ethanol, R, = 0.47); benzylmethylsulfide (developed in chloroform, R, = 0.50); S-methyl phenylmercaptan (developed in chloroform, R, = 0.52). In each case, radioactivity migrating beyond the origin depended on the presence of both enzyme and thiol substrate. Protein was determined by the method of Lowry et al. (11) with human serum albumin as a standard. After the initial purification stages, protein was estimated by measurement of absorbance at 280 nm (Egg = 10).
Standard Enzyme Assay The standard assay used for following purification of the enzyme was performed with 2thioacetanilide as the thiol in a glass-stoppered, conical, 15-ml centrifuge tube containing the following in a final volume of 350 ~1: 0.1 M potassium phosphate at pH 7.9, 1 mM EDTA, and 0.5% Triton X-100. To this mixture were added the following in sequence: 20 ~1 of 0.1 M 2-thioacetanilide (prepared fresh daily in 95% ethanol), 20 ~1 of 5 PM S-adenosyl-L-[m&yl-3H]methionine in 10 mM sulfuric acid and 10 ~1 of enzyme solution. The solution was mixed by vortexing for approximately 1 s. After incubation at 37°C for 10 min, the reaction was stopped with 1 ml of 2 M potassium borate at pH 10. The borate solution was applied as a jet from a 2-ml Cornwall syringe fitted with a 21-gauge needle; this procedure satisfactorily stopped the reaction in less than 15 s. The resultant mixture was extracted with 6 ml of toluene by shaking vigorously for 10 s, separating the phases by brief centrifugation, and removing 5 ml of the toluene layer for determination of radioactivity. Under these conditions, greater than 99% of the radioactive reaction product is obtained by a single extraction. Interference by catechol-0-methyltransferase in the early stages of purification is prevented by the presence of EDTA in the incubation mixture. Triton X-100 is included to decrease enzyme inactivation during the dilution and mixing steps; it does not otherwise affect the reaction rate. As described, the assay resulted
in activity as a linear function of protein concentration under conditions in which less than 10% of S-adenosylmethionine was converted to product. The method is extraordinarily sensitive allowing accurate measurement of as little as 1 ng of purified enzyme.
Other Enzyme Assays The time course of the reaction was followed by removing 200~~1samples from a single assay mixture containing the same constituents as the standard system and adding them to 1 ml of the above noted borate buffer. Toluene, 6 ml, was added and the mixture shaken immediately. Kinetic constants for thiol substrates were determined at 3o”C, rather than at 37”C, to minimize thermal inactivation of the enzyme over the time course of the assay. Radioactive product was measured for each substrate concentration at 2 and 12 min after initiation of the reaction; the difference between these values is the rate measurement used for the data of Table I. In each case the extraction efficiency of toluene for the radioactive product was determined under the outlined conditions and the assay value corrected for this factor (Table I). With ethylthiocarbamate and with thioglycolate, the reaction was stopped with 0.1 ml of 1 N HCl rather than with borate buffer so as to allow extraction of product from acid solution. With none of the substrates examined was there evidence for a nonenzyme catalyzed reaction. In the absence of a thiol substrate from the standard incubation mixture, extractable radioactivity was proportional to the total amount of S-adenosylmethionine and independent of the time of incubation. Since this assay procedure is not applicable to glutathione and cysteine, products of reaction with these compounds were sought by subjecting aliquots of appropriate reaction mixtures to thin layer chromatography on silica gel sheets (Eastman, No. 13181). Authentic samples of the S-methyl derivatives had an R, of 0.4 for S-methylcysteine and 0.2 for S-methylglutathione after development of chromatograms with methanol. PURIFICATION OF THE S-METHYLTRANSFERASE
Solubilixation The thiol methyltransferase from rat liver is associated with the microsomal fraction (1, 8). However, we noted that prior freezing of rat liver to -70°C causes up to 30% of the total activity to remain in the supernatant liquid on subsequent homogenization and centrifugation for 1 h at 106,OOOg. Incubation of the homogenate for 2 h at 20°C prior to centrifugation did not significantly increase the portion of the activity found in the supernatant fraction nor reduce the total activity present. Thus, time-
THIOL S-METHYLTRANSFERASE
FROM RAT LIVER
633
TABLE I SUBSTRATESOF THIOL METHYLTRANSFERASE
Substrate Parachlorothiophenol Phenyl sulfide 4-Nitrothiophenol Diethylthiocarbamylsulfide 2-Thioacetanilide 2-Benzimidazole thiol Thioglycolic acid L-Cysteine methyl ester N-Acetyl-L-cysteine 6-Propyl-2-thiouracil 2-Mercaptopropionic acid 1-Methylimidazole-2-thiol 2,3-Dimercaptopropanol 3-Mercaptopropionic acid methyl ester 2-Mercaptoethanol Dithiothreitol L-Cysteine Glutathione a The linear (0.96), L-cysteine b Percentage e No activity
Apparent K,” mf)
Apparent V mea
(nmoUmg/min)
Extraction efficiencyb (%)
0.00054 0.0011 0.0028
6.2 6.1 7.5
0.99 0.99 0.99
0.012 0.043 0.11 0.19 0.21 0.40 1.0 1.2 1.4 1.6
3.5 7.6 2.4 3.7 1.4 1.0 6.4 3.1 2.0 5.4
0.98 0.99 0.92 0.98 0.54 0.66 0.66 0.99 0.95 0.78
4.7 8.1 -
3.2 5.4
0.99 0.65 -
regression correlation constant was greater than 0.99 except for diethylthiocarbanylsulfide methyl ester (0.97), and N-acetyl+cysteine (0.97). extracted by toluene under the conditions described under Methods. at the limits of detection noted.
dependent processes such as partial proteolysis are unlikely to be responsible. The duration of freezing was also unimportant: Livers that were frozen in powdered Dry Ice and then immediately homogenized gave similar patterns to livers frozen for 2 months at -70°C. (1) Homogenization. Male Sprague-Dawley rats (200 to 300 g) were killed by carbon dioxide asphyxiation and the livers frozen with Dry Ice and stored at -70°C until used. Approximately 50 livers, 600 g, were thawed by repeated washes with water at room temperature until the wash was no longer grossly red. The livers were then homogenized in batches of 150 g each for 30 s in a total of 1100 ml of ice-cold water with a Waring Blendor. After centrifugation for 1 h at 100,OOOg the clear, red supematant liquid was carefully aspirated from the pellet. In this and all subsequent steps it was found essential to minimize foaming and other vigorous agitation. Thus, test tube fractions were routinely pooled by drawing the contents into a 10 ml pipet and carefully allowing the liquid to flow directly into the liquid pool without splashing. In all ultrafiltration steps, pressure within an Amicon apparatus was maintained below
10 psi so as to avoid later foaming when the pressure was released; the stirring bar was maintained below the surface of the concentrating liquid and turned at the minimum rate (less than 2 Hz). These precautions became increasingly important with increasing purity of the enzyme. (2) DEAh’-cellulose. The extract was adjusted to pH 7.7 with 1 M Tris base and was diluted with an approximately equal volume of water until a conductivity of 1 mmho was reached. The solution was charged onto a column of DEAE-cellulose (11 cm high and 14 cm diameter) which had been prepared by adjusting 1 kg of DE-42 cellulose (Whatman) to pH 7.7 while suspended in 0.05 M potassium phosphate. The ion exchanger was equilibrated with 10 liters of a buffer consisting of 20 mM Tris at pH 7.7 (4°C) and 0.01 mM EDTA; conductivity at 4°C was 0.7 mmho. After adding enzyme, the column was washed with an additional 4 liters of the starting buffer and eluted with a linear, 4-liter gradient of 0 to 0.2 M NaCl in starting buffer. Fractions of 25 ml were collected and active fractions were pooled as shown in Fig. 1A. (3) Ammonium sulfate concentration. The pooled eluate, 1 to 1.4 liters, was made 80% saturated in am-
634
WEISIGER AND JAKOBY FRACTION
NUMBER
2
1
. . . ...’
1
SEPHADEX
G75
.-L8 0 p2 D 1 1.0 = 0
m
FIG. 1. El&ion patterns for several steps in the purification procedure. Thiol S-methyltransferase activity is designated by solid lines, protein by dashed lines, and conductivity measured at 4°C by dotted lines. The fractions pooled are shown by brackets. monium sulfate (516 g/liter) and, after 1 h at 4”C, the precipitate was separated by centrifugation at 24,000g for 1 h. The paste was suspended in 100 ml of 0.1 M potassium phosphate containing 0.1 mu EDTA, pH 7.9, and dialyzed sequentially against 20 vol of the following solutions for the indicated periods: 0.1 mM d&odium EDTA for 4 h, 0.1 mM disodium EDTA for 10 h, and 5 mM potassium phosphate containing 0.1 mM EDTA, pH 6.7, for 4 h. The suspension was clarified by centrifugation. (4) Hydroqapatite (pH 6.7). The preparation was adjusted to 20 mM potassium phosphate at pH 6.7 by addition of a molar solution of the same buffer. The enzyme solution was applied to a column of hydroxyapatite (5 x 12 cm), previously equilibrated with 0.01 M potassium phosphate containing 0.1 mM EDTA at pH 6.7, and eluted with a linear, 3-liter gradient of 0.01 to 0.2 M potassium phosphate at pH 6.7 containing 0.1 mM EDTA. ,Fractions of 25 ml were collected (Fig. 1B) and concentrated with an Amicon PM-10 membrane to 40 ml. (5) Hydroaqaputite (pH 6.2). The concentrate was dialyzed overnight against 0.01 M potassium phosphate0.1 mM EDTA, pH 6.2, and layered onto a hydroxyapatite column (1.5 x 7 cm) previously equilibrated with the same buffer. The column was developed with a 600-ml gradient of 0.01 to 0.2 Y potassium phosphate at pH 6.2 containing 0.1 mM EDTA. Fractions of 7 ml were collected (Fig. 10.
(6) Preparative isoelectric focusing. The enzyme solution was subjected to isoelectric focusing in a llO-ml column containing 2% of pH 5 to 7 ampholines (LKB) and stabilized by a 0 to 40% sucrose gradient. Focusing proceeded for 72 h at up to 1000 V. Fractions of 1 ml were collected and those active fractions near pH 6.2 and at the center of the activity peak, representing about 60% of the activity present, were pooled. Taking a larger proportion of the activity, especially if fractions at the lower pH region were used, was found to result in an unacceptable degree of contamination by other proteins. (7) Sephxw!esgel chromatography. The pooled fractions, about 5.6 ml, were charged directly onto a Sephadex G-75 (Superfine) column (1.5 x 90 cm) and eluted with 0.1 M sodium chloride, 10 mM Tris, and 0.1 mM EDTA at pH 7.5. Thiol methyltransferase eluted as a single peak of constant specific activity that was well separated from a larger leading peak (Fig. 1D). The results of the purification procedure are summarized in Table II. RESULTS
Homogeneity
and Molecular
Weight
Thiol S-methyltransferase was observed to yield a single band upon subjection to
THIOL
S-METHYLTRANSFERASE TABLE
FROM
635
RAT LIVER
II
THIOL METHYLTRANSFERASE:SUMMARYOF PURIFICATION (600 g LIVER)
Step (1) High-speed supernatant (2) DEAE-cellulose (3) Salt concentration (4) Hydroxyapatite (pH 6.7) (5) Hydroxyapatite (pH 6.2) (6) Isoelectric focusing (7) Sephadex G-75
Volume (ml) 1,150 740 85 73 7
Total protein (mg) 76,400 4,570 3,400 168 49
4.1
1.9
4.7
0.28
sodium dodecyl sulfate gel electrophoresis when a load of 50 pg of protein was applied. Calibration of the gel system with human serum albumin, rabbit muscle pyruvate kinase, yeast alcohol dehydrogenase, and human carbonic anhydrase allowed the estimation of a subunit molecular weight of 27,500; this value was not altered by omission of p-mercaptoethanol in the treatment of the protein prior to electrophoresis. From gel filtration studies with a column (1.5 x 90 cm) of Sephadex G-75 (Superfine) and calibration with horse cytochrome c, bovine chymotrypsinogen A, hen ovalbumin, and bovine serum albumin, a molecular weight of 28,000 was estimated for the protein.
Total activity (nmol min-‘)
Specific activity (nmol min-* mg-I) 0.001
114 78 96 27 15 3.4 2.8
0.017 0.028 0.16 0.31 1.8
10
Under the assay conditions used, the optimum pH was at 7.5 when tested in a buffer composed of 100 InM acetic acid, 100 mM potassium dihydrogen phosphate, and amounts of glycylglycine necessary to achieve the desired pH (Fig. 2). Halfmaximal activity was observed at pH 6.6 and 8.6. Thiol Substrates and Their Products Kinetic parameters for the several compounds that were examined were obtained from the steady-state rate of product formation at 30°C (Table I). It will be evident that the specificity of the enzyme is broad although two naturally occurring thiols,
Stability and pH Optimum At each stage of purification the enzyme is highly unstable under all conditions encountered. The homogeneous protein at 4°C in 140 InM sodium chloride and 10 mM Trischloride at pH 7.4, loses activity in a firstorder reaction with a half-life of 4 days; these are the conditions under which the enzyme was stored. All attempts at freezing the enzyme, particularly after the initial stages of purification, and despite efforts to freeze directly with liquid nitrogen, resulted in destruction of activity. At pH 7.9 and 3O”C, in the absence of its nucleotide substrate, the enzyme has a half-life of less than 1 min. Attempts at stabilization of the enzyme at 4°C with glycerol, variation in pH, or the addition of glutathione were only of minimal value.
0it-. 69’.
1 7
\
I
,l\...-,
8
9
10
PH
FIG. 2. Thiol S-methyltransferase activity as a function of pH. Buffers below pH 8.8 contained 100 mM acetic acid, 100 mM potassium phosphate, 0.1 mM EDTA, 0.6% Triton X-100, and were adjusted to the appropriate pH at 20°C with KOH. Buffers above pH 8.8 differed in that 100 rnM glycylglycine was substituted for acetic acid. Incubation time: 10 min.
636
WEISIGER AND JAKOBY
cysteine and glutathione, do not serve as substrates. Nevertheless, the methyl ester and N-acetyl derivatives of cysteine are S-methylated. Nucleophilic groups other than thiol did not act as methyl acceptors: Phenol, aniline, and pyridine were not substrates. Since most of the tested compounds and their products were volatile it was difficult to identify the products of the reaction. However, the products from four thiols for which the S-methyl derivatives were available for comparison, i.e., S-mercaptobenzothiazole, 6-thiopurine, thiophenol, and benzyl mercaptan, were examined after incubation with enzyme. In each case, the radioactive product had an identical Rf and ran coincidentally with the authentic S-methyl derivative upon chromatography on silica gel. The K, for S-adenosylmethionine in the otherwise standard reaction mixture, measured from the steady state rate, varied as a function of temperature with values of
4-
TABLE III
TURNOVERTIMEOFTHIOLS-METHYLTRANSFERASE ASAFUNCTIONOFTEMPERATURE" T (“C)
Burst phase* (min)
Steady state phase’ (min)
37 30 25 20 15 10 5 0
1.3 1.6 1.9 2.1 2.5 3.0 3.6 4.7
1.8 7.4 12 19 26 34 45 57
a The enzyme was incubated at the given temperature with 225 nM S-adenosyl-L-methionine and 5 mM 2-thiolacetanilide at pH 7.9. In each instance, turnover time is expressed as the number of minutes required for the enzyme to cycle once. * Data taken from the rate during the first minute of reaction. c Data taken from the rate attained between 5 and 10 min.
1 PM at 37”C, 0.6 FM at 3O”C, and 42 nM at 15°C. Kinetics
FIG. 3. Product formation as a function of time and protein concentration. All reaction vessels, at 15”C, contained 90 mM potassium phosphate at pH 7.9, 5 mM2-thioacetanalid, 50 mMS-adenosyl-~[methyl-3H]methionine, 1 mM EDTA, and 0.5% Triton X-100. No activity was present in the absence of enzyme. Curve 1: time course with 50 ng of the purified transferase; Curve 2: same as 1 but with 100ng of transferaae; Curve 3: same as 1 with the addition of 50 ng of transferase after 5 min; Curve 4: same as 1 except for the incubation of the enzyme with 1 @MS-adenosylhomocysteine prior to lOOO-fold dilution into the assay mixture (final concentration of the inhibitor was 1 nM).
The course of the enzyme-catalyzed reaction of S-adenosylmethionine and 2-thioacetanilide is biphasic, displaying an initially rapid formation of products that is followed by a slower linear rate (Fig. 3, curve 1). We refer to these two portions of the curve as “burst” and “steady state” rate, respectively. This behavior was exhibited despite a range of substrate concentrations, at eight temperatures between 0°C and 37°C and with each of the thiols tested including 2-mercaptoethanol, phenylsulfide, and 2-mercaptopropionic acid, i.e., with substrates having a wide range of apparent K, values for the transferase (Table I). The decrease in rate during the burst phase is in the form of a first-order decay to the steady state rate which has a to 5 of 2 min under the conditions used in Fig. 3. The value of t0.5 varies with temperature and is presented in Table III as a function of the turnover time for the transferase. Extrapolation of the linear portion, i.e., the steady state rate, to zero time indicates that the burst represented about 0.5 mol
THIOL
S-METHYLTRANSFERASE
of each product per mole of enzyme. Doubling the enzyme concentration doubled product formation at each time point (Fig. 3, curve 2) whereas adding a second, equal amount of enzyme after completion of the burst phase led to a second burst superimposed on the linear rate of the initial reaction (curve 3). Although the thioether product of the reaction is not inhibitory at 5 mM, the second product, S-adenosylhomocysteine, is recognized as an effective inhibitor of the methyltransferases in general (12). Maximal formation of products for the conditions of Fig. 3 is of the order of a few picomoles per milliliter, but this concentration did not produce noticeable inhibition of either phase of the reaction. Thus, classical reversible product inhibition seems unlikely as an explanation. However, when the enzyme was incubated in 1 mM S-adenosylhomocysteine at 0°C for 10 min and then diluted lOOO-fold in a standard assay medium, the burst phase was entirely eliminated while the steady state rate remained unchanged (Fig. 3, curve 4). These results are consistent with a mechanism by which the intermediate product complex, S-adenosylhomocysteine-enzyme, is slow to dissociate so that dissociation of product is rate limiting. Classical inhibition studies with S-adenosylhomocysteine reveal competitive inhibition with S-adenosylmethionine for the steady-state phase (K, = 12 nM at 15°C; 20 nM at 30°C). DISCUSSION
Thiol S-methyltransferase is a microsomal enzyme which may be isolated in soluble form from frozen rat liver. Solubilization is associated with a change in the kinetics of the transferase-catalyzed reaction. Although microsomal preparations from freshly excised livers exhibit normal kinetic behavior under standard conditions, the soluble preparation, at all stages of purification, have a biphasic rate curve which we ascribe to the slow dissociation of S-adenosyl homocysteine from the enzyme-product complex. In support of this contention we point to the elimination of the burst phase by incubation with S-adenosyl homocysteine. The role of the enzyme in detoxication has been strengthened by the recent dis-
FROM
RAT LIVER
637
covery of cysteine conjugate /3-lyase (13), an enzyme that catalyzes the formation of thiols from thioethers of cysteine. Such thioethers commonly arise in the course of mercapturic acid formation (14). As with other enzymes proposed as active in detoxification, e.g., the cytochrome P-450 oxidation system (15), the glutathione transfer&es (14), and epoxide hydrase (16), the thiol S-methyltransferase confines its specificity to the donor molecule while allowing a broad spectrum of mainly hydrophobic compounds as acceptor substrates (17). REFERENCES 1. BREINER, J., AND GREENBERG, D. M. (1961) Biochim. Biophys. Acta 46, 217-224. 2. JAKUBOWSKI, M., AND GESSNER, T. (1972) Biothem. Pharmacol. 21, 3073-3076. 3. COBBY, J., MAYERSOHN, M., AND SELLIAH, S. (1977) Life Sci. 21, 937-942. 4. GESSNER, T., AND JAKUBOWSKI, M. (1972) Biohem. Pharmacol. 21,219~230. 5. REMY, C. M. (1963) J. Biol. Chem. 238, 10781084. 6. LINDSAY, R. H., HULSEY, B. S., AND ABOULENEIN, H. Y. (1975) B&hem. Pharmacol. 24, 463-468. 7. BORCHARDT, R. T., CHENG, C. F., COOKE, P. H., AND CREVELING, C. R. (1974) Lifi Sci. 14, 1089-1100. 8. BORCHARDT, R. T., AND CHENG, C. F. (1978) Biochim. Biophys. Acta 522, 340-353. 9. LEVIN, 0. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 5, pp. 27-32, Academic Press, New York. 10. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-4412. 11. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193,265-275. 12. AXELROD, J. (1971) in Concepts in Biochemical Pharmacology (Bredie, B. B., and Giiette, J. R., eds.), Vol. 2, pp. 609-619, Springer-Verlag, Berlin. 13. TATEISHI, M., SUZUKI, S., AND SHIMIZU, H. (1978) J. Biol. Chem. 253, 8854-8859. 14. JAKOBY, W. B. (1978) Advan. Ewzymol. 46,383414. 15. ESTABROOK, R. W. (1971) in Concepts in Biechemical Pharmacology (Bredie, B. B., and Gillette, J. R., eds.), Vol. 2, pp. 264-284, Springer-Verlag, Berlin. 16. Lu, A. Y. H., JERINA, D. M., AND LEVIN, W. (1977) J. Biol. Chem. 252,3715-3723. 17. JAKOBY, W. B., AND KEEN, J. M. (1978) Trends Biochem. Sci. 2, 229-231.
Thiol S-Methyltransferase RICHARD A. WEISIGER’
AND
from Rat Liver WILLIAM
B. JAKOBY
Section on Enzymes and Cellular Biochemistry, Laboratory of Biochemistry and Metabolism, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014 Received March 12, 1979; revised April 12, 1979 The thiol S-methyltransferase from rat liver has been solubilized and prepared in homogeneous form. The enzyme exists in a monomer of M, 28,000although enzyme activity is highly unstable with a half-life of 4 days under the best conditions of storage. The reaction requires S-adenosylmethionine as methyl donor but, as is the case with many enzymes active in detoxification, a large variety of lipophilic compounds can serve as acceptors. Acceptor activity is limited to thiols. The naturally occurring hydrophilic thiols, glutathione and cysteine, act neither as substrates nor as inhibitors. The course of the reaction is biphasic with an initial rapid formation of product that is followed by a slower linear rate. The suggestion is offered that this behavior reflects the slow dissociation of an enzyme-product complex composed of enzyme and S-adenosyl-homocysteine.
The S-adenosylmethionine-mediated methylation of thiols was observed by Bremer and Greenberg in the microsomal fractions of liver, kidney, and lung of the rat as well as in the livers of other mammals (1). The enzyme, thiol methyltransferase (EC 2.1.1.9>, displayed little specificity for the thiol substrate: Mercaptoethanol, mercaptoacetate, methylmercaptan, and 2,3-dimercaptopropanel, among others, were all active as substrates. Although the assay used (1) was unsuitable for testing compounds such as cysteine and GSH as substrates, these two physiological thiols were not inhibitory in the methylation of mercaptoethanol. Subsequent work with particulate systems has implicated thiol S-methyltransferase in the metabolism of several drugs including diethyldithiocarbamate (Antabuse) (2-4), Z-thiouracil (5, 6), 6-thiopurine (5), and 6-propyl-2-thiouracil (6). The enzyme has been solubilized with Triton X-100 from rat liver microsomes (7) and a subsequent 20-fold purification has been achieved (8). 1 Present address: Department of Medicine, University of California School of Medicine, San Francisco, Calif.
S-Adenosylmethionine
+ RSH +
S-adenosylhomocysteine + RSCH,.
[l]
This report presents the details of solubilization, purification to homogeneity, and partial characterization of the thiol methyltransferase from rat liver. MATERIALS Frozen rat livers were obtained from ARS SpragueDawley rats and stored at -70°C for as long as 3 months prior to use. Hydroxyapatite for chromatography was prepared (9) for us by David Rogerson of this Institute. S-Adenosyl-L&&hyl-3H]methionine (9 to 15 Wmmol) was obtained from Amersham and diluted, when necessary, in 10 mM sulfuric acid. Mercaptans and their derivatives were obtained from the following sources: N-acetyl+cysteine, S-adenosyl-L-homocysteine, L-cysteine, L-cysteine methyl ester, 2,3-dimercaptopropanol, diethyldithiocarbamate glutathione, pmercaptoethanol, 2-mercap topropionic acid, S-methyl+cysteine, S-methyl-glutathione, and thioglycolic acid from Sigma Chemical; dithiothreitol from Schwarz Mann; 2-mercaptoethylamine and 2-amino-Bmercaptopurine from Calbiochem; 2-benzimidazolethiol, 2-mercaptoacetanalide, and 2methylthiobenzothiazole &om Eastman: benzylmethyl sulfide, 2-mercaptobenzothiazole, 4-nitrothiophenol, and thioanisole from Aldrich. 6-Propyl-2-thiouracil and
631
0003-9861/79/100631-07$02.00/O Copyright 0 1979by AcademicPress, Inc. All rights of reproductionin any form reserved.
632
WEISIGER AND JAKOBY
1-methylimidazole-2-thiol (Tappazole) were gifts from Jan Wolff of this Institute. METHODS Radioactivity was measured with a Beckman LB-8100 scintilation spectrometer using 0.75% 2,5-diphenyloxazole and 0.015% 1,4-bis-2-(5-phenyloxazolyl)benzene in toluene as the fluor. Electrophoresis was performed in sodium dodecyl sulfate gels as described by Weber and Osborne (10). Thin layer chromatography of the methylation products of thiols was carried out with Eastman silica coated gel plates (No. 13181) and compared with the authentic derivatives: S-methyl-2-mercaptobenzothiazole (developed in 95% ethanol, R, = 0.54); S-methyl6-thiopurine (developed in 95% ethanol, R, = 0.47); benzylmethylsulfide (developed in chloroform, R, = 0.50); S-methyl phenylmercaptan (developed in chloroform, R, = 0.52). In each case, radioactivity migrating beyond the origin depended on the presence of both enzyme and thiol substrate. Protein was determined by the method of Lowry et al. (11) with human serum albumin as a standard. After the initial purification stages, protein was estimated by measurement of absorbance at 280 nm (Egg = 10).
Standard Enzyme Assay The standard assay used for following purification of the enzyme was performed with 2thioacetanilide as the thiol in a glass-stoppered, conical, 15-ml centrifuge tube containing the following in a final volume of 350 ~1: 0.1 M potassium phosphate at pH 7.9, 1 mM EDTA, and 0.5% Triton X-100. To this mixture were added the following in sequence: 20 ~1 of 0.1 M 2-thioacetanilide (prepared fresh daily in 95% ethanol), 20 ~1 of 5 PM S-adenosyl-L-[m&yl-3H]methionine in 10 mM sulfuric acid and 10 ~1 of enzyme solution. The solution was mixed by vortexing for approximately 1 s. After incubation at 37°C for 10 min, the reaction was stopped with 1 ml of 2 M potassium borate at pH 10. The borate solution was applied as a jet from a 2-ml Cornwall syringe fitted with a 21-gauge needle; this procedure satisfactorily stopped the reaction in less than 15 s. The resultant mixture was extracted with 6 ml of toluene by shaking vigorously for 10 s, separating the phases by brief centrifugation, and removing 5 ml of the toluene layer for determination of radioactivity. Under these conditions, greater than 99% of the radioactive reaction product is obtained by a single extraction. Interference by catechol-0-methyltransferase in the early stages of purification is prevented by the presence of EDTA in the incubation mixture. Triton X-100 is included to decrease enzyme inactivation during the dilution and mixing steps; it does not otherwise affect the reaction rate. As described, the assay resulted
in activity as a linear function of protein concentration under conditions in which less than 10% of S-adenosylmethionine was converted to product. The method is extraordinarily sensitive allowing accurate measurement of as little as 1 ng of purified enzyme.
Other Enzyme Assays The time course of the reaction was followed by removing 200~~1samples from a single assay mixture containing the same constituents as the standard system and adding them to 1 ml of the above noted borate buffer. Toluene, 6 ml, was added and the mixture shaken immediately. Kinetic constants for thiol substrates were determined at 3o”C, rather than at 37”C, to minimize thermal inactivation of the enzyme over the time course of the assay. Radioactive product was measured for each substrate concentration at 2 and 12 min after initiation of the reaction; the difference between these values is the rate measurement used for the data of Table I. In each case the extraction efficiency of toluene for the radioactive product was determined under the outlined conditions and the assay value corrected for this factor (Table I). With ethylthiocarbamate and with thioglycolate, the reaction was stopped with 0.1 ml of 1 N HCl rather than with borate buffer so as to allow extraction of product from acid solution. With none of the substrates examined was there evidence for a nonenzyme catalyzed reaction. In the absence of a thiol substrate from the standard incubation mixture, extractable radioactivity was proportional to the total amount of S-adenosylmethionine and independent of the time of incubation. Since this assay procedure is not applicable to glutathione and cysteine, products of reaction with these compounds were sought by subjecting aliquots of appropriate reaction mixtures to thin layer chromatography on silica gel sheets (Eastman, No. 13181). Authentic samples of the S-methyl derivatives had an R, of 0.4 for S-methylcysteine and 0.2 for S-methylglutathione after development of chromatograms with methanol. PURIFICATION OF THE S-METHYLTRANSFERASE
Solubilixation The thiol methyltransferase from rat liver is associated with the microsomal fraction (1, 8). However, we noted that prior freezing of rat liver to -70°C causes up to 30% of the total activity to remain in the supernatant liquid on subsequent homogenization and centrifugation for 1 h at 106,OOOg. Incubation of the homogenate for 2 h at 20°C prior to centrifugation did not significantly increase the portion of the activity found in the supernatant fraction nor reduce the total activity present. Thus, time-
THIOL S-METHYLTRANSFERASE
FROM RAT LIVER
633
TABLE I SUBSTRATESOF THIOL METHYLTRANSFERASE
Substrate Parachlorothiophenol Phenyl sulfide 4-Nitrothiophenol Diethylthiocarbamylsulfide 2-Thioacetanilide 2-Benzimidazole thiol Thioglycolic acid L-Cysteine methyl ester N-Acetyl-L-cysteine 6-Propyl-2-thiouracil 2-Mercaptopropionic acid 1-Methylimidazole-2-thiol 2,3-Dimercaptopropanol 3-Mercaptopropionic acid methyl ester 2-Mercaptoethanol Dithiothreitol L-Cysteine Glutathione a The linear (0.96), L-cysteine b Percentage e No activity
Apparent K,” mf)
Apparent V mea
(nmoUmg/min)
Extraction efficiencyb (%)
0.00054 0.0011 0.0028
6.2 6.1 7.5
0.99 0.99 0.99
0.012 0.043 0.11 0.19 0.21 0.40 1.0 1.2 1.4 1.6
3.5 7.6 2.4 3.7 1.4 1.0 6.4 3.1 2.0 5.4
0.98 0.99 0.92 0.98 0.54 0.66 0.66 0.99 0.95 0.78
4.7 8.1 -
3.2 5.4
0.99 0.65 -
regression correlation constant was greater than 0.99 except for diethylthiocarbanylsulfide methyl ester (0.97), and N-acetyl+cysteine (0.97). extracted by toluene under the conditions described under Methods. at the limits of detection noted.
dependent processes such as partial proteolysis are unlikely to be responsible. The duration of freezing was also unimportant: Livers that were frozen in powdered Dry Ice and then immediately homogenized gave similar patterns to livers frozen for 2 months at -70°C. (1) Homogenization. Male Sprague-Dawley rats (200 to 300 g) were killed by carbon dioxide asphyxiation and the livers frozen with Dry Ice and stored at -70°C until used. Approximately 50 livers, 600 g, were thawed by repeated washes with water at room temperature until the wash was no longer grossly red. The livers were then homogenized in batches of 150 g each for 30 s in a total of 1100 ml of ice-cold water with a Waring Blendor. After centrifugation for 1 h at 100,OOOg the clear, red supematant liquid was carefully aspirated from the pellet. In this and all subsequent steps it was found essential to minimize foaming and other vigorous agitation. Thus, test tube fractions were routinely pooled by drawing the contents into a 10 ml pipet and carefully allowing the liquid to flow directly into the liquid pool without splashing. In all ultrafiltration steps, pressure within an Amicon apparatus was maintained below
10 psi so as to avoid later foaming when the pressure was released; the stirring bar was maintained below the surface of the concentrating liquid and turned at the minimum rate (less than 2 Hz). These precautions became increasingly important with increasing purity of the enzyme. (2) DEAh’-cellulose. The extract was adjusted to pH 7.7 with 1 M Tris base and was diluted with an approximately equal volume of water until a conductivity of 1 mmho was reached. The solution was charged onto a column of DEAE-cellulose (11 cm high and 14 cm diameter) which had been prepared by adjusting 1 kg of DE-42 cellulose (Whatman) to pH 7.7 while suspended in 0.05 M potassium phosphate. The ion exchanger was equilibrated with 10 liters of a buffer consisting of 20 mM Tris at pH 7.7 (4°C) and 0.01 mM EDTA; conductivity at 4°C was 0.7 mmho. After adding enzyme, the column was washed with an additional 4 liters of the starting buffer and eluted with a linear, 4-liter gradient of 0 to 0.2 M NaCl in starting buffer. Fractions of 25 ml were collected and active fractions were pooled as shown in Fig. 1A. (3) Ammonium sulfate concentration. The pooled eluate, 1 to 1.4 liters, was made 80% saturated in am-
634
WEISIGER AND JAKOBY FRACTION
NUMBER
2
1
. . . ...’
1
SEPHADEX
G75
.-L8 0 p2 D 1 1.0 = 0
m
FIG. 1. El&ion patterns for several steps in the purification procedure. Thiol S-methyltransferase activity is designated by solid lines, protein by dashed lines, and conductivity measured at 4°C by dotted lines. The fractions pooled are shown by brackets. monium sulfate (516 g/liter) and, after 1 h at 4”C, the precipitate was separated by centrifugation at 24,000g for 1 h. The paste was suspended in 100 ml of 0.1 M potassium phosphate containing 0.1 mu EDTA, pH 7.9, and dialyzed sequentially against 20 vol of the following solutions for the indicated periods: 0.1 mM d&odium EDTA for 4 h, 0.1 mM disodium EDTA for 10 h, and 5 mM potassium phosphate containing 0.1 mM EDTA, pH 6.7, for 4 h. The suspension was clarified by centrifugation. (4) Hydroqapatite (pH 6.7). The preparation was adjusted to 20 mM potassium phosphate at pH 6.7 by addition of a molar solution of the same buffer. The enzyme solution was applied to a column of hydroxyapatite (5 x 12 cm), previously equilibrated with 0.01 M potassium phosphate containing 0.1 mM EDTA at pH 6.7, and eluted with a linear, 3-liter gradient of 0.01 to 0.2 M potassium phosphate at pH 6.7 containing 0.1 mM EDTA. ,Fractions of 25 ml were collected (Fig. 1B) and concentrated with an Amicon PM-10 membrane to 40 ml. (5) Hydroaqaputite (pH 6.2). The concentrate was dialyzed overnight against 0.01 M potassium phosphate0.1 mM EDTA, pH 6.2, and layered onto a hydroxyapatite column (1.5 x 7 cm) previously equilibrated with the same buffer. The column was developed with a 600-ml gradient of 0.01 to 0.2 Y potassium phosphate at pH 6.2 containing 0.1 mM EDTA. Fractions of 7 ml were collected (Fig. 10.
(6) Preparative isoelectric focusing. The enzyme solution was subjected to isoelectric focusing in a llO-ml column containing 2% of pH 5 to 7 ampholines (LKB) and stabilized by a 0 to 40% sucrose gradient. Focusing proceeded for 72 h at up to 1000 V. Fractions of 1 ml were collected and those active fractions near pH 6.2 and at the center of the activity peak, representing about 60% of the activity present, were pooled. Taking a larger proportion of the activity, especially if fractions at the lower pH region were used, was found to result in an unacceptable degree of contamination by other proteins. (7) Sephxw!esgel chromatography. The pooled fractions, about 5.6 ml, were charged directly onto a Sephadex G-75 (Superfine) column (1.5 x 90 cm) and eluted with 0.1 M sodium chloride, 10 mM Tris, and 0.1 mM EDTA at pH 7.5. Thiol methyltransferase eluted as a single peak of constant specific activity that was well separated from a larger leading peak (Fig. 1D). The results of the purification procedure are summarized in Table II. RESULTS
Homogeneity
and Molecular
Weight
Thiol S-methyltransferase was observed to yield a single band upon subjection to
THIOL
S-METHYLTRANSFERASE TABLE
FROM
635
RAT LIVER
II
THIOL METHYLTRANSFERASE:SUMMARYOF PURIFICATION (600 g LIVER)
Step (1) High-speed supernatant (2) DEAE-cellulose (3) Salt concentration (4) Hydroxyapatite (pH 6.7) (5) Hydroxyapatite (pH 6.2) (6) Isoelectric focusing (7) Sephadex G-75
Volume (ml) 1,150 740 85 73 7
Total protein (mg) 76,400 4,570 3,400 168 49
4.1
1.9
4.7
0.28
sodium dodecyl sulfate gel electrophoresis when a load of 50 pg of protein was applied. Calibration of the gel system with human serum albumin, rabbit muscle pyruvate kinase, yeast alcohol dehydrogenase, and human carbonic anhydrase allowed the estimation of a subunit molecular weight of 27,500; this value was not altered by omission of p-mercaptoethanol in the treatment of the protein prior to electrophoresis. From gel filtration studies with a column (1.5 x 90 cm) of Sephadex G-75 (Superfine) and calibration with horse cytochrome c, bovine chymotrypsinogen A, hen ovalbumin, and bovine serum albumin, a molecular weight of 28,000 was estimated for the protein.
Total activity (nmol min-‘)
Specific activity (nmol min-* mg-I) 0.001
114 78 96 27 15 3.4 2.8
0.017 0.028 0.16 0.31 1.8
10
Under the assay conditions used, the optimum pH was at 7.5 when tested in a buffer composed of 100 InM acetic acid, 100 mM potassium dihydrogen phosphate, and amounts of glycylglycine necessary to achieve the desired pH (Fig. 2). Halfmaximal activity was observed at pH 6.6 and 8.6. Thiol Substrates and Their Products Kinetic parameters for the several compounds that were examined were obtained from the steady-state rate of product formation at 30°C (Table I). It will be evident that the specificity of the enzyme is broad although two naturally occurring thiols,
Stability and pH Optimum At each stage of purification the enzyme is highly unstable under all conditions encountered. The homogeneous protein at 4°C in 140 InM sodium chloride and 10 mM Trischloride at pH 7.4, loses activity in a firstorder reaction with a half-life of 4 days; these are the conditions under which the enzyme was stored. All attempts at freezing the enzyme, particularly after the initial stages of purification, and despite efforts to freeze directly with liquid nitrogen, resulted in destruction of activity. At pH 7.9 and 3O”C, in the absence of its nucleotide substrate, the enzyme has a half-life of less than 1 min. Attempts at stabilization of the enzyme at 4°C with glycerol, variation in pH, or the addition of glutathione were only of minimal value.
0it-. 69’.
1 7
\
I
,l\...-,
8
9
10
PH
FIG. 2. Thiol S-methyltransferase activity as a function of pH. Buffers below pH 8.8 contained 100 mM acetic acid, 100 mM potassium phosphate, 0.1 mM EDTA, 0.6% Triton X-100, and were adjusted to the appropriate pH at 20°C with KOH. Buffers above pH 8.8 differed in that 100 rnM glycylglycine was substituted for acetic acid. Incubation time: 10 min.
636
WEISIGER AND JAKOBY
cysteine and glutathione, do not serve as substrates. Nevertheless, the methyl ester and N-acetyl derivatives of cysteine are S-methylated. Nucleophilic groups other than thiol did not act as methyl acceptors: Phenol, aniline, and pyridine were not substrates. Since most of the tested compounds and their products were volatile it was difficult to identify the products of the reaction. However, the products from four thiols for which the S-methyl derivatives were available for comparison, i.e., S-mercaptobenzothiazole, 6-thiopurine, thiophenol, and benzyl mercaptan, were examined after incubation with enzyme. In each case, the radioactive product had an identical Rf and ran coincidentally with the authentic S-methyl derivative upon chromatography on silica gel. The K, for S-adenosylmethionine in the otherwise standard reaction mixture, measured from the steady state rate, varied as a function of temperature with values of
4-
TABLE III
TURNOVERTIMEOFTHIOLS-METHYLTRANSFERASE ASAFUNCTIONOFTEMPERATURE" T (“C)
Burst phase* (min)
Steady state phase’ (min)
37 30 25 20 15 10 5 0
1.3 1.6 1.9 2.1 2.5 3.0 3.6 4.7
1.8 7.4 12 19 26 34 45 57
a The enzyme was incubated at the given temperature with 225 nM S-adenosyl-L-methionine and 5 mM 2-thiolacetanilide at pH 7.9. In each instance, turnover time is expressed as the number of minutes required for the enzyme to cycle once. * Data taken from the rate during the first minute of reaction. c Data taken from the rate attained between 5 and 10 min.
1 PM at 37”C, 0.6 FM at 3O”C, and 42 nM at 15°C. Kinetics
FIG. 3. Product formation as a function of time and protein concentration. All reaction vessels, at 15”C, contained 90 mM potassium phosphate at pH 7.9, 5 mM2-thioacetanalid, 50 mMS-adenosyl-~[methyl-3H]methionine, 1 mM EDTA, and 0.5% Triton X-100. No activity was present in the absence of enzyme. Curve 1: time course with 50 ng of the purified transferase; Curve 2: same as 1 but with 100ng of transferaae; Curve 3: same as 1 with the addition of 50 ng of transferase after 5 min; Curve 4: same as 1 except for the incubation of the enzyme with 1 @MS-adenosylhomocysteine prior to lOOO-fold dilution into the assay mixture (final concentration of the inhibitor was 1 nM).
The course of the enzyme-catalyzed reaction of S-adenosylmethionine and 2-thioacetanilide is biphasic, displaying an initially rapid formation of products that is followed by a slower linear rate (Fig. 3, curve 1). We refer to these two portions of the curve as “burst” and “steady state” rate, respectively. This behavior was exhibited despite a range of substrate concentrations, at eight temperatures between 0°C and 37°C and with each of the thiols tested including 2-mercaptoethanol, phenylsulfide, and 2-mercaptopropionic acid, i.e., with substrates having a wide range of apparent K, values for the transferase (Table I). The decrease in rate during the burst phase is in the form of a first-order decay to the steady state rate which has a to 5 of 2 min under the conditions used in Fig. 3. The value of t0.5 varies with temperature and is presented in Table III as a function of the turnover time for the transferase. Extrapolation of the linear portion, i.e., the steady state rate, to zero time indicates that the burst represented about 0.5 mol
THIOL
S-METHYLTRANSFERASE
of each product per mole of enzyme. Doubling the enzyme concentration doubled product formation at each time point (Fig. 3, curve 2) whereas adding a second, equal amount of enzyme after completion of the burst phase led to a second burst superimposed on the linear rate of the initial reaction (curve 3). Although the thioether product of the reaction is not inhibitory at 5 mM, the second product, S-adenosylhomocysteine, is recognized as an effective inhibitor of the methyltransferases in general (12). Maximal formation of products for the conditions of Fig. 3 is of the order of a few picomoles per milliliter, but this concentration did not produce noticeable inhibition of either phase of the reaction. Thus, classical reversible product inhibition seems unlikely as an explanation. However, when the enzyme was incubated in 1 mM S-adenosylhomocysteine at 0°C for 10 min and then diluted lOOO-fold in a standard assay medium, the burst phase was entirely eliminated while the steady state rate remained unchanged (Fig. 3, curve 4). These results are consistent with a mechanism by which the intermediate product complex, S-adenosylhomocysteine-enzyme, is slow to dissociate so that dissociation of product is rate limiting. Classical inhibition studies with S-adenosylhomocysteine reveal competitive inhibition with S-adenosylmethionine for the steady-state phase (K, = 12 nM at 15°C; 20 nM at 30°C). DISCUSSION
Thiol S-methyltransferase is a microsomal enzyme which may be isolated in soluble form from frozen rat liver. Solubilization is associated with a change in the kinetics of the transferase-catalyzed reaction. Although microsomal preparations from freshly excised livers exhibit normal kinetic behavior under standard conditions, the soluble preparation, at all stages of purification, have a biphasic rate curve which we ascribe to the slow dissociation of S-adenosyl homocysteine from the enzyme-product complex. In support of this contention we point to the elimination of the burst phase by incubation with S-adenosyl homocysteine. The role of the enzyme in detoxication has been strengthened by the recent dis-
FROM
RAT LIVER
637
covery of cysteine conjugate /3-lyase (13), an enzyme that catalyzes the formation of thiols from thioethers of cysteine. Such thioethers commonly arise in the course of mercapturic acid formation (14). As with other enzymes proposed as active in detoxification, e.g., the cytochrome P-450 oxidation system (15), the glutathione transfer&es (14), and epoxide hydrase (16), the thiol S-methyltransferase confines its specificity to the donor molecule while allowing a broad spectrum of mainly hydrophobic compounds as acceptor substrates (17). REFERENCES 1. BREINER, J., AND GREENBERG, D. M. (1961) Biochim. Biophys. Acta 46, 217-224. 2. JAKUBOWSKI, M., AND GESSNER, T. (1972) Biothem. Pharmacol. 21, 3073-3076. 3. COBBY, J., MAYERSOHN, M., AND SELLIAH, S. (1977) Life Sci. 21, 937-942. 4. GESSNER, T., AND JAKUBOWSKI, M. (1972) Biohem. Pharmacol. 21,219~230. 5. REMY, C. M. (1963) J. Biol. Chem. 238, 10781084. 6. LINDSAY, R. H., HULSEY, B. S., AND ABOULENEIN, H. Y. (1975) B&hem. Pharmacol. 24, 463-468. 7. BORCHARDT, R. T., CHENG, C. F., COOKE, P. H., AND CREVELING, C. R. (1974) Lifi Sci. 14, 1089-1100. 8. BORCHARDT, R. T., AND CHENG, C. F. (1978) Biochim. Biophys. Acta 522, 340-353. 9. LEVIN, 0. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 5, pp. 27-32, Academic Press, New York. 10. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-4412. 11. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193,265-275. 12. AXELROD, J. (1971) in Concepts in Biochemical Pharmacology (Bredie, B. B., and Giiette, J. R., eds.), Vol. 2, pp. 609-619, Springer-Verlag, Berlin. 13. TATEISHI, M., SUZUKI, S., AND SHIMIZU, H. (1978) J. Biol. Chem. 253, 8854-8859. 14. JAKOBY, W. B. (1978) Advan. Ewzymol. 46,383414. 15. ESTABROOK, R. W. (1971) in Concepts in Biechemical Pharmacology (Bredie, B. B., and Gillette, J. R., eds.), Vol. 2, pp. 264-284, Springer-Verlag, Berlin. 16. Lu, A. Y. H., JERINA, D. M., AND LEVIN, W. (1977) J. Biol. Chem. 252,3715-3723. 17. JAKOBY, W. B., AND KEEN, J. M. (1978) Trends Biochem. Sci. 2, 229-231.