S-methylation of 2-mercaptopyrazine in rat liver microsomes and cytosol

Article abstract of DOI:10.1080/004982599238155

1. 2-(Allylthio)pyrazine (2-AP) has been demonstrated to protect the liver against toxicants by inhibiting CYP2E1 activity. Since 2-mercaptopyrazine (2-MP) is presumed to be a metabolite of 2-AP, the experiments were performed to determine whether rat liver microsomal and/or cytosolic preparations could catalyse the S-methylation of 2-MP. 2. It was found that both rat liver microsomes and cytosol could catalyse the S-methylation of 2-MP. The microsomal activity displayed biphasic substrate kinetics, with apparent K(m) = 8.44 ± 2.68 and 417 ± 74 μM for the high- and low-affinity activities respectively. The high-affinity activity had an apparent K(m) for S-adenosyl-L-methionine (Ado-Met) of 3.52 μM. The cytosolic activity also displayed biphasic substrate kinetics, with apparent K(m) of 3.26 ± 0.62 and 91.6 ± 23.1 μM for the high- and low-affinity activities respectively. 3. The microsomal S-methylation of 2-MP was inhibited by 2,3-dichloro-α-methylbenzylamine (DCMB), SKF-525A and benzylamine, known microsomal thiol methyltransferase (TMT) inhibitors, whereas cytosolic activity was inhibited by anisic acid and 3-chlorobenzoate, which also inhibit cytosolic thiopurine methyltransferase (TPMT). Both activities were inhibited by S-adenosyl-L-homocysteine (Met-Hcy). 4. These results suggest that both TMT and TPMT may be involved in the in vivo methylation of 2-MP.

Full text of DOI:10.1080/004982599238155

xenobiotica , 1999, vol. 29, no. 9, 909±916
S-methylation of 2-mercaptopyrazine in rat liver
microsomes and cytosol
D. H. LEE and D. H. KIM*
* Bioanalysis and Biotransformation Research Center, Korea Institute of Science and
Technology, PO Box 131, Chungryang, Seoul, Korea
Received 6 January 1999
1
. 2-(Allylthio)pyrazine (2-AP) has been demonstrated to protect the liver against
toxicants by inhibiting CYP2E1 activity. Since 2-mercaptopyrazine(2-MP) is presumed to
be a metabolite of 2-AP, the experiments were performed to determine whether rat liver
microsomal and}or cytosolic preparations could catalyse the S-methylation of 2-MP.
2
. It was found that both rat liver microsomes and cytosol could catalyse the S-
methylation of 2-MP. The microsomal activity displayed biphasic substrate kinetics, with
apparent K
5 8.4462.68 and 417674 lm for the high- and low-aænity activities
respectively. The high-aænity activity had an apparent K for S-adenosyl-l-methionine
Ado-Met) of 3.52 lm. The cytosolic activity also displayed biphasic substrate kinetics,
m
m
(
with apparent K
respectively.
m
of 3.2660.62 and 91.6623.1 lm for the high- and low-aænity activities
3
. The microsomal S-methylation of 2-MP was inhibited by 2,3-dichloro-a-methyl-
benzylamine (DCMB), SKF-525A and benzylamine, known microsomal thiol methyl-
transferase(TMT)inhibitors, whereascytosolic activity was inhibited by anisic acid and 3-
chlorobenzoate, which also inhibit cytosolic thiopurine methyltransferase (TPMT). Both
activities were inhibited by S-adenosyl-l-homocysteine (Met-Hcy).
4. These results suggest that both TMT and TPMT may be involved in the in vivo
methylation of 2-MP.
Introduction
S-Adenosyl-l-methionine (Ado-Met)-dependent sulphur methylation plays a
major role in the metabolism and elimination of aliphatic and aromatic sulphydryl
drugs such as 6-mercaptopurine, captopril and disulphiram (Remy 1963, Drummer
et al. 1982, Glauser et al. 1993). Thiol methylation in mammals is catalysed by two
separate enzymes: cytosolic thiopurine methyltransferase (TPMT) (EC 2.1.1.67)
and microsomal thiol methyltransferase (TMT) (EC 2.1.1.9). These two enzymes
diåer in substrate speci®cities and inhibitor sensitivities, and regulation. TPMT is
selectively inhibited by benzoic acid derivatives and is involved in the methylation
of aromatic and heterocyclic sulphydryl compounds (Remy 1963, Woodson and
Weinshilboum 1983, Woodson et al. 1983). In contrast, TMT preferentially
catalyses the S-methylation of aliphatic sulphydryl compounds, and is inhibited by
2
1
,3-dichloro-a-methylbenzylamine(DCMB) and SKF-525A (Weinshilboum et al.
979, Keith et al. 1984, Glauser et al. 1992).
2
-(Allylthio)pyrazine (2-AP) has been reported to protect the liver against toxic
agents (acetaminophen and carbon tetrachloride) by selectively inhibiting hepatic
cytochrome P4502E1 (Kim et al. 1997) as well as by directly scavenging oxygen
species (Kim et al. 1996). This compound also reduced vinyl carbamate- or vinyl
carbamate epoxide-induced hepatotoxicity, mutagenicity and tumorigenicity (Surh
et al. 1998). 2-AP is extensively metabolized and no parent compoundwas detected
*
Author for correspondence; E-mail: dhkim!kist.re.kr
Xenobiotica ISSN 0049±8254 print}ISSN 1366±5928 on line ’ 1999 Taylor & Francis Ltd
http:}}www.tandf.co.uk}JNLS}xen.htm http:}}www.taylorandfrancis.com}JNLS}xen.htm

9
10
D. H. Lee and D. H. Kim
in urine and bile (Han and Lee 1999). In the preliminary experiments, S-methyl-2-
mercaptopyrazine was tentatively identi®ed in the urine from the 2-AP treated rat,
suggesting that 2-mercaptopyrazine (2-MP) is generated in the course of the
metabolism of 2-AP. To understand the fate of 2-MP in the rat is of importance
because it is pharmacologically active (Kang and Kim 1998). Accordingly, the
purpose of the present investigation was to characterize the S-methylationof 2-MP,
a metabolite intermediate generated from 2-AP, by microsomal TMT and cytosolic
TPMT to evaluate further the potential role of TMT and TPMT in the in vivo
methylation of 2-MP.
Materials and methods
Materials
2
-MP with a chemical purity "98% was provided by Professor Nak Doo Kim, Seoul National
University. [" C-methyl]Ado-Met (spec. act. 59 lCi lmol) was from New England Nuclear (Boston,
MA, USA). SKF-525A and DCMB were from Research Biochemicals, Inc. (Natrick, MA, USA). Ado-
Met, Ado-Hcy, benzylamine, 3,4-dimethoxybenzoate, m-anisic acid and 3-chlorobenzoate were from
Sigma (St Louis, MO, USA). All other chemicals used were highest grade commercially available.
%
}
Preparation of subcellular fraction.
Male Sprague±Dawley rats (200±220 g) from Dae Han (Taejon, Korea) were used for the preparation
of subcellular fractions. Immediately following perfusion of livers with phosphate-buåered saline, the
livers were removed, washed twice and suspended in 0.1 m Tris-acetate (pH 7.4) containing 0.1 m KCl,
1
mm EDTA and 20 lm butylated hydroxytoluene to a ®nal concentration of 30% (v}v). The mixture
was homogenized (Ultra-turrax T25, IKA Lab) and centrifuged at 10000g for 30 min at 4 C. The
supernatant was re-centrifuged at 100000g for 60 min at 4 C (CENTRIKON T-2180, Kontron,
Milano, Italy) and the cytosolic fraction was recovered. The microsomal pellet was resuspended in
.1 m potassium pyrophosphate (pH 7.4) containing 1 mm EDTA and 20% (w}v) glycerol and
centrifuged again at 100 000g for 60 min at 4 C. The resulting pellet was suspended in 10 mm Tris-
acetate (pH 7.4) containing 1 mm EDTA and 20% (w}v) glycerol and aliquots were stored at ±80
C until
use. Pooled microsomes and cytosol from 10 diåerent rats were used for 2-MP methyltransferase assay.
Ê
Ê
0
Ê
Ê
2
-MP methyltransferase assay
-MP methyltransferase activity was assayed by measuring the conversion of 2-MP to radioactively
2
"
%
labelled Me-MP (®gure 1) with [ C-methyl]Ado-Met as the methyl donor, as described by Glauser et
al. (1993) with slight modi®cation. In the microsomal assay, the reaction mixture consisted of 0.1 m
potassium phosphate buåer (pH 7.5) containing 0.1 mg microsomal protein, 30 lm Ado-Met (9
lCi}lmol) and various concentrations of 2-MP in a ®nal volume of 200 ll. Samples without 2-MP were
used as blank for the assay. In the cytosolic assay, 0.1 mg cytosolic protein was added to the reaction
mixture instead of microsomes. The mixtures were incubated for 30 min at 37 C and the reaction was
Ê
terminated by the addition of 200 ll 0.1 N HCl. The radioactive product was separated from ["% C-
methyl]Ado-Met by solvent extraction with 2 vols toluene, and radioactivity was measured in a liquid
scintillation counter (2000CA, Packard, Meriden, CT, USA).
Hplc analysis of methylated products
The residue extracted with toluene was analysed on a reverse-phase C") hplc column (Ultrasphere
ODS, 4.63 250 mm, 5 lm; Beckman), using a Waters hplc system. The column was eluted at a ¯ow rate
of 1.0 ml}min with 10% methanol (v}v) in distilled water with a linear gradient to 70% methanol over
2
6 min. The eluent was monitored at 320 nm and radioactivity was monitored using a û-RAM
radioactivity ¯ow detector (IN}US Corporation, Tampa, FL, USA) equipped with solid phase cell.
Kinetic analysis
Initially the kinetic data were analysed using the Eadie±Hofstee equation. Owing to the non-linear
nature of 2-MP methylation, Michaelis±Menten constant (K
calculated using a non-linear regression model derived from the Michaelis±Menten equation:
1 [S])1 Vmax #[S]}(K #
m
) and maximum velocity (Vmax ) were
V
total 5 Vmax "[S]}(K "
m
m
1 [S]),
where Vtotal and [S] are the rate of S-methylation and substrate concentration respectively.

2-Mercaptopyrazine S-methylation
911
Figure 1. Scheme for 2-MP S-methylation. Ado-Met is S-adenosyl-l-methionine; Ado-Hcy is S-
adenosyl-l-homocysteine.
Regression analyses were performed using WinNonlin (v.4.2; Scienti®c Consults, Lexington, KN,
USA).
Results
Identi®cation of methylation products
The product of the 2-MP methyltransferase reaction catalysed by rat liver
microsomes was identi®ed by hplc. Me-MP eluted at 19.3 min and the radioactive
reaction product co-eluted at the same retention time (®gure 2). The radioactive
peak was fractionated and its structure was con®rmed on the basis of mass spectral
data (data not shown). Of the radioactivity applied to the column, 95% was
recovered and 98% of the recovered radioactivity was associated with Me-MP.
S-methylation of 2-MP by rat liver microsomes
The eåect of 2-MP concentration on hepatic microsomal 2-MP methyl-
transferase activity was investigated to determine kinetic pro®le of the rat enzyme
(®gure 3A). As shown in the Eadie±Hofstee plot (®gure 3B), S-methylationof 2-MP
in the presence of rat liver microsomes displayed biphasic kinetics at 2-MP
concentrations from 2 to 2000 lm. Low and high Km calculated by non-linear
regression were 8.4462.68 and 417674 lm respectively with correspondingV
max
5
0
.17560.035 and 1.21960.048 nmol}min}mg protein. The eåects of Ado-Met
concentration on 2-MP methylation were determined in the presence of 5 lm 2-MP
and an Eadie±Hofstee plot of the data is shown in ®gure 4. The apparent Km for
Ado-Met was 3.5260.42 lm. A similar K was calculated when the reaction was
m
analyzed in the presence of 500 lm 2-MP.
S-methylation of 2-MP by rat liver cytosol
To determine whether S-methylation of 2-MP occurred in rat liver cytosol,
cytosolic 2-MP methyltransferase activity was assayed with various concentrations
of 2-MP (®gure 5A) and an Eadie±Hofstee plot of the data is shown ®gure 5B. S-
methylation of 2-MP in the presence of rat liver cytosol also displayed biphasic
kinetics at 2-MP concentrations from 2 to 2000 lm. Low and high K calculated by
m
non-linear regression were 3.2660.62 and 91.6623.1 lm respectively with cor-
max
responding V 5 0.02560.002 and 0.05660.004 nmol}min}mg protein. The
eåects of Ado-Met concentration on 2-MP methylation were determined in the
presence of 5 lm 2-MP and the apparent K for Ado-Met was 4.3460.63 lm, which
m
is similar to that in microsomes. Cytosolic 2-MP methyltransferase activity was
C 9% compared with microsomal activity (table 1).

9
12
D. H. Lee and D. H. Kim
Figure 2. Hplc radio-chromatogram of the product of 2-MP S-methylation catalysed by rat liver
microsomes.
Figure 3. Rat liver microsomal 2-MP S-methylation. (A) Eåect of 2-MP concentration on 2-MP
S-methylation. (B) Eadie±Hofstee plot of the data shown in (A). Each point is the mean of at least
three determinations.
Figure 4. Eadie±Hofstee plot of rat liver microsomal 2-MP S-methylation. Each point is the mean of
at least three determinations.

2-Mercaptopyrazine S-methylation
913
Figure 5. Rat liver cytosolic 2-MP S-methylation. (A) Eåect of 2-MP concentration on 2-MP
S-methylation. (B) Eadie±Hofstee plot of the data shown in (A). Each point is the mean of at least
three determinations.
Table 1. Subcellular distribution of 2-MP S-methylation
Speci®c activity
(nmol}min}mg)
% Microsomal
activity
Protein source (lg)
n
Cytosol 100
Microsomes 100
8
8
0.03960.001
0.41760.015
9.3
100
Microsomal and cytosolic 2-MP S-methyltransferaseactivities were measured
at a concentration of 100lM 2-MP
Table 2. Eåects of various methyltransferase inhibitors on 2-MP S-methylation mediated by rat liver
microsomes and cytosol.
a
Microsomes
Cytosol
Concentration
(mm)
Inhibitors
High-aænity Low-aænity High-aænity Low-aænity
b
DCMB
1.0
0.5
1.0
1.0
1.0
1.0
1.0
0.1
21.762.2
63.366.2
48.567.8
45.166.5
101.863.4
87.363.0
95.2610.6
22.567.4
23.462.5
56.2618.8
73.7613.9
95.267.1
86.261.7
95.769.2
88.166.5
33.868.0
78.164.7
96.362.8
86.862.1
96.163.3
22.863.7
11.463.8
22.563.9
8.367.8
60.364.6
102.6610.8
95.060.6
90.466.0
41.862.5
23.868.3
37.165.5
16.461.8
SKF-525A
benzylamine
aniline
3
3
,4-dimethoxybenzoate
-chlorobenzoate
m-anisic acid
Ado-Hey
c
aValues are % basal activity .
Microsomal high- and low-aænity 2-MP methyltransferase activities were measured at 5 and 500
lm 2-MP respectively; cytosolic activities were measured at 5 and 200 lm 2-MP respectively.
b
Data are mean6SD of three determinations.
c
The basal activities at a substrateconcentrationof 5 lm were 0.065 and 0.016 nmol}min}mg protein
for microsomes and cytosol respectively.
Eåects of methyltransferase inhibitors
DCMB, benzylamine and SKF-525A are eåective inhibitors of microsomal
TMT (Glauser et al. 1993)and benzoicacid derivatives and anisic acid preferentially
inhibit cytosolic TPMT (Woodson et al. 1983). Ado-Hcy, a substrate analogue,
completelyblocksall Ado-Met-mediatedS-methylationreactions (Borchardt 1977).
The S-methylation of 2-MP was monitored in the presence of various inhibitors
known to inhibit TMT and TPMT activities to evaluate the role of TMT and
TPMT in this reaction and the results were summarized in table 2. Ado-Hcy, 0.1
mm, inhibited high- and low-aænity microsomal methylation activities of 2-MP by

9
14
D. H. Lee and D. H. Kim
7
9
7 and 66% respectively. Cytosolic 2-MP methylation activity was abolished by
2% in the presence of 0.1 mm Ado-Hcy. DCMB, SKF-525A, benzylamine and
aniline inhibited 22±78% of the microsomal activity, whereas the cytosolic activity
was only inhibited by DCMB to a lesser degree. 3,4-Dimethoxybenzoate, 3-
chlorobenzoate and m-anisic acid selectively inhibited the cytosolic S-methylation
of 2-MP. These compounds in the experiments did not aåect the microsomal
activity.
Discussion
2
-AP is a pyrazine derivative of allylsulphide synthesized for use as a chemo-
preventive agent. This compound was an eæcient and selective inhibitor of
cytochromeP4502E1 and, hence, was eåective in protecting against hepatic damage
induced by chemical toxicants (Kim et al. 1997). 2-MP, a potential metabolic
intermediate of 2-AP, is presumed to be pharmacologically active. The suppression
of lipopolysaccharide (LPS)-induced expression of nitric oxide synthase and liver
damage by 2-MP have been characterized (Kang and Kim 1998)
2
-AP is known to be extensively metabolized in rat and S-methyl mercapto-
pyrazine has been tentatively identi®ed in the urine from the 2-AP-treated rat (Han
and Lee 1999). Therefore, the in vitro S-methylation of 2-MP was examined to
understand the in vivo metabolism and enzymes involved in its metabolism. Hplc
analysis con®rmed that the radiolabelled product from the incubation of 2-MP with
rat liver microsomes or cytosol in the presence of "% C-Ado-Met was methyl 2-MP.
Under the hplc conditions used, recovery of radioactivity was quantitative and no
other signi®cant metabolites were found. Therefore, rat liver microsomes and
cytosol are capable of catalysing the conversion of 2-MP to S-methyl-2-MP in vitro.
The subcellular distribution of 2-MP methyltransferase activity revealed that C
9
0% of the activity was found in the microsomes, suggesting that microsomal TMT
preferentially catalyses the S-methylation of 2-MP. This results is somewhat
unexpected because cytosolic TPMT is primarily involved in the S-methylation of
aromatic and heterocyclic sulphydryl compoundsin human (Remy 1963, Woodson
et al. 1983) and there may be species diåerence in thiol methyltransferse enzymes.
The microsomal 2-MP methyltransferase activity displayed biphasic substrate
kinetics. The phenomenon of biphasic substrate kinetics has been evaluated
extensively with both human liver microsomes and human RBC membrane TMT
activities (Weinshilboum et al. 1979, Keith et al. 1983, Glauser et al. 1992). This
phenomenon was also demonstrated in the S-methylation of DDC by rat liver
microsomes (Lill et al. 1996). The Km for Ado-Met of high- and low-aænity
microsomal 2-MP methyltransferase were similar, indicating that no diåerences
exist in the aænity for Ado-Met of the two microsomal components of methyl-
transferase. The cytosolic 2-MP methyltransferase activity also displayed biphasic
substrate kinetics with a methyl acceptor substrate but not with Ado-Met. This
behaviour has not been described with other methyl acceptor substrates either in
human liver cytosol or in rat liver cytosol, perhaps because high concentrations of
substrates had not been tested.
Eåects of various methyltransferase inhibitors of TMT and TPMT were
assessed to determine the role of TMT and TPMT in the S-methylation of 2-MP.
The in vitro methylation of diethyldithiocarbamateby human liver microsomes was
inhibited by 100% using 1 mm DCMB (Glauser et al. 1993), whereas maximum

2-Mercaptopyrazine S-methylation
915
inhibition of methylation of diethyldithiocarbamate by rat liver microsomes was C
0% at the same concentration of DCMB (Lill et al. 1996). The present results
6
revealed that DCMB did not completely inhibit both high- and low-aænity
microsomal S-methylation of 2-MP, consistent with the degree of inhibition of
DDC methylation by rat liver microsomal TMT (Lill et al. 1996). SKF-525A, a
selective inhibitor of TMT, only marginally inhibited the cytosolic S-methylation
of 2-MP, whereas it inhibited 60% of the high- and low-aænity microsomal S-
methylation of 2-MP. The S-methylation of 2-MP by rat liver cytosol was very
sensitive to benzoic acid derivatives in the present experiments. These results are
consistent with benzoic acid derivative-induced inhibition of human and mouse
TPMT (Woodson et al. 1983).
The results collectively demonstrate that 2-MP could be methylated by both rat
liver microsomes and cytosol with a methyldonor,Ado-Met, and the S-methylation
of 2-MP could be mediated by TMT and TPMT.
Acknowledgement
The authors thank Dr Yong-Bok Lee, Chunnam University, for performing the
kinetic analysis. This work was supported by the Korea Science and Engineering
Foundation (KOSEF) through The Research Center for New Drug Development,
Seoul National University.
References
Borchardt, R. T., 1977, Synthesis and biological activity of analogs of adenosylhomocysteine as
inhibitors of methyltransferases.In F. Salvatore, E. Borek, V. Zappia, Williams-Ashman and F.
Schlenk (eds), Biochemistry of Adenosylmethionine (New York: Columbia University Press), pp.
1
51±171.
Drummer , O. H., Jarrott, B. and Louis , W. H., 1982, Demonstration of a S-methyl metabolite of
captopril in patients undergoing chronic captopril therapy. Clinical and Experimental Phar-
macology and Physiology, 7 (suppl.), 81±96.
Glauser, T. A., Kerremans , A. L. and Weinshilboum , R. M., 1992, Human hepatic microsomal thiol
methyltransferase assay conditions, biochemical properties, and correlation studies. Drug
Metabolism and Disposition, 20, 247±255.
Glauser, T. A., Nelson , A. N., Zembower , D. E., Lipsky , J. J. and Weinshilboum , R. M., 1993,
Diethyldithiocarbamate S-methylation: Evidence for catalysis by human liver thiol methyl-
transferase and thiopurine methyltransferase. Journal of Pharmacology and Experimental
Therapeutics, 266, 23±32.
Han, G. S. and Lee, M. G., 1999, Pharmacokinetics of a chemoprotective agent, 2-(allylthio)pyrazine,
after intravenous and oral administration to rats: Liver, stomach ®rst-pass eåects. Drug
Metabolism and Disposition, 27, 221±226.
Kang, K. W. and Kim , N. D., 1998, Eåects of mercaptopyrazine, a metabolite of 2-(allylthio)pyrazine,
on LPS-induced sepsis model. Presented at the 50th Annual Meeting of The Korean Society of
Pharmacology, 5±6 November, Muju, Korea.
Kim , S. G., Cho, J. Y., Kim , S. H. and Kim , N. D., 1996, Protective eåects of 2-(allylthio)pyrazine on
retinoyl palmitate- and pyridine-potentiated carbon tetrachloride-induced hepatotoxicity: eåect
on Ux-174 DNA strand breakage. Yakhak Hoeji, 40, 723±733.
Kim , N. D., Kwak, M. K. and Kim , S. G., 1997, Inhibition of cytochrome P450 2E1 expression by 2-
(
allylthio)pyrazine, a potential chemoprotective agent: hepatoprotective eåects. Biochemical
Pharmacology, 53, 261±269.
Keith , R. A., Jardine , I., Kerremans , A. and Weinshilboum , R. M., 1984, Human erythrocytes
membrane thiol methyltransferase: S-methylation of captopril, N-acetylcysteine, and 7-a-thio-
spirolactone. Drug Metabolism and Disposition, 12, 717±724.
Lill , J. S., Mays, D. C. and Lipsky , J. J., 1996, S-methylation of diethyldithiocarbamic acid in rat liver
microsomes. Xenobiotica, 26, 1025±1033.
Remy , C. N. 1963, Metabolism of thiopyrimidines and thiopurines: S-methylation with S-adenosyl-
methionine transmethylase and catabolism in mammalian tissue. Journal of Biological Chemistry,
2
38, 1078±1084.

9
16
2-Mercaptopyrazine S-methylation
Surh, Y. J., Kim , S. G., Park, K. K., Shon , Y., Lee J. M., Kim , N. D. and Miller , J. A., 1998,
Chemopreventive eåects of 2-(allylthio)pyrazine on hepatic lesion, mutagenesis, and tumori-
genesis induced by vinyl carbamate or vinyl carbamate epoxide. Carcinogenesis, 19, 1263±1267.
Weinshilboum , R. M., Sladek, S. and Klumpp , S., 1979, Human erythrocyte thiol methyltransferase:
Radiochemical microassay and biochemical properties. Clinica et Chimica Acta, 97, 59±71.
Woodson , L. C., Ames , M. M., Selassie , C. D., Hansch, C. and Weinshilboum , R. M., 1983,
Thiopurine methyltransferase: Aromatic thiol substrates and inhibition by benzoic acid
derivatives. Molecular Pharmacology, 24, 471±478.
Woodson , L. C. and Weinshilboum , R. M., 1983, Human kidney thiopurine methyltransferase:
puri®cation and biochemical properties. Biochemical Pharmacology, 32, 819±826.