A sensitive fluorometric assay was developed for alcohol sulfotransferase (AST).
This was the first continuous fluorometric assay reported for AST. It utilized 3’-phosphoadenosine 5’-phosphosulfate (PAPS) regenerated from 3’-phosphoadenosine 5’-phosphate (PAP) by a recombinant phenol sulfotransferase (PST) using 4-methylumbelliferyl sulfate (MUS) as the sulfuryl group donor. The recombinant PST did not use the alcohol substrate under the designed condition, and the sensitivity for AST activity was found comparable to that of radioactive assay as reported in the literature. The change of fluorescence intensity of 4-methylumbelliferone (MU) corresponded directly to the amount of active AST and was sensitive enough to measure ng or picomole amount of the enzyme activity. This fluorometric assay was used to determine the activities of AST as purified form and in crude extracts of pig liver, rat liver, and Escherichia coli. Some properties of hDHEA-ST were determined by this method and were found comparable to published data. Under similar assay conditions, the contaminated activities of arylsulfatase in crude extracts were also determined. This method not only is useful for the routine and detailed kinetic study of this important class of enzymes but also has the potential for the development of a high-throughput procedure using microplate reader.
`
Introduction
Sulfotransferases (STs) are a large family of enzymes that catalyze the transfer of sulfuryl group from PAPS to numerous endogenous and exogenous compounds (Jakoby and Ziegler, 1990). Cytosolic STs catalyze the sulfonation of steroid hormones, catecholamines/neurotransmitters, drugs and environmental chemicals, and are involved in hormone homeostasis and metabolic detoxication/activation of xenobiotics (Coughtrie et al., 1998; Duffel and Guengerich, 1997; Falany et al., 1993;
Mulder and Jakoby, 1990; Weinshilboum et al., 1994). In contrast, Golgi membrane-bound STs catalyze the sulfonation of macromolecules including glycosaminoglycans and proteins, and play important roles in the modulation of receptor binding, intercellular communication, and signaling processes (Bowman and Bertozzi, 1999; Weinshilboum et al., 1997).
As a member of cytosolic STs, AST catalyzes the sulfonation of various steroids and their derivatives as well as many xenobiotic alcohols (Falany, 1997;
Weinshilboum et al., 1997). Substrates of human AST include hydroxysteroids such as dehydroepiandrosterone (DHEA), testosterone, β-estradiol, and many other endogenous steroids (Chen et al., 1996; Kakuta et al., 1998). Steroid sulfonation has been recognized as an important function for maintaining steroid hormone level in vivo (Chang et al., 2004). A prominent example is that the bulk of DHEA produced by adrenal glands is sulfonated and secreted into circulation, which served as a precursor for the androgenic and estrogenic steroids in extra-adrenal tissues (Kroboth et al.,
products (Ramaswamy and Jakoby, 1987), which involve stopping the reactions by heat treatment after a fixed time interval, centrifugation to remove precipitates formed, and changing the solvent system prior to thin-layer or paper chromatography. These procedures are tedious for routine and detailed kinetic studies of AST enzymes. Other reported AST assays are also end-point analyses requiring the determination of PAP using high-performance liquid chromatography (Sheng and Duffel, 2001). Routine spectrophotometric assays, however, have been available only for PST (Ramaswamy and Jakoby, 1987). Despite the considerable progress made in recent years on AST enzymes, several fundamental issues concerning the role of neurosteroids in neuron morphogenesis, regulation, and physiological involvement still remain to be fully elucidated. The present study was prompted by an attempt to develop a convenient assay in order to address theses important issues.
Here we report the development of a fluorometric assay for AST (Figure 1). In this assay, the regeneration of PAPS from PAP with MUS as the sulfuryl group donor was catalyzed by PAP-free PST in a reverse physiological reaction (Lin and Yang, 1998). This reaction was coupled to AST, where purified human dehydroepiandrosterone sulfotransferase (hDHEA-ST) was used. The product, MU, served as fluorometric indicator to monitor hDHEA-ST activity.
Experimental Procedures
Materials. MUS, MU, DHEA, PAP, PAPS, tris[hydroxymethyl]aminomethane (Tris), 2-[N-morpholino]ethanesulfonic acid (MES), phenylmethylsulfonyl fluoride (PMSF), [ethylenedinitrilo] tetracetic acid (EDTA), glutathione (reduced form) and dithiothreitol (DTT) were purchased from Sigma (St. Louis, MO). Potassium phosphate (dibasic), glycine, and sodium dodecyl sulfate (SDS) were obtained from J.
T. Baker (Phillipsburg, NJ 08865 U.S.A.). Glutathione S-transferase Sepharose fast flow was obtained from Amersham Pharmacia Biotech Asia Pacific (Hong Kong). All other chemicals were of the highest purity commercially available.
Preparation of PAP-free PST. The β-form of recombinant PST (Yang et al., 1996) was used as the PAP-free enzyme. Recombinant mutant PST, K65ER68G, was cloned into an expression vector, pET3c, and transformed into Escherichia coli BL21 (DE3) (Chen et al., 1992). The enzyme isolation procedure was the same as described earlier (Yang et al., 1996), which resulted in a homogeneous protein as determined by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970).
Preparation of hDHEA-ST. Recombinant hDHEA-ST was cloned into an expression vector, pGEX-2TK and transformed into Escherichia coli BL21 (DE3).
The expression and purification of hDHEA-ST was described previously (Chang et al., 2001) and a homogeneous protein was obtained as determined by
absorbency at 280 nm (1.7 ml/mg cm-1 and 2.4 ml/mg cm-1, respectively) (Gill and von Hippel, 1989) with an UV/Vis spectrophotometer (Hitachi UV/Vis-3300, Japan).
PST assay. The activity of PAP-free PST, K65ER68G, was determined based on the change of fluorescence due to the production of MU from MUS as measured using a spectrofluorometer (Hitachi F-4500, Japan). The excitation and emission wavelengths were 360 nm and 450 nm, respectively. The reaction mixture included 5 mM 2-mercaptoethanol, 4 mM MUS, 20 µM PAP, 100 mM potassium phosphate buffer (pH 7.0), and 0.5-2 µg K65ER68G. This assay was also referred to as a reverse physiological reaction catalyzed by K65ER68G. One unit was defined as 1 µmol of PAP converted to PAPS per minute with 4-methylumbelliferyl sulfate under the reaction conditions described above.
Coupled-enzyme assay for AST. Reaction mixture (1ml) contained 100 mM potassium phosphate buffer (pH 7.0), 5 mM 2-mercaptoethanol, 20 µM PAPS, 4 mM MUS, 5 µM DHEA, and 3.2 mU (5.4 µg) K65ER68G. AST (hDHEA-ST or crude extract) was added following a preincubation period so as to start the reaction at 37°C.
Because commercial PAPS contained significant amounts of PAP which is an inhibitor of sulfotransferase (Duffel and Jakoby, 1981; Yang, et al., 1996) the assay mixture was preincubated for 15 min prior to the addition of DHEA to ensure that all PAP had been converted to PAPS by K65ER68G before complete enzymatic reaction was started. The production of MU was monitored by fluorescence as previously described. The change of fluorescence was linear for over 20 min of initial reaction time.
prepared from 20 g each of frozen rat or pig liver which was mixed with 20 ml buffer A (10 mM Tris-HCl at pH 7.4 plus 125 mM sucrose, 10% glycerol, 1 mM DTT, l.5 mM PMSF, and 1 mM EDTA) and homogenized with liquid nitrogen. Bacterial cell extract was prepared from about 1 g Escherichia coli pelleted from 250 ml cell culture, which was mixed with 20 ml buffer A and sonicated three times, then collect supernatants.
Results and Discussion
K65ER68G as catalyst for the regeneration of PAPS. In this proposed
coupled-enzyme assay, PST was used to regenerate PAPS from PAP and MUS as illustrated in Figure 1. Under the similar conditions, arylsulfatase activity not only could be determined in the presence of only MUS but PST activity could be also determined in the absence of AST or its substrate. Two characteristics of wild-type PST, however, might prevent the effective production of PAPS. It had been shown that wild-type PST contained tightly bound PAP (Yang et al., 1996), a sulfotransferase inhibitor which exists in all sulfotransferase-catalyzed reactions. Moreover, phenols were also inhibitors of PST (Duffel and Jakoby, 1981). Under reducing conditions, the activity of PST could be significantly affected by the presence of PAP and/or phenol in the proposed PAPS regenerating system. A ternary complex of PST, PAP, and phenol might form to slow down the regeneration of PAPS from PAP (Marshall et al., 2000; Vakiani et al., 1998; Whittemore et al., 1985). Fortunately, previous studies had demonstrated that a PST mutant, K65ER68G, was free of the complications mentioned above (Yang et al., 1996). Preliminary studies showed that K65ER68G could efficiently catalyze the production of PAPS, and PAP and MU did not inhibit the reaction under the conditions adopted in this report. As shown in Table 1, Km for MUS determined was not significantly affected by the pH of the solution. In contrast, Vmax of the same reaction was significantly decreased at pH 9.0. These data were useful for the design of coupled-enzyme assay.
Selection of excitation and emission wavelengths for the coupled-enzyme assay.
To bring about a virtually irreversible auxiliary reaction with regard to the initial
concentration of MUS (4 mM) was used to saturate the PAPS regenerating system based on data compiled in Table 1. A suitable excitation wavelength was determined to prevent the “inner filter effect” (Lackowicz, 1983) caused by the absorption of MUS. Briefly, due to different absorption spectra of MUS and MU the excitation (absorption) wavelength of MU must be selected carefully to avoid absorption from MUS. The excitation wavelength, therefore, was selected according to the absorption spectra of MU and MUS as shown in Figure 2. The suitable excitation wavelength was chosen by comparing the absorption spectra of the two compounds. Therefore, AST activity was in effect determined by the increase of fluorescence of MU at 450 nm upon excitation at 360 nm. The relative emission coefficient of the fluorescence at 450 nm was determined under different conditions as shown in Table 2. The sensitivity of this method could thus reach nM range since the pmole amount of MU could be reliably determined. The intensity of the fluorescence was significantly dependent on the pH value ranging from 6 to 10, primarily due to the deprotonation of MU at alkaline pH (pKa of MU = 7.8 (Sun et al., 1998)). The temperature effect was less significant as shown in Table 2.
Coupled-enzyme assay. It is expected that the reaction rate of AST, which was in the range of sub-µmole/min/mg (Chang et al., 2001), could be easily monitored using this coupled-enzyme system. It is worth pointing out that the major requirements for this assay were the adequate amount of MUS, an excess of K65ER68G activity, and a saturating PAPS concentration.
hDHEA-ST. Some background fluorescence due to the auxiliary reaction was observed. This was probably because of the presence as an impurity of PAP in commercially available PAPS (Duffel and Jakoby, 1981; Yang et al., 1996;). The fluorescence background was, however, low in the absence of PAPS, K65ER68G, or MUS. Under these conditions, the auxiliary reaction did not take place. No effects were observed on the measured rates by raising the concentration of PAPS (data not shown), which indicated that PAPS concentration was saturating enough for the coupling system to reach its maximum activity. Taking together the results shown in Figure 3, it was concluded that the continuous changes in fluorescence were specifically attributable to hDHEA-ST activity.
To determine the linear range of AST assay at a given K65ER68G concentration (3.2 mU), the rate of MUS reduction was measured using a concentration range (0.07-1.35 µg) of hDHEA-ST as shown in Figure 4. Although this linear range and sensitivity of hDHEA-ST assay could be further extended, it was suitable for our needs under the present situation.
To test our coupled-enzyme assay and compare the results with the data appeared in the literature, the hDHEA-ST was characterized by this fluorometric assay as shown in Figures 5 to 7. These results fitted perfectly with previously reported data using radioactive assay procedures (Chang et al., 2001). Figure 5 shows the pH-dependency of the DHEA-sulfonating activity. The optimum pH spanned from 7 through 9. The enzyme activity at pH 6.0 was approximately 50% of that in the optimum pH range. Virtually no activity was detected at pH 10.0. The effects of temperature on the DHEA-sulfonating activity was examined over 25-50℃ as shown in Figure 6. Maximum sulfonating activity was observed at approximately 40℃. The enzyme activity at 25℃ and 50℃ were approximately 50％ of maximal activity at
DHEA concentration on the DHEA-sulfonating activity. Significant substrate inhibition was observed as previously reported (Chang et al, 2004; Duffel and Jakoby, 1981). Substrate inhibition leading to the formation of a nonproductive enzyme-PAP-substrate complex (Marchall et al, 2000; Vakiani et al, 1998;
Whittemore et al, 1985) in fact is a rather common feature among member of the sulfotransferase family (Falany et al, 1989; Marcus et al, 1980; Otterness et al, 1992).
These results compare favorably to published Km values determined by a noncoupled, radioisotopic thin-layer chromatography assay (Chang et al, 2001; 2004). DHEA were reported to yield Km and Kis values of 2.1 and 3.8 µM, respectively (Chang et al, 2001). This coupled assay determined these values to be 4.7 and 4.3 µM, respectively.
We suggest that the discrepancy between these values is due to the inherent inaccuracy of the radioisotopic assay. This coupled-enzyme assay allows the continuous measurement of initial reaction velocity more accurately than end-point assays. Besides, it was found that the sensitivity of this fluorometric assay was comparable to that of the radioactive assay for AST reported in the literature (Chang et al, 2001; 2004). The activity of the amount of enzyme used (100 ng) previously could be easily determined by the present method as shown in Figure 4.
Determination of AST activity in biological samples. We further demonstrated the feasibility of present assay for measuring AST activity in biological samples. In addition, several other enzyme activities associated with sulfonation/desulfonation could also be determined under similar conditions. Sulfatase, alcohol and phenol
could not be observed in the absence of PAPS, and therefore reaction condition II gave a background activity exhibited mainly by arylsulfatase. The sulfatase activity was further confirmed and quantified under the reaction conditions that contained only MUS and buffer (or in the absence of DHEA and PAPS, data not shown). PST activity could also be determined using a similar procedure (data was not shown since it did not interfere with the AST activity). In the absence of MUS, the fluorescence observed was close to the experimental error (data not shown) and represented the background derived from the interference of biological samples. Thus, the AST activity could be calculated simply by subtracting arylsulfatase activity from that measured in the complete coupled-enzyme reaction. Based on this method, the AST activity in rat liver was found to be significantly higher than that in pig liver or E. coli (Table 3). Moreover, the AST activity extracted from transformed E. coli cells (with pGEX-2TK) was approximately 100 times than that in untransformed cells.
Conclusion
We developed in this study a continuous fluorometric AST assay whose sensitivity was comparable to that of the end-point radioactive assay reported in the literature.
This method was demonstrated to be useful for the determination of AST activities associated with homogeneous AST or those present in crude extracts from biological samples. This new assay procedure could be adapted for high-throughput assay using a microplate reader.
Acknowledgement
This work was supported by National Science Foundation, under Grant NSC 92-2311-B-009-003 and the Brain Research Center of the University System of Taiwan, under Grant 91B-711 and 92B-711.
Table 1
Production of PAPS from PAP and MUS catalyzed by K65ER68Ga
pH Km (µM) Vmax (nmol min-1 mg-1)
6.0 161 ± 14 204 ± 7
7.0 183 ± 15 399 ± 9
8.0 196 ± 21 142 ± 5
9.0 167 ± 51 2.9 ± 0.2
a The reaction mixture included 5 mM β-mercaptoethanol, 20 µM PAP, and MUS (50 µM - 3.2 mM) plus 0.55 µg enzyme in 100 mM buffer (MES at pH 6.0, potassium phosphate at pH 7.0, and Tris at pH 8.0 and pH 9.0). The Km and Vmax were obtained using nonlinear regression by SigmaPlot 2001, V7.0 and Enzyme Kinetics Module, V1.1 (SPSS Inc., Chicago, IL).
Table 2
The Relative Emission Coefficient of MU at 450 nma
Relative Emission coefficient (cm-1nM-1) Temperature (℃)
pH
25 30 35 37 40 45 50
6.0 0.12
7.0 0.4 0.43 0.45 0.48 0.50 0.52 0.55
7.5 0.64
8.0 1.14
9.0 2.14
10.0 4.10
a The fluorescence of MU (100, 200, and 400 nM) in 100 mM buffer (MES at pH 6.0, potassium phosphate at pHs 7.0 and 7.5, Tris-base at pHs 8.0 and 9.0 and glycine at pH 10.0) was determined with a spectrofluorometer (Hitachi F-4500, Japan). The excitation and emission wavelengths were at 360 and 450 nm, respectively.
Table 3
Activities of AST and arylsulfatase in biological samples a
Rat liver Pig liver E. coli E. coli ( with pGEX-2TK)
a Detailed procedures were described under Coupled-enzyme assay for AST in Materials and Method except that purified hDHEA-ST was replaced by the extract of biological sample. Specific activity referred to MU produced following the addition of extract whose protein concentration was determined by absorption at A280. Total activity referred to MU produced with one gram of wet cell or liver.
b AST activity was eliminated in the absence of PAPS as shown in Figure 3.
O O
Figure 1. Scheme for the determination of AST activity. This assay was based on the regeneration of PAPS from PAP catalyzed by a recombinant PST (K65ER68G) using MUS as the sulfuryl group donor. In coupled-enzyme assay, PST represented an auxiliary enzyme, and the product, MU, was used as a fluorescent indicator of enzyme turnover.
Moreover, the biosynthesis of PAPS from ATP and SO4
2- was catalyzed by hPAPS synthetase, which was a bifunctional enzyme with activity of ATP sulfurylase and APS kinase.
A B
pH 6.0
Wavelength (nm)
250 300 350 400 450
Absorbance
250 300 350 400 450
Absorbance
250 300 350 400 450
Absorbance
250 300 350 400 450
Absorbance
Figure 2. Absorption spectra of MUS and MU. The spectra were obtained in 1ml aqueous solutions contained 100 µM MUS (○) or MU (●) and 100 mM buffer (MES at pH 6.0, potassium phosphate at pH 7.0, and Tris-base at pH 8.0 and pH 9.0.)
Time (sec)
0 50 100 150 200 250 300
RFU
0 200 1200 1300 1400
Figure 3. Progress curves of the coupled-enzyme assay for AST. Complete reaction (●) and control reactions without PAPS (○), DHEA (▼), MUS (▽), K65ER68G (■), or hDHEA-ST(□) of coupled-enzyme assay for AST were conducted at pH 7.0, 37°C.
Detailed procedures were described under Coupled-enzyme assay for AST in Materials and Methods.
hDHEA-ST (ng)
0 300 600 900 1200 1500
∆ Fl/min
-5 0 5 10 15 20 25 30
Figure 4. Effective range of fluorometric assay for hDHEA-ST. Complete reaction (●) and control reactions without PAPS (○), DHEA (▼), MUS (▽), or K65ER68G (■) of coupled-enzyme assay were run in a total volume of 1000 µl at 37°C. Detailed procedures were described under Coupled-enzyme assay for AST in Materials and Methods. Each point was determined from triplicate assay data and standard error was obtained by SigmaPlot 2001, V7.0 (SPSS Inc., Chicago, IL).
pH value
5 6 7 8 9 10 11
Specific activity (nmol/min/mg) 0
20 40 60 80 100 120
Figure 5. pH profile of recombinant hDHEA-ST. The enzymatic assays were carried out with about 0.5 µg recombinant hDHEA-ST under the condition of Coupled-enzyme
assay for AST as described in Materials and Methods using different buffer systems (100 mM MES at pH 6.0, potassium phosphate at pH 7.0, Tris-base at pH 8.0 or 9.0, and glycine at pH 10.0). Each point was determined from triplicate assay data and standard error was obtained by SigmaPlot 2001, V7.0 (SPSS Inc., Chicago, IL).
Temperature (oC)
20 25 30 35 40 45 50 55
Specific activity (nmol/min/mg) 0
20 40 60 80 100 120
Figure 6. Temperature effect on the activity of recombinant hDHEA-ST. The enzymatic assays were carried out with about 0.5 µg recombinant hDHEA-ST under the condition of coupled-enzyme assay for AST at pH 7.0 in different temperature. Each point was determined from triplicate assay data and standard error was obtained by SigmaPlot 2001, V7.0 (SPSS Inc., Chicago, IL).
[DHEA] (µM)
0 10 20 30 40 50 60
Specific activity (nmol/min/mg) 0
20 40 60 80 100 120
Figure 7. Substrate inhibition of DHEA on recombinant hDHEA-ST. The enzymatic assays were carried out under the condition of coupled-enzyme assay for AST at pH 7.0 with about 0.5 µg recombinant hDHEA-ST and different DHEA concentrations (0.3 ~ 50.0 µM). The kinetic parameters were obtained using the substrate inhibition equation v
= V[S]/{Km + [S](1 + [S]/Kis)}by SigmaPlot 2001, V7.0 and Enzyme Kinetics Module, V1.1 (SPSS Inc., Chicago, IL)
Chapter 2 Effects of Quaternary structure on Cytosolic Sulfotransferase Function
Cytosolic sulfotransferases (STs) catalyze the sulfonation of small molecules including xenobiotics, bioamines, hormones, and steroids. The enzymes, including mammalian phenol sulfotransferase (PST) and alcohol sulfotransferase (AST), are generally homodimers in solution and play a role in detoxication, hormone homeostasis and signal transduction, such as neurotransmission. Previous structural characterization of mutations within the dimer interface of cytosolic ST have demonstrated that KTVE motif are the common protein-protein interaction motif that mediates their homo- as well as heterodimerization. Through site-directed mutagenesis, and gel filtration, we also confirm that a single mutation of Val266 to Glu was sufficient to convert rat PST (rPST) to a monomer by breaking hydrophobic KTVE motif. The similar result was found in AST by mutation of Val260 to Glu. First, kinetic constants of substrates (pNPS and DHEA) and cofactors (PAP and PAPS) were determined to identify the effect of dimerization on cytosolic ST catalysis. Second, due to stimulatory and inhibitory effects of metal ions on cytosolic STs, metal effect on dimeric and monomeric STs was also compared. No significant difference on catalysis between dimer and monomer was observed either in PST and AST. Therefore, thermal inactivation was investigated into the thermostability in dimeric and monomeric STs. Although the importance of dimerization
Cytosolic sulfotransferases (STs) catalyze the sulfonation of small molecules including xenobiotics, bioamines, hormones, and steroids. The enzymes, including mammalian phenol sulfotransferase (PST) and alcohol sulfotransferase (AST), are generally homodimers in solution and play a role in detoxication, hormone homeostasis and signal transduction, such as neurotransmission. Previous structural characterization of mutations within the dimer interface of cytosolic ST have demonstrated that KTVE motif are the common protein-protein interaction motif that mediates their homo- as well as heterodimerization. Through site-directed mutagenesis, and gel filtration, we also confirm that a single mutation of Val266 to Glu was sufficient to convert rat PST (rPST) to a monomer by breaking hydrophobic KTVE motif. The similar result was found in AST by mutation of Val260 to Glu. First, kinetic constants of substrates (pNPS and DHEA) and cofactors (PAP and PAPS) were determined to identify the effect of dimerization on cytosolic ST catalysis. Second, due to stimulatory and inhibitory effects of metal ions on cytosolic STs, metal effect on dimeric and monomeric STs was also compared. No significant difference on catalysis between dimer and monomer was observed either in PST and AST. Therefore, thermal inactivation was investigated into the thermostability in dimeric and monomeric STs. Although the importance of dimerization