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Studies on rat hepatic hydroxysteroid sulfotransferase — immunochemistry, development and pI variants
Chemico-Biological Interoctions ELSEVIER
Chemico-Biological Interactions 92 (1994) 15-24
Studies on rat hepatic hydroxysteroid sulfotransferase - - immunochemistry, development and pI variants Hiroshi Homma*, Izumi Nakagome, Minoru Kamakura, Masami Hirota, Mie Takahashi, Michio Matsui Kyoritsu College of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105, Japan Received 16 August 1993; revision received 4 February 1994; accepted 14 February 1994
Abstract
Rat hepatic hydroxysteroid sulfotransferase which sulfoconjugates androsterone (androsterone-sulfating sulfotransferase) is an oligomer consisting of several subunits with distinct p l values but with the same molecular mass (pl variants). N-terminal amino acid sequences of the p I variants are all identical. The enzyme is exclusively present in the liver, in which its Iobular localization is sex-dependent. The localization of the enzyme is markedly different from that of an isoenzyme of phenol sulfotransferase. In weanling and adult female rats, the relative abundance of the pI variants is different. During development from weanling stage to adulthood, the amounts of acidic variants increase, whereas the relative levels of alkaline variants remain constant. Keywords: Sulfotransferase; Hydroxysteroid; pI variants; Development; Immunohistochemistry
I. Introduction
The activity o f rat hepatic h y d r o x y s t e r o i d sulfotransferase t o w a r d a n d r o s t e r o n e (androsterone-sulfating sulfotransferase~ A D - S T ) exhibits a characteristic alteration during p o s t n a t a l d e v e l o p m e n t (Fig. 1). It increases after birth in parallel in b o t h * Corresponding author. Abbreviations: AD-ST, androsterone-sulfating sulfotransferase; PAP, 3'-phosphoadenosine 5'phosphate; 2DGE, two-dimensional gel electrophoresis; P-ST, phenol sulfotransferase.
0009-2797/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSD1 0009-2797(94)03286-H
t6
H, Homma et al, / Chem.-Biol. Interact. 92 (1994) 15-24
0.4
>.
0.3 ¸
> L) 6o
0.2
~E _~ m~
0.1
0.0 0
20
40
60
80
days Fig. 1. Postnatal development of androsterone-sulfating sultbtransferase (AD-ST) in hepatic cytosols of male and female rats. Hepatic cytosols were prepared from male (0) and female (O) rats of various ages and AD-ST activity was determined and expressed as nmol/min per mg protein, Data were means of three to ten animals and vertical lines represent S.D. in animals at 20-80 days of age. Data from animals at I and 10 days of age were obtained from the pooled liver ,samples. Details are described previously [1],
sexes until weanling stage. The activity thereafter decreases in males, while in females it declines temporarily and increases again to a maximal level in adult females. The activity in adult females is approx. 15-fold higher than in adult males. The activities in both sexes demonstrate age-related characteristic changes during postnatal development. In the following three parts of the present paper, we describe the molecular basis of the age- and gender-related changes of the AD-ST activity; (i) molecular characterization of the AD-ST enzyme, (ii) immunochemical characterization of the enzyme and (iii) molecular characterization of changes of the enzyme during postnatal development. Age-dependent changes in the expression of hydroxysteroid ST isoenzymes in rat liver have been characterized at the molecular level [2-4]. In the present paper, it is described that the AD-ST comprises several subunits with distinct pI values but with the same molecular mass. In addition, the changes in the relative amounts of the subunits during the postnatal development are characterized. 2. Molecular characterization of AD-ST enzyme
We have purified a major isoenzyme of AD-ST from hepatic cytosols of adult female rats in virtue of DEAE-cellulose column chromatography and PAP-agarose
H. H o m m a et al. ~Chem.-Biol. Interact. 92 (1994) 15-24
17
0,3
0.2
E E
cO
O
(xJ
E c
v
._>
l
01
CO
c~ <
O to t'~
<
-'•'•
5O
AA/~/~ A
lO0
150
200
time (min) Fig. 2. HPLC gel filtration chromatography of hepatic cytosols of female rats. Molecular size of the native form of AD-ST was determined by applying rat hepatic cytosols to HPLC gel filtration chromatography. Cytosolic fractions were prepared from female rats of 8 weeks of age and 5.3 mg protein (200#1) was injected onto a column of TSK gel G3000SW (two i.d. 7.5 × 600 mm stainless steel columns in tandem). The column was eluted at a flow rate of 0.4 ml/min with 50 mM Tris-HC1 (pH7.4), 3 mM 2mercaptoethanol, 0.1 mM EDTA, 0.2 M NaCI and 250 mM sucrose. Fractions of 0.4 ml were collected and assayed for AD-ST activity ( ~ ). Elution of proteins was monitored by measuring the absorbance at 280 nm ( - - ). Void volume, (V0) and positions of elutions of standard proteins (ferritin; 440kDa, aldolase; 158 kDa, BSA; 67 kDa) are also indicated. Details are similar to those in the previous report [6].
affinity column chromatography. The enzyme was purified 122-fold over the activity in the cytosols and the purified enzyme revealed a single protein band with a subunit molecular mass of 30 kDa on SDS-polyacrylamide gel electrophoresis [5,6]. In order to determine the molecular mass of the native form of the AD-ST, the cytosolic fraction of rat liver was applied to HPLC gel filtration chromatography (Fig. 2). The activity was eluted in the fraction which corresponded to molecular mass of about 200 kDa, based on the calculation with the standard proteins. The AD-ST enzyme is therefore an oligomer which consists of subunits with a molecular mass of 30 kDa.
18
H. Homma et al. / Chem.-Biol. Interact. 92 (1994) 15-24 Isoelectrofi)~'using y
L;
30kDa
pl
1,2
6.7
6.1 5.8
Fig. 3. Two-dimensional gel electrophoresis of purified AD-ST. In the first dimension, a 4% polyacrylamide isoelectrofocusing gel was run under denaturing condition in the presence of 2% nonidet P-40, 8.5 M urea and 2% pharmalyte 3-10. In the second dimension, SDS-polyacrylamide (12.5%) gel electrophoresis was carried out. Protein spots were visualized by Coomassie blue staining. The pl values of each spots were determined using internal pl markers. Details were described previously [5]. (Reproduced from ref. 5, with permission.)
0.5
0.4 ~__~ 0.3 "~
0.2
0.1
Q-
0.0
liver
lung
kidney
brain
stomach
intestine
Fig. 4. Tissue distribution of AD-ST. The lung, kidneys, brain, stomach and intestine were excised from male (1~) and female (m) rats of 9 weeks of age and homogenates were prepared using a Polytron homogenizer. Cytosolic fractions were obtained by centrifugation (105 000 × g for 60 min) of the homogenates. AD-ST activity was determined by incubating the reaction mixture (500/A) at 37°C, which contained 100 mM Tris-HCI (pH 7.4), l0 mM MgCI2, 20 #M EDTA, 100 ~M PAPS, 86 #M [3H]androsterone (approx. 350 dpm/nmol) and a cytosolic fraction.
H. Homma et a l . / Chem.-Biol• Interact. 92 (1994) 15-24
19
Two dimensional gel electrophoresis (2DGE) was carried out with the purified AD-ST. The enzyme was focused mainly into four spots which have isoelectric points of 7.2, 6.7, 6.1 and 5.8 (Fig. 3). The most abundant components have pI values of 7.2 and 6.7, while minor constituents are pI 6.1 and 5.8 spots. These protein spots were designated as pl variants. As described below, a rabbit polyclonal antiserum which was raised against the purified AD-ST detected these pI variants in fresh liver cytosols of female rats, indicating that the variants are not artifacts during purification procedure but authentic or real components of AD-ST oligomer. The AD-ST enzyme therefore is an oligomer which comprises several pI variants with the same molecular mass of 30 kDa. 3. Immunochemical characterization of AD-ST enzyme In Fig. 4, the content of AD-ST enzyme was investigated in several extrahepatic tissues of adult male and female rats using a rabbit polyclonal antiserum which was prepared previously [5]. Little activity of AD-ST was detected in the cytosols of the lung, kidney, brain, stomach and intestine (Fig. 4). Immunoblot analysis revealed a faint band with a subunit molecular mass of approx. 35kDa in the kidney cytosols
Adults 2-
Weanlings 02
3
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~
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=
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vc
0.5 :
A
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o
0 50
100 Fraction No.
150
0
50
100
150
Fraction No.
Fig. 5. DEAE-cellulose column chromatography of hepatic cytosols of weanling and adult female rats. Cytosolic fractions of weanling and adult female rats were fractionated on a DEAE-cellulose column. The fractions (indicated by bars) which had the highest AD-ST activity and only little 4-nitrophenol ST activity were pooled and subjected to chromatofocusing as shown in Fig. 6. Details are described elsewhere [9]. (Reproduced from ref. 9, with permission.)
20
H. Homma et al. ~Chem.-Biol. Interact. 92 (1994) 15-24
and several very faint bands in other tissues. These bands were very weak and their identities are not known at present. The liver is therefore abundant in AD-ST enzyme and other tissues so far examined had little amounts of the enzyme. The localization of AD-ST enzyme in rat liver was immunohistochemically examined [7]. The liver sections of adult male and female rats were stained with the antiAD-ST antiserum and fluorescent FITC-labeled anti-rabbit IgG. The enzyme is not present in sinusoidal cells but present in parenchymal cells. In the sections of female rats, all the hepatocytes are stained uniformly for the AD-ST enzyme throughout the liver, whereas in male rats, the enzyme is localized in the hepatocytes proximal to the portal vein. It is of note that AD-ST activity is high in female rats in which the enzyme molecule is present in all the hepatocytes, while the activity is low in male rats in which the enzyme is localized. We have isolated another isoenzyme of sulfotransferase, P-STg, which is an isoform of phenol sulfotransferase [8]. The localization of P-STg in the liver was also determined by using a rabbit polyclonal anti-P-STg antiserum. In contrast with ADST activity, phenol sulfotransferase activity is higher in male rats than in female rats. The results of immunohistochemical staining for P-STg revealed a marked contrast to those observed for AD-ST. In male rats, P-STg enzyme is detected in all the hepatocytes, whereas in female rats, the enzyme is localized in the hepatocytes proximal
Weanlings
t
Adults
-----
'
0
a
3-
I
II
t
i
2
,
I
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4
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~a
-3
~
~
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E
vc
<~o5
10
20
30 40 Fraction No
50
0 60
10
20
30 40 Fraction No
50
60
Fig, 6. Chromatofocusing of AD-ST fractions obtained by DEAE-cellulose column chromatography. The pooled AD-ST fractions obtained from weanling and adult female rats as described in Fig.5 were subjected to chromatofocusing in which the pH gradient was generated from 8.4 to 5.0. The fractionated samples at pH 7.8-7.2 and pH 6.6-5.5 were designated as fraction I and II, respectively (indicated by bars). These fractions were separately pooled and subsequently applied onto a PAP-agarose affinity column. Details were described elsewhere [9]. (Reproduced from ref. 9, with permission.)
21
H. Homma et aL ~Chem.-Biol. Interact. 92 (1994) 15-24
to the central vein. It is evident that the localization of sulfotransferase is sexdependent and also dependent on the type of isoenzyme.
4. Molecular characterization development
of changes of AD-ST enzyme during postnatal
As illustrated in Fig. 1, the AD-ST activity revealed a biphasic change during postnatal development of female rats. We have carried out immunoblot analysis and compared the pl variants of AD-ST in weanling (20 days of age) and adult (110 days of age) female rats [5]. The relative amounts of pl variants are different between these two stages of age. In weanling rats, the intensity of pI 7.2 spot is greater than that of pI 6.7 spot, whereas in adult rats, the intensities of these two spots are almost the same [5]. In order to define this difference, AD-ST was isolated from liver cytosols of rats of these two stage of age. The elution profiles of AD-ST activity during DEAE-cellulose column chromatography of liver cytosols of weanling and adult female rats are given in Fig. 5. In weanling rats, only one major peak of the activity was evident. On the other hand, the activity of adult rats was apparent in several peaks and the major peak was eluted at the same sodium chloride concentration as the activity of weanling rats. The major activity in adult rats was broader than that in weanling rats. The AD-ST fractions of weanling and adult rats were pooled and subjected to chromatofocusing as shown in Fig. 6.
Weanlings
Adults
A Fraction
I
~
~ */2
6.'/
6.1
-3okDo
~.
~
-pl
7.2
6.7
6.l
-~0.
l?
~J " P !
..pl
B
Fraction II
Fig. 7. Two-dimensional gel electrophoresis of purified AD-ST from weanling and adult female rats. The purified AD-ST from fraction I of weanling (A) and adult (B) rats as well as from fraction I1 of weanling (C) and adult rats (D) were subjected to two-dimensional gel electrophoresis and pl variants were detected by Coomassie blue staining. The amounts of protein employed were (A), 37#g; (B), 9t~g; (C), 17/~g; (D), 12#g. (Reproduced from ref. 9, with permission.)
22
H. Homma et al. ~Chem.-Biol. Interact. 92 (1994) 15-24
Table 1 N-terminal amino acid sequences of pl variants of AD-ST pl variants
N-Terminal amino acid sequence 1
10
20
30
40
PDYTWFEGIP
FPAFGIPKET
LQNVCNKFVV
KEEDLILLTY
PK - - SGT
45
Weanlings Fraction I pl 7.2 pl 6.7 pl 6.1 Adults Fraction I pl 7.2 pl 6.7 pl 6.1 Fraction II pl 6.7 pl 6.1
~m
Purified AD-ST preparations from weanling and adult female rats were subjected to two-dimensional gel electrophoresis as indicated in Fig. 3 and electrotransferred to a poly(vinylidene difluoride) (PVDF) membrane filter. After Coomassie blue staining, protein spots were excised and applied directly to a protein sequencer. The pl variants in fraction II of weanlings and pl 7.2 variant in fraction II of adults were not analyzed, since they were not obtained in sufficient quantity. Asterisks represent residue ambiguity. Dashes are identical with those of pl 7.2 variant of fraction I from weanlings. Details were described elsewhere [9]. (Reproduced from ref. 9, with permission)
With both age groups, major amounts of activity were eluted in the pH gradient from pH 7.8 to 7.2 (fraction I). However, activity eluting at pH 6.6-5.5 (fraction II) was much higher with adult rats. Fractions I and II from rats of each age group were pooled and purified by PAP-agarose affinity chromatography. The purified AD-STs were then analyzed by 2DGE in order to determine the composition of the pI variants as shown in Fig. 7. In fraction I of weanling and adult rats, the relative intensities of the pI 7.2 and 6.7 spots were both high and the intensity of the pI 6.1 spot was low. On the other hand, in fraction II of both age groups the relative intensities of the pI 6.7 and 6.1 spots were high and the intensity of the pl 7.2 spot was low. These observations were consistent with the chromatofocusing elution profiles (Fig. 6). Therefore, these lines of evidence indicate that in weanling rats, AD-ST is primarily composed of the pI 7.2 and 6.7 subunits and that during development from the weanling stage to adulthood, the amounts of the pI 6.7 and 6.1 variants are increased and an acidic form(s) of AD-ST (fraction II) is increased. N-terminal amino acid sequences of the pI variants were determined as indicated
H. Homma et al. ~Chem.-Biol. Interact. 92 (1994) 15-24
23
30 kDa pl 7.2
6.7
Fig. 8. Two-dimensional gel electrophoresis of recombinant product of ST-40, a cDNA clone of rat hepatic hydroxysteroid sulfotransferase. ST-40 was isolated from a rat liver cDNA library with anti-AD-ST antiserum. The nucleotide sequence of its coding region was determined to be identical with the reported sequence of ST-40 [10]. The entire coding region was amplified by polymerase chain reaction with appropriate primers that contained EcoRl sites in the 5' end. After digestion of the amplified product with EcoRl, the coding region was inserted into the EcoRl site of the prokaryote expression vector, pKK223-3. No mutations were generated during amplification with Taq DNA polymerase. Transformants (JMI09) with this recombinant DNA were cultivated and a homogenate was prepared by sonication in 10 mM Tris-HCI, 250 mM sucrose, 0.1 mM EDTA and 3 mM 2-mercaptoethanol (pH 7.4). A soluble fraction was prepared by centrifugation and 40/~g protein of this fraction was subjected to two-dimensional gel electrophoresis and transferred to a nitrocellulose membrane filter. The blot was probed with anti-AD-ST antiserum (1:5000).
in Table 1. The sequences are identical for all the variants within the area determined. The sequence is also completely identical with the sequence deduced from the nucleotide sequence of rat liver hydroxysteroid sulfotransferase cDNA clone, ST-40 [10], except for the N-terminal methionine. These results indicate that the p/variants may have originated from posttranslational modification of a single subunit or that the plvariants may be highly homologous proteins with distinct primary structures outside the N-terminal region. We have isolated several cDNA clones including ST-40 from a rat liver cDNA library by using anti-AD-ST antiserum. The recombinant product of ST-40 expressed in E.coli cells revealed AD-ST activity and it demonstrated two protein spots with distinct pI values on 2DGE (Fig. 8). This result suggests that some of the pl variants are derived from a single subunit by posttranslational modification but details remain to be known. 5. References M. Matsui and K.H. Watanabe, Developmental alteration of hepatic UDP-glucuronosyltransferase and sulphotransferase toward androsterone and 4-nitrophenol in Wistar rats, Biochem. J., 204 (1982) 441-447.
24 2
H. Homma et al. ~Chem.-Biol. Interact. 92 (1994) 15-24
B. Chatterjee, M.A. Mancini and A.K. Roy, The senescence marker protein (SMP-2) of the rat liver: purification, immunochemical characterization and age-dependent regulation, Biochim. Biophys. Acta, 1034 (1990) 162-169. 3 C-S. Song, J.M. Kim, A.K. Roy and B. Chatterjee, Structural and regulation of the senescence marker protein 2 gene promotor, Biochemistry, 29 (1990) 542-551. 4 M. Runge-Morris and J. Wilusz, Age and gender-related gene expression of hydroxysteroid sulfotransferase-a in rat liver, Biochem. Biophys. Res. Commun., 175 (1991) 1051-1056. 5 H. Homma, 1. Nakagome, M. Kamakura and M. Matsui, lmmunochemical characterization of developmental changes in rat hepatic hydroxysteroid sulfotransferase, Biochim. Biophys. Acta, 1121 (1992) 69-74. 6 H. Homma, T. Sasaki and M. Matsui, Properties of androsterone-sulfating sulfotransferase in female rat liver, Chem. Pharm. Bull., 39 (1991) 1499-1503. 7 H. Homma, I. Nakagome and M. Matsui, Differential localization of sulfotransferase isoenzymes in rat liver, Biochem. Biophys. Res. Commun., 183 (1992) 872-878. 8 H. Homma, M. Kamakura, 1. Nakagome and M. Matsui, Purification of a rat liver phenol sulfotransferase (P-STG) with the aid of guanidine hydrochloride treatment, Chem. Pharm. Bull., 39 (1991) 3307-3312. 9 M. Takahashi, H. Homma and M. Matsui, Developmental changes in the isoelectric variants of rat hepatic hydroxysteroid sulphotransferase, Biochem. J., 293 (1993) 795-800. 10 K. Ogura, J. Kajita, H. Narihata, T. Watabe, S. Ozawa, K. Nagata, Y. Yamazoe and R. Kato, cDNA cloning of the hydroxysteroid sulfotransferase STa sharing a strong homology in amino acid sequence with the senescence marker protein SMP-2 in rat livers, Biochem. Biophys. Res. Commun., 166 (1990) 1494-1500.
Chemico-Biological Interactions 92 (1994) 15-24
Studies on rat hepatic hydroxysteroid sulfotransferase - - immunochemistry, development and pI variants Hiroshi Homma*, Izumi Nakagome, Minoru Kamakura, Masami Hirota, Mie Takahashi, Michio Matsui Kyoritsu College of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105, Japan Received 16 August 1993; revision received 4 February 1994; accepted 14 February 1994
Abstract
Rat hepatic hydroxysteroid sulfotransferase which sulfoconjugates androsterone (androsterone-sulfating sulfotransferase) is an oligomer consisting of several subunits with distinct p l values but with the same molecular mass (pl variants). N-terminal amino acid sequences of the p I variants are all identical. The enzyme is exclusively present in the liver, in which its Iobular localization is sex-dependent. The localization of the enzyme is markedly different from that of an isoenzyme of phenol sulfotransferase. In weanling and adult female rats, the relative abundance of the pI variants is different. During development from weanling stage to adulthood, the amounts of acidic variants increase, whereas the relative levels of alkaline variants remain constant. Keywords: Sulfotransferase; Hydroxysteroid; pI variants; Development; Immunohistochemistry
I. Introduction
The activity o f rat hepatic h y d r o x y s t e r o i d sulfotransferase t o w a r d a n d r o s t e r o n e (androsterone-sulfating sulfotransferase~ A D - S T ) exhibits a characteristic alteration during p o s t n a t a l d e v e l o p m e n t (Fig. 1). It increases after birth in parallel in b o t h * Corresponding author. Abbreviations: AD-ST, androsterone-sulfating sulfotransferase; PAP, 3'-phosphoadenosine 5'phosphate; 2DGE, two-dimensional gel electrophoresis; P-ST, phenol sulfotransferase.
0009-2797/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSD1 0009-2797(94)03286-H
t6
H, Homma et al, / Chem.-Biol. Interact. 92 (1994) 15-24
0.4
>.
0.3 ¸
> L) 6o
0.2
~E _~ m~
0.1
0.0 0
20
40
60
80
days Fig. 1. Postnatal development of androsterone-sulfating sultbtransferase (AD-ST) in hepatic cytosols of male and female rats. Hepatic cytosols were prepared from male (0) and female (O) rats of various ages and AD-ST activity was determined and expressed as nmol/min per mg protein, Data were means of three to ten animals and vertical lines represent S.D. in animals at 20-80 days of age. Data from animals at I and 10 days of age were obtained from the pooled liver ,samples. Details are described previously [1],
sexes until weanling stage. The activity thereafter decreases in males, while in females it declines temporarily and increases again to a maximal level in adult females. The activity in adult females is approx. 15-fold higher than in adult males. The activities in both sexes demonstrate age-related characteristic changes during postnatal development. In the following three parts of the present paper, we describe the molecular basis of the age- and gender-related changes of the AD-ST activity; (i) molecular characterization of the AD-ST enzyme, (ii) immunochemical characterization of the enzyme and (iii) molecular characterization of changes of the enzyme during postnatal development. Age-dependent changes in the expression of hydroxysteroid ST isoenzymes in rat liver have been characterized at the molecular level [2-4]. In the present paper, it is described that the AD-ST comprises several subunits with distinct pI values but with the same molecular mass. In addition, the changes in the relative amounts of the subunits during the postnatal development are characterized. 2. Molecular characterization of AD-ST enzyme
We have purified a major isoenzyme of AD-ST from hepatic cytosols of adult female rats in virtue of DEAE-cellulose column chromatography and PAP-agarose
H. H o m m a et al. ~Chem.-Biol. Interact. 92 (1994) 15-24
17
0,3
0.2
E E
cO
O
(xJ
E c
v
._>
l
01
CO
c~ <
O to t'~
<
-'•'•
5O
AA/~/~ A
lO0
150
200
time (min) Fig. 2. HPLC gel filtration chromatography of hepatic cytosols of female rats. Molecular size of the native form of AD-ST was determined by applying rat hepatic cytosols to HPLC gel filtration chromatography. Cytosolic fractions were prepared from female rats of 8 weeks of age and 5.3 mg protein (200#1) was injected onto a column of TSK gel G3000SW (two i.d. 7.5 × 600 mm stainless steel columns in tandem). The column was eluted at a flow rate of 0.4 ml/min with 50 mM Tris-HC1 (pH7.4), 3 mM 2mercaptoethanol, 0.1 mM EDTA, 0.2 M NaCI and 250 mM sucrose. Fractions of 0.4 ml were collected and assayed for AD-ST activity ( ~ ). Elution of proteins was monitored by measuring the absorbance at 280 nm ( - - ). Void volume, (V0) and positions of elutions of standard proteins (ferritin; 440kDa, aldolase; 158 kDa, BSA; 67 kDa) are also indicated. Details are similar to those in the previous report [6].
affinity column chromatography. The enzyme was purified 122-fold over the activity in the cytosols and the purified enzyme revealed a single protein band with a subunit molecular mass of 30 kDa on SDS-polyacrylamide gel electrophoresis [5,6]. In order to determine the molecular mass of the native form of the AD-ST, the cytosolic fraction of rat liver was applied to HPLC gel filtration chromatography (Fig. 2). The activity was eluted in the fraction which corresponded to molecular mass of about 200 kDa, based on the calculation with the standard proteins. The AD-ST enzyme is therefore an oligomer which consists of subunits with a molecular mass of 30 kDa.
18
H. Homma et al. / Chem.-Biol. Interact. 92 (1994) 15-24 Isoelectrofi)~'using y
L;
30kDa
pl
1,2
6.7
6.1 5.8
Fig. 3. Two-dimensional gel electrophoresis of purified AD-ST. In the first dimension, a 4% polyacrylamide isoelectrofocusing gel was run under denaturing condition in the presence of 2% nonidet P-40, 8.5 M urea and 2% pharmalyte 3-10. In the second dimension, SDS-polyacrylamide (12.5%) gel electrophoresis was carried out. Protein spots were visualized by Coomassie blue staining. The pl values of each spots were determined using internal pl markers. Details were described previously [5]. (Reproduced from ref. 5, with permission.)
0.5
0.4 ~__~ 0.3 "~
0.2
0.1
Q-
0.0
liver
lung
kidney
brain
stomach
intestine
Fig. 4. Tissue distribution of AD-ST. The lung, kidneys, brain, stomach and intestine were excised from male (1~) and female (m) rats of 9 weeks of age and homogenates were prepared using a Polytron homogenizer. Cytosolic fractions were obtained by centrifugation (105 000 × g for 60 min) of the homogenates. AD-ST activity was determined by incubating the reaction mixture (500/A) at 37°C, which contained 100 mM Tris-HCI (pH 7.4), l0 mM MgCI2, 20 #M EDTA, 100 ~M PAPS, 86 #M [3H]androsterone (approx. 350 dpm/nmol) and a cytosolic fraction.
H. Homma et a l . / Chem.-Biol• Interact. 92 (1994) 15-24
19
Two dimensional gel electrophoresis (2DGE) was carried out with the purified AD-ST. The enzyme was focused mainly into four spots which have isoelectric points of 7.2, 6.7, 6.1 and 5.8 (Fig. 3). The most abundant components have pI values of 7.2 and 6.7, while minor constituents are pI 6.1 and 5.8 spots. These protein spots were designated as pl variants. As described below, a rabbit polyclonal antiserum which was raised against the purified AD-ST detected these pI variants in fresh liver cytosols of female rats, indicating that the variants are not artifacts during purification procedure but authentic or real components of AD-ST oligomer. The AD-ST enzyme therefore is an oligomer which comprises several pI variants with the same molecular mass of 30 kDa. 3. Immunochemical characterization of AD-ST enzyme In Fig. 4, the content of AD-ST enzyme was investigated in several extrahepatic tissues of adult male and female rats using a rabbit polyclonal antiserum which was prepared previously [5]. Little activity of AD-ST was detected in the cytosols of the lung, kidney, brain, stomach and intestine (Fig. 4). Immunoblot analysis revealed a faint band with a subunit molecular mass of approx. 35kDa in the kidney cytosols
Adults 2-
Weanlings 02
3
t2
l
t
.,--"" i f Im
• 0.1
~
'0.1
<
~. z
0 1.0
0
-4 -
r.l-"
c,l
z
1.5-
02
-
-3 ~
1.0
=
~
0.5-
"2
vc
0.5 :
A
1
o
0 50
100 Fraction No.
150
0
50
100
150
Fraction No.
Fig. 5. DEAE-cellulose column chromatography of hepatic cytosols of weanling and adult female rats. Cytosolic fractions of weanling and adult female rats were fractionated on a DEAE-cellulose column. The fractions (indicated by bars) which had the highest AD-ST activity and only little 4-nitrophenol ST activity were pooled and subjected to chromatofocusing as shown in Fig. 6. Details are described elsewhere [9]. (Reproduced from ref. 9, with permission.)
20
H. Homma et al. ~Chem.-Biol. Interact. 92 (1994) 15-24
and several very faint bands in other tissues. These bands were very weak and their identities are not known at present. The liver is therefore abundant in AD-ST enzyme and other tissues so far examined had little amounts of the enzyme. The localization of AD-ST enzyme in rat liver was immunohistochemically examined [7]. The liver sections of adult male and female rats were stained with the antiAD-ST antiserum and fluorescent FITC-labeled anti-rabbit IgG. The enzyme is not present in sinusoidal cells but present in parenchymal cells. In the sections of female rats, all the hepatocytes are stained uniformly for the AD-ST enzyme throughout the liver, whereas in male rats, the enzyme is localized in the hepatocytes proximal to the portal vein. It is of note that AD-ST activity is high in female rats in which the enzyme molecule is present in all the hepatocytes, while the activity is low in male rats in which the enzyme is localized. We have isolated another isoenzyme of sulfotransferase, P-STg, which is an isoform of phenol sulfotransferase [8]. The localization of P-STg in the liver was also determined by using a rabbit polyclonal anti-P-STg antiserum. In contrast with ADST activity, phenol sulfotransferase activity is higher in male rats than in female rats. The results of immunohistochemical staining for P-STg revealed a marked contrast to those observed for AD-ST. In male rats, P-STg enzyme is detected in all the hepatocytes, whereas in female rats, the enzyme is localized in the hepatocytes proximal
Weanlings
t
Adults
-----
'
0
a
3-
I
II
t
i
2
,
I
15-
4
II
~a
-3
~
~
2"
E
vc
<~o5
10
20
30 40 Fraction No
50
0 60
10
20
30 40 Fraction No
50
60
Fig, 6. Chromatofocusing of AD-ST fractions obtained by DEAE-cellulose column chromatography. The pooled AD-ST fractions obtained from weanling and adult female rats as described in Fig.5 were subjected to chromatofocusing in which the pH gradient was generated from 8.4 to 5.0. The fractionated samples at pH 7.8-7.2 and pH 6.6-5.5 were designated as fraction I and II, respectively (indicated by bars). These fractions were separately pooled and subsequently applied onto a PAP-agarose affinity column. Details were described elsewhere [9]. (Reproduced from ref. 9, with permission.)
21
H. Homma et aL ~Chem.-Biol. Interact. 92 (1994) 15-24
to the central vein. It is evident that the localization of sulfotransferase is sexdependent and also dependent on the type of isoenzyme.
4. Molecular characterization development
of changes of AD-ST enzyme during postnatal
As illustrated in Fig. 1, the AD-ST activity revealed a biphasic change during postnatal development of female rats. We have carried out immunoblot analysis and compared the pl variants of AD-ST in weanling (20 days of age) and adult (110 days of age) female rats [5]. The relative amounts of pl variants are different between these two stages of age. In weanling rats, the intensity of pI 7.2 spot is greater than that of pI 6.7 spot, whereas in adult rats, the intensities of these two spots are almost the same [5]. In order to define this difference, AD-ST was isolated from liver cytosols of rats of these two stage of age. The elution profiles of AD-ST activity during DEAE-cellulose column chromatography of liver cytosols of weanling and adult female rats are given in Fig. 5. In weanling rats, only one major peak of the activity was evident. On the other hand, the activity of adult rats was apparent in several peaks and the major peak was eluted at the same sodium chloride concentration as the activity of weanling rats. The major activity in adult rats was broader than that in weanling rats. The AD-ST fractions of weanling and adult rats were pooled and subjected to chromatofocusing as shown in Fig. 6.
Weanlings
Adults
A Fraction
I
~
~ */2
6.'/
6.1
-3okDo
~.
~
-pl
7.2
6.7
6.l
-~0.
l?
~J " P !
..pl
B
Fraction II
Fig. 7. Two-dimensional gel electrophoresis of purified AD-ST from weanling and adult female rats. The purified AD-ST from fraction I of weanling (A) and adult (B) rats as well as from fraction I1 of weanling (C) and adult rats (D) were subjected to two-dimensional gel electrophoresis and pl variants were detected by Coomassie blue staining. The amounts of protein employed were (A), 37#g; (B), 9t~g; (C), 17/~g; (D), 12#g. (Reproduced from ref. 9, with permission.)
22
H. Homma et al. ~Chem.-Biol. Interact. 92 (1994) 15-24
Table 1 N-terminal amino acid sequences of pl variants of AD-ST pl variants
N-Terminal amino acid sequence 1
10
20
30
40
PDYTWFEGIP
FPAFGIPKET
LQNVCNKFVV
KEEDLILLTY
PK - - SGT
45
Weanlings Fraction I pl 7.2 pl 6.7 pl 6.1 Adults Fraction I pl 7.2 pl 6.7 pl 6.1 Fraction II pl 6.7 pl 6.1
~m
Purified AD-ST preparations from weanling and adult female rats were subjected to two-dimensional gel electrophoresis as indicated in Fig. 3 and electrotransferred to a poly(vinylidene difluoride) (PVDF) membrane filter. After Coomassie blue staining, protein spots were excised and applied directly to a protein sequencer. The pl variants in fraction II of weanlings and pl 7.2 variant in fraction II of adults were not analyzed, since they were not obtained in sufficient quantity. Asterisks represent residue ambiguity. Dashes are identical with those of pl 7.2 variant of fraction I from weanlings. Details were described elsewhere [9]. (Reproduced from ref. 9, with permission)
With both age groups, major amounts of activity were eluted in the pH gradient from pH 7.8 to 7.2 (fraction I). However, activity eluting at pH 6.6-5.5 (fraction II) was much higher with adult rats. Fractions I and II from rats of each age group were pooled and purified by PAP-agarose affinity chromatography. The purified AD-STs were then analyzed by 2DGE in order to determine the composition of the pI variants as shown in Fig. 7. In fraction I of weanling and adult rats, the relative intensities of the pI 7.2 and 6.7 spots were both high and the intensity of the pI 6.1 spot was low. On the other hand, in fraction II of both age groups the relative intensities of the pI 6.7 and 6.1 spots were high and the intensity of the pl 7.2 spot was low. These observations were consistent with the chromatofocusing elution profiles (Fig. 6). Therefore, these lines of evidence indicate that in weanling rats, AD-ST is primarily composed of the pI 7.2 and 6.7 subunits and that during development from the weanling stage to adulthood, the amounts of the pI 6.7 and 6.1 variants are increased and an acidic form(s) of AD-ST (fraction II) is increased. N-terminal amino acid sequences of the pI variants were determined as indicated
H. Homma et al. ~Chem.-Biol. Interact. 92 (1994) 15-24
23
30 kDa pl 7.2
6.7
Fig. 8. Two-dimensional gel electrophoresis of recombinant product of ST-40, a cDNA clone of rat hepatic hydroxysteroid sulfotransferase. ST-40 was isolated from a rat liver cDNA library with anti-AD-ST antiserum. The nucleotide sequence of its coding region was determined to be identical with the reported sequence of ST-40 [10]. The entire coding region was amplified by polymerase chain reaction with appropriate primers that contained EcoRl sites in the 5' end. After digestion of the amplified product with EcoRl, the coding region was inserted into the EcoRl site of the prokaryote expression vector, pKK223-3. No mutations were generated during amplification with Taq DNA polymerase. Transformants (JMI09) with this recombinant DNA were cultivated and a homogenate was prepared by sonication in 10 mM Tris-HCI, 250 mM sucrose, 0.1 mM EDTA and 3 mM 2-mercaptoethanol (pH 7.4). A soluble fraction was prepared by centrifugation and 40/~g protein of this fraction was subjected to two-dimensional gel electrophoresis and transferred to a nitrocellulose membrane filter. The blot was probed with anti-AD-ST antiserum (1:5000).
in Table 1. The sequences are identical for all the variants within the area determined. The sequence is also completely identical with the sequence deduced from the nucleotide sequence of rat liver hydroxysteroid sulfotransferase cDNA clone, ST-40 [10], except for the N-terminal methionine. These results indicate that the p/variants may have originated from posttranslational modification of a single subunit or that the plvariants may be highly homologous proteins with distinct primary structures outside the N-terminal region. We have isolated several cDNA clones including ST-40 from a rat liver cDNA library by using anti-AD-ST antiserum. The recombinant product of ST-40 expressed in E.coli cells revealed AD-ST activity and it demonstrated two protein spots with distinct pI values on 2DGE (Fig. 8). This result suggests that some of the pl variants are derived from a single subunit by posttranslational modification but details remain to be known. 5. References M. Matsui and K.H. Watanabe, Developmental alteration of hepatic UDP-glucuronosyltransferase and sulphotransferase toward androsterone and 4-nitrophenol in Wistar rats, Biochem. J., 204 (1982) 441-447.
24 2
H. Homma et al. ~Chem.-Biol. Interact. 92 (1994) 15-24
B. Chatterjee, M.A. Mancini and A.K. Roy, The senescence marker protein (SMP-2) of the rat liver: purification, immunochemical characterization and age-dependent regulation, Biochim. Biophys. Acta, 1034 (1990) 162-169. 3 C-S. Song, J.M. Kim, A.K. Roy and B. Chatterjee, Structural and regulation of the senescence marker protein 2 gene promotor, Biochemistry, 29 (1990) 542-551. 4 M. Runge-Morris and J. Wilusz, Age and gender-related gene expression of hydroxysteroid sulfotransferase-a in rat liver, Biochem. Biophys. Res. Commun., 175 (1991) 1051-1056. 5 H. Homma, 1. Nakagome, M. Kamakura and M. Matsui, lmmunochemical characterization of developmental changes in rat hepatic hydroxysteroid sulfotransferase, Biochim. Biophys. Acta, 1121 (1992) 69-74. 6 H. Homma, T. Sasaki and M. Matsui, Properties of androsterone-sulfating sulfotransferase in female rat liver, Chem. Pharm. Bull., 39 (1991) 1499-1503. 7 H. Homma, I. Nakagome and M. Matsui, Differential localization of sulfotransferase isoenzymes in rat liver, Biochem. Biophys. Res. Commun., 183 (1992) 872-878. 8 H. Homma, M. Kamakura, 1. Nakagome and M. Matsui, Purification of a rat liver phenol sulfotransferase (P-STG) with the aid of guanidine hydrochloride treatment, Chem. Pharm. Bull., 39 (1991) 3307-3312. 9 M. Takahashi, H. Homma and M. Matsui, Developmental changes in the isoelectric variants of rat hepatic hydroxysteroid sulphotransferase, Biochem. J., 293 (1993) 795-800. 10 K. Ogura, J. Kajita, H. Narihata, T. Watabe, S. Ozawa, K. Nagata, Y. Yamazoe and R. Kato, cDNA cloning of the hydroxysteroid sulfotransferase STa sharing a strong homology in amino acid sequence with the senescence marker protein SMP-2 in rat livers, Biochem. Biophys. Res. Commun., 166 (1990) 1494-1500.