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Immunochemical characterization of developmental changes in rat hepatic hydroxysteroid sulfotransferase
Biochimica et BiophysicaActa, 1121 (1992)69-74
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
69
BBAPRO 34195
lmmunochemical characterization of developmental changes in rat hepatic hydroxysteroid sulfotransferase Hiroshi Homma, Izumi Nakagome, Minoru Kamakura and Michio Matsui Kyoritsu Collegeof Pharmacy, Tokyo (Japan)
(Received 30 September 1991)
Key words: Sulfotransferase;Androsterone; Development;lsoenzyme; lsoelectricvariant; (Rat liver) A major isoenzyme of hepatic androsterone-sulfating sulfotransferase (AD-ST) was purified from adult female rats. The activity was purified ! 22-fold over that found in the cytosol and showed a single protein band with a subunit molecular mass of 30 kDa after sodium dodecyI sulfate polyacrylamide gel electrophoresis. The purified enzyme exhibited four isoelectric variants of subunits on denaturing isoelectrofocusing gels (pl = 5.8, 6.1, 6.7 and 7.2). Rabbit antiserum raised against the enzyme specifically detected AD-ST polypcptide in rat liver cytosoi, lmmunoblot analysis of liver cytosol from female and male rats at various ages showed good correlation between the levels of AD-ST activity and AD-ST polypeptide. Significant levels of AD-ST activity and polypeptide were detected in senescent male rats, though normal adult male rats have very low levels of AD-ST activity and protein. The relative content of the isoelectric variants of AD-ST were different in liver cytosol of weanling and adult females, indicating that age- and gender-related alterations of hepatic AD-ST activity are primarily determined by the levels of AD-ST polypeptide and the relative amounts of the four isoelectric variants of the enzyme.
Introduction Sulfotransferase (ST) catalyzes the transfer of sulfate group from 3'-phosphoadenoslne 5'-phosphosulfate (PAPS) to a variety of drugs, xenobiotics and endogenous compounds. Previous investigations revealed heterogeneity and multiplicity of STs in rat liver cytosol as well as in human liver and platelets [1-4]. Rat liver hydroxysteroid STs, which have high catalytic activity for steroid hormones, alcohols and bile acids were shown in purification studies to comprise several isoenzymes [2--4]. These activities are known to be under the regulation of gonadal and adrenal steroids [4] as well as pituitary growth hormone [5]. We have previously reported the following features of ST activity in rat liver cytosol using androsterone (AID) as a substrate. (1) The activity in adult female rats is 15-fold higher than in adult males [6]. Such a marked sex-related difference was not observed in ST activity with hydroxysteroids [5,7]. (2) AD-ST activity in male rats increases upon administration of estrogen [8]. Abbreviations: ST, sulfotransferase; AD-ST androsterone-sul[ating sulfotransferase; PAPS, Y-phosphoadenosine 5'-phosphosulfate; PAP, Y-phosphoadenosine5'-phosphate; SDS-PAGE, sodium dodecyi sulfate polyacrylamide gel electrophoresis. Correspondence: M. Matsui, Kyoritsu College of Pharmacy, Shibakoen, Minato-ku,Tokyo 105, Japan.
(3) The activity exhibits a characteristic alteration in male and female rats during postnatal development [6]. it increases after birth in parallel in both sexes until the weanling stage when it reaches a maximum and subsequently decreases in males. In females, on the other hand, AD-ST activity is biphasic; it declines temporarily and increases again to a high level in adult females. (4) A major isoenzyme of AD-ST was partially purified from female rat liver. It is an oligomer consisting of several subunits of the same molecular mass (30 kDa) but with distinct p l values [9]. In order to understand the molecular basis of the gender- and age-related changes in AD-ST activity, we further purified a major isoenzyme of AD-ST from adult female rats and characterized it. We also raised rabbit polyclonal antiserum against the purified enzyme and used it for immunochemical characterization of AD-ST. Materials and Methods Materials
[9,11-3H]Androsterone (60 Ci/mmol) and [1-14C]4 nitrophenol (56 mCi/mmol) were obtained from New England Nuclear (Boston, MA, USA) and Amersham International, (Buckinghamshire, UK), respectively. DEAE-eellulose DE-52 is a product of Whatman Biosystems (Maidstone, Kent, UK). Agarose adenosine 3',5'-bisphosphate type 2 (PAP-agarose)and Pharmalyte 3-10 were purchased from Pharmacia (Uppsala,
70 Sweden). 3'-Phosphoadenosine 5'-phosphate (PAP), 5'AMP, 3'-AMP, horse heart myoglobin and bovine milk /3-1actoglobulin A were obtained from Sigma (St. Louis, MO, USA). Urea and Nonidet P-40 were purchased from Wako Chemical (Osaka, Japan) and from iwai Chemical (Tokyo, Japan), respectively. Complete adjuvant H37 Ra and Freund's incomplete adjuvant are products of Difco Laboratories (Detroit, MI, USA). Peroxidase-conjugated affinipure goat anti-rabbit IgG (H + L) and Block Ace (a blocking reagent for immunoblot analysis) were obtained from Jackson Immunoresearch Laboratories (West Grove, PA, USA) and Dainippon Seiyaku (Osaka, Japan), respectively. 3'-Phosphoadenosine 5'-phosphosulfate ( P A P S ) w a s prepared by the method of Singer [10]. All other reagents were of the highest grade available.
ers used for SDS-PAGE were phosphorylase b (94 kDa), BSA (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and fl-lactoglobulin (14.4 kDa) in the Electrophoresis Calibration Kit (Pharmacia). Two-dimensional gel electrophoresis was carried out as described previously [9]. Briefly, in the first dimension, a 4% l~olyacrylamide isoelectrofocusing gel was run under denaturing conditions in the presence of 2% Nonidet P-40, 8.5 M urea and 2% Pharmalyte 3-10. In the second dimension, SDS-PAGE was performed using 12.5% polyacrylamide gels. Horse heart myoglobin (pl = 6.8 and 7.2, subunit molecular mass = 16950) and bovine milk fllactoglobulin A ( p l = 5 . 1 , subunit molecular mass = 18400) were used as internal p l markers.
Preparation of anti-AD-ST antiserum Purification of AD-ST Preparation of the cytosolic fraction of female rat liver (8 weeks of age) and determinations of AD-ST and 4-nitrophenol ST activities were carried out as previously described [9,11]. The cytosolic fractions of rat lung, kidney, brain, stomach and small intestine were prepared in a manner similar to that used for liver cytosol. Protein content was measured by the method of Bradford [ 12].
Step 1. DEAE-celkdose chromatography of AD-ST. DEAE-cellulose column chromatography of the cytosolic fraction of female rat liver was conducted as described previously [9,11] and fractions which contained the highest activity of AD-ST and little or no 4nitrophenol ST activity were pooled. These fractions were concentrated by dialysis against 10% poly(ethylene glycol) 20000 in 10 mM Tris-HCI, 250 mM sucrose, 0.1 mM EDTA and 3 mM 2-mercaptoethanol (pH 7.4), then dialyzed against 5 mM sodium phosphate (pH 8.0) (buffer A). Step 2. PAP-agarose affinity chromatography. A portion of the concentrated AD-ST fractions was subsequently subjected to a PAP-agarose affinity column chromatography following the method of Barnes et al. [131. A column of PAP-agarose (7 x 96 ram) was preequilibrated in bufier A. The sample applied to the column contained 3-6 mg protein/ml resin. After application, the column was washed at a flow rate of 46 m l / h sequentially with 50 ml of buffer A, 50 ml of 50 mM sodium phosphate (pH 8.0), 20 ml of 5 mM 5'-AMP in buffer A and 20 ml of 5 mM 3'-AMP in buffer A. The AD-ST activity was then eluted with 20 ml of 0.1 mM PAP in buffer A and 1 ml fractions were collected.
Electrophoresis SDS-PAGE was carried out by the method of Laemmli [14] and the proteins were visualized with Coomassie blue staining. The molecular weight mark-
One male New Zealand White rabbit was administered approx. 100 #g of purified AD-ST in complete adjuvant subcutaneously along the back. 4 weeks later, the rabbit received a booster injection containing the same amount of the purified enzyme in Freund's incomplete adjuvant. After 10 days, the animal was bled for preparation of the antiserum. Preimmune serum was also prepared as a control.
lmmunoblot analysis Proteins separated by SDS-PAGE were transferred from the gel to a nitrocellulose filter (Bio-Rad, Richmond, CA, USA) with a Bio-Rad Trans-Blot SD apparatus according to the procedure described by the manufacturer. The amounts of protein used in each experiment are indicated in the figure legends. When extrahepatic tissues were used, those samples contained 20 ttg of cytosolic protein. The nitrocellulose filter was blocked overnight at 4*(2 with a blocking reagent, Block Ace. A 1 : 10000 dilution of rabbit antiserum and a 1:1000 dilution of peroxidase-conjugated goat anti-rabbit IgG antibody were routinely used as primary and secondary antibody, respectively. The immunocomplex was IocaIized using a color generating reaction: the oxidation of 3,3'-diaminobenzidine tetrachloride by hydrogen peroxide. Results
Purification of androsterone-sulfating sulfotransferase
(AD-ST) The cytosolic fraction of liver cells of female rats (8 weeks of age) was subjected to DEAE-cellulose column chromatography for the initial purification of ADST as described previously [9,11]. This chromatography step yielded fractions containing the highest activity of AD-ST and lowest activity of 4-nitrophenol-ST and cortisoI-ST [11]. These fractions were pooled, concentrated and subsequently applied to a PAP-agarose
71 TABLE I
7
t~,ri]ication of A D - S T from female rat lirer cytoso!
6
C
E 0
One unit represents 1 nmoi of androsterone conjugated/rain. 5'
Protein
E
c
4.
>
3
(mg)
Cytosol 3169 DEAEcellulose 109.5
IB
g
2
PAPagarose :'
|
- l ~
1" 0
0
s
~
i
S
10
15
Fraction
20
affinity column. Affinity chromatography was then conducted and the elution pattern of the AD-ST activity from the column is shown in Fig. 1. SDS-PAGE analysis of the final preparation revealed a single protein band with a subunit molecular mass of 30 kDa (Fig. 2). The specific activity of the purified enzyme was 47.3
kDa
2
1230
0.388
1
239
2.18
5.6
15.3
47.3
122
A portion of the DEAE-cellulose fraction (22 mg protein) was applied to PAP-agarose affinity chromatography.
no.
Fig. 1. PAP-agarose affinity chromatography of AD-ST. The pooled fractions containing AD-ST activity, obtained after DEAE-cellulo~ chromatography, were concentrated and a portion of this 122 mg protein in 17 ml buffer) was applied to a PAP-agarose affinity column (7 × q6 mm). The column was washed with v~rious buffers as described in Materials and Methods and the AD-ST activity was specifically eluted with 0.1 mM PAP, The activities eluted in the various buffers were as follows: 0.07 nmol/min per ml in flow through fraction, O.! in buffer A, (I.(14 in 50 mM ,sodium phosphate buffer, 0.04 in 5'-AMP-containing buffer and 0 in 3'-AMP-containing buffer. Fractions 3-9 were pooled for the final purification step.
1
0,324
Activity Specific activity Purification (nmol/min) (nmol/min per mg) (fold)
3 "
67'
43
nmol/min per mg protein, representing a 122-fold purification over its activity in the cytosol (Table I). The final preparation of AD-ST contains PAP, since it is included in the elution medium of the affinity chromatography step. It is well known that STs have very high affinity for PAP [2]. The presence of PAP in the preparation appears to stabilize the purified enzyme, since no significant loss of the enzyme activity was observed over a month of storage at 4°C. PAP is also a competitive inhibitor of ST activity [2] (the final concentration of PAP in the assay medium is less than 10 ~tM). Thus the specific activity and purification fold should be regarded as minimal values. As shown in Fig. 3, purified AD-ST was subsequently subjected to two-dimensional gel eIectrophoresis, in which isoelectrofocusing was carried out under denaturing conditions in the first dimension, followed by SDS-PAGE in the second dimension. The purified enzyme focused mainly into four protein spots with p l values of 7.2, 6.7, 6.1 and 5.8, determined on thc basis of calculations using internal p l markers. The relative amounts of the p l 6.7 and 7.2 spots are similar, whereas the relative level of the p l 6.1 spot is low and the p l 5.8 spot is very weak (Fig. 3).
3O l
20.1 "'~r . , . ' . ~ k :k
$.1 U Fig. 2. SDS-polyacrylamide gel eleclrophoresis of AD-ST. Sample preparations of AD-ST were analyzed al each purification step by SDS-PAGE and visualized by Coomassie blue staining. Lane 1. standard markers; lane 2, cytosolic fraction (9 tLg); lane 3, pooled fractions after DEAE-cellulose chromatography {8 p,g); lane 4, purified AD-ST after PAP-agaros¢ affinity chromatography (0.3 t.tg).
Fig. 3. Two-dimensional gel electrophoresis of purified AD-ST. Purified AD-ST was analysed by two dimensional gel electrophoresis and was visualized by Coomassie blue staining. The pl value of each spot is shown and was determined using internal p l markers, as described in Materials and Methods.
72
lmmunochemical characterization of AD-ST expression in male and female rat liver We prepared rabbit polyclonal antiserum against purified AD-ST and used it for immunochemical analysis of AD-ST levels. The antiserum precipitated about 90% of the purified AD-ST enzyme activity in immunoprecipitation reactions with protein A-Sepharose (data not shown). It also recognized purified AD-ST polypeptide (Fig. 4a), however, it did not cross react significantly with purified phenol ST from male rat liver (P-STc~) [15] (data not shown). Immunoblot analysis was carried out on cytosolic fractions of rat liver taken from males a~d females at various ages. In liver preparations from female rats of 60 and 110 days of age, the antiserum recognized a polypeptide with a subunit molecular mass of 30 kDa, corresponding to the molecular mass of the purified AD-ST subunit (Fig. 4a). In contrast, very low levels of the 30 kDa polypeptide were present in the liver cy(a)
P
I, ,l 20
~ 60
I I 110
I I 550
Age (days)
(b)
- 0,0 t o.so i
[] b.-~
"'°1/
:~ 0,$01
9( 0.10] 0.0020
•
•
60
110
550
Age (days) Fig. 4. Age-related expression of AD-ST in male and female ral liver. (a) Cytosolic fractions containing 0.5 /~g protein were subjected to 12.5% SDS-PAGE and transferred to a nitrocellulose filter. This blot was probed with anti-AD-ST antiserum (1:20000 dilution) and the immunoreactive polypeptides were visualized as described in Materials and Methods. Each pair of lanes shows samples taken from male rats in the left lane and female rats in the right lane. Lane P contains purified AD-ST (0.13/~g). (b) Liver cytosolic fractions were prepared from male and female rats at 20, 60, I l0 and 550 days of age and the AD-ST activity in each fraction was determined.
2 0 days
pl
dull"
--
7.|
it.1'
-"
",-
-30kDa
ill
$.11
6.1
5.8
1 10 days
•
"
pl
T.2
8.?
~30kDa
Fig. 5. C o m p a r i ~ n of AD-ST in liver cylosolic fractions of weanling and adult female rats. The liver cytosolic fractions (0.5/zg protein) taken from female rats at 20 and i l 0 days of age were subjected to two-dimensional gel electrophoresis and transferred to nitrocellulose filters. These blots were probed with anti-AD-ST antiserum (t : 10000 dilution) and the immunoreactive proteins were visualized as described in Materials and Methods. Additional spots visible in the sample of the rat at I l0 days of age are indicated ( A, ,~ ).
tosol of male rats of the .same ages (Fig. 4a). This is consistent with the low levels of AD-ST activity observed in liver preparations from male rats of these ages (Fig. 4b). In contrast to the AD-ST levels found in older animals, the livers of male and female rats of 20 days of age contained similar amounts of AD-ST protein as well as high AD-ST activity (Fig. 4a, b). It is noteworthy that significant levels of AD-ST protein and AD-ST activity are present in the livers of male rats which are 550 days old. In addition to the AD-ST subunit, a very weak 34 kDa polypeptide was also visible throughout the immunochemically tested samples irrespective of age and sex (Fig. 4a). The identity of this protein is unknown at present, but may represent other forms of Sl'. Purified AD-ST is comprised of several isoelectric variants (Fig. 3), so we analyzed the cytosolic fraction of female rat liver by two-dimensional gel electrophoresis and followed that with immunoblot analysis using the anti-AD-ST antiserum. Four protein spots with p l values of 7.2, 6.7, 6.1 and 5.8 were present in the liver cytosol of adult female rats (Fig. 5). The relative amounts of the isoelectric variants of AD-ST were compared in weanling (20-days-old) and adult (ll0-days-old) female rats. As shown in Fig. 5, the relative amounts of the immunoreactive isoelectric variants change with age. in 20-day-old females, the amount of the pI 7.2 spot is greater than that of the p l 6.7 variant, while in ll0-day-old rat, the levels of these
73 two variants are comparable to one another. Little difference was observed in the relative levels of the weaker spots, p l 6.1 and 5.8 between liver samples taken at different ages. In the adult rat, two additional spots are visible (one is obvious, while the other is very faint, Fig. 5); the identity of these are uncertain at present. Since significant levels of AD-ST are obviously present in the liver cytosolie fraction of 550-day-old male rats (Fig. 4), we also investigated the isoelectric variant composition in these animals and found it to be similar to that seen in weanling females (data not shown). The AD-ST content was also measured in several extrahepatic tissues. Little AD-ST activity was found in the cytosolic fractions of the lung, kidney, brain, stomach or small intestine taken from adult male or female rats. Without exception, no immunoreactive polypeptide with a subunit molecular mass of 30 kDa was observed in any of these tissues (data not shown). Discussion
We previously reported age-related alterations in AD-ST activity in male and female rat liver [6]. Gender- and age-dependent changes were also observed in other ST activities in rat liver [5,7,16]. In order to clarify the molecular mechanism of these age-dependent changes of AD-ST in male and female rats, we purified a major isoenzyme of AD-ST [9,11] from the liver cytosol of adult females. PAP-agarose affinity chromatography according to the method of Barnes et at. [13], provided more effective purification than the method used in our previous studies [9,11]. Using the Barnes method, AD-ST was purified 122-fold over its activity in the liver cytosol. Several isoenzTmes of hydroxysteroid STs were isolated from female rat liver [3,4,13,17]. Purification fold of our final preparation is comparable to the results described in these reports. As described above, our final AD-ST preparation contains PAP, which is very effective in stabilizing the purified enzyme and also a competitive inhibitor of ST activity. Thus the specific activity and purification fold of the final preparation should be regarded as minimal values. The subunit molecular mass of purified AD-ST is 30 kDa and is in the range of values (28 kDa-32 kDa) reported for other hydroxysteroid STs [2-4]. The identity of this AD-ST with respect to previously isolated STs is uncertain at present. When the purified AD-ST was subjected to two dimensional gel electrophoresis, several isoelectric variants were observed, as previously described for a partially purified preparation [9]. Since these variants were present in freshly prepared liver cytosol as detected by immunoblot analysis with anti-AD-ST antiserum, they are assumed to be authentic and not to
have arisen as artifacts of purification. AD-ST is thought to be an oligomer of large molecular weight [9] and composed of several subunits of similar molecular weight, but distinct p l values. This is the first demonstration that a hydroxysteroid ST is composed of several isoelectric variants with the same subunit molecular weight. It is however, still unknown whether the different p l values are due to different primary structures or to posttranslational modification of the subunits. Immunoblot analysis showed that adult female rat liver cytosol contains much higher levels of AD-ST than does that of adult males. This is consistent with the previous observation [6] that AD-ST activity in adult females is 15-fold higher than in adult males. lmmunoblot analysis of the liver cytosol of male and female rats at various ages demonstrated good correlations between the AD-ST polypeptide content and activity levels. These results indicate that age- and gender-related differences in AD-ST activity in rats are determined primarily by the level of AD-ST polypeptide. in previous reports using monoclonai antibody against rat hepatic bile acid ST [18,19], it was demonstrated that gender differences and responses to gonadal hormones in the ST activity were correlated with the enzyme protein content. Rat hepatic ST activities towards hydroxysteroids are under endocrine control [4,5]. AD-ST activity also responds to gonadal hormone [8]. The molecular basis for control mechanism of the level of AD-ST polypeptide remains to be understood. Recently, Runge-M~;rris and Wilusz [20] measured the hepatic levels of a hydroxysteroid ST mRNA using an oligonucleotide probe of 21 base pairs, which was based on the nucleotide sequence of the eDNA encoding a rat hepatic hydroxysteroid ST (STa) [21,22]. A marked difference was observed in the mRNA levels in prepubescent male and female rats (22-30-days-old). In males, the mRNA level was 8-10-fold higher at puberty (42--45-days-old) than at prepubescent ages (22-26-days-old). These results are in marked contrast with the observations described in this study and those reported previously [6]. This contrast may indicate that the expression of these STs is controlled at the translational a n d / o r posttranslational level as reported for P-45011E1, a drug-metabolizing enzyme in phase 1 [23251. It has been reported that ST activities towards steroids and bile acids are high in the liver cytosol of senescent male rats [26-28]. Recently it was reported that the amino acid sequences of steroid STs of rat liver [21:22] and of bovine placenta [29], which were deduced from the corresponding cDNAs have considerable homology with the senescence marker protein-2 (SMP-2) [30], the function of which is unknown. The SMP-2 is significantly expressed in the livers of senescent male rats, while its level in young adult males is
74 very low [30]. On the basis of these studies, we examined AD-ST in liver cytosol of a senescent male rat and detected significant activity and protein levels. The specific expression of AD-ST in liver as described in this study and its changes during the life span of male rats appear to be regulated in a manner similar to that of SMP-2 [30]. In liver cytosol of female rats, AD-ST activity alternates biphasically from postnatal development through adulthood [6]. Similar developmental changes in the activity and isoenzyme pattern have been reported for rat hepatic steroid ST [7] and bile acid ST [16]. As described above, AD-ST is composed of subunits with distinct p/ values. We compared the isoelectric variants of AD-ST subunits in weanling (20-days-old) and adult (ll0-days-old) female rats by immunoblot analysis (Fig. 5). The differences observed in the relative amounts of the AD-ST isoelectric variants suggests that the levels of the subunits change during the temporary decline in AD-ST activity, which occurs at a ~ u t 40 days of age. It is of interest that the relative levels of these isoelectric variants in senescent male rats is similar to that in weanling females. The functional significance of the changes in the isoelectric variants during development remains to be clarified. Studies on the catalytic properties of the enzyme at different ages may reveal the significance of these various forms of AD-ST. Molecular cloning of STa cDNA using anti-STa antibody [21,22] yielded many clones from a rat liver cDNA library and nucleotide sequencing revealed that two of the clones shared significant homology. The genomic cloning of SMP-2 [30] produced two distinct clones, which had sequence homology and originated from two separate genes. It is possible that the isoelectric variants of AD-ST originate from separate genes and that their expression is regulated during aging. The multiplicity and heterogeneity of hydroxysteroid STs and regulation of their expression must be further studied at the molecular level. References I Falany, C.N. (1991) Trends Pharmacol. Sci. 12, 255-259. 2 Mulder, GJ. and Jakoby, W.B. (1990) in Conjugalion Reactions in Drug Metabolism (Mulder, G.J., ed.), pp. 107-161, Taylor & Francis. London.
3 Jakoby, W.B., Duffel, M.W., Lyon, E.S. and Ramaswamy, S. (1984) in Progre~q in Drug Metabolism, Vol. 8 (Bridges, J.W. and Chasseaud, L.F., eds.), pp. 1 !-33, Taylor & Francis, London. 4 Singer, S.S. (1985) in Biochemical Pharmacology and Toxicology, Vol. 1 (Zakim, D. and Vesscy, D.A., eds.), pp. 95-159, John Wiley & Sons, New T'ork. 5 Yamazoe, Y., Gong, D., Murayama, N., Abu-Zcid, M. and Kalo, R. (1989) Mol. Pharmacol. 35, 707-712. 6 Matsui, M. and Watanabe, H.K. (1982) Biochem. J. 204, 441-447. 7 Singer, S.S., Giera, D., Johnson, J. and Sylvester, S. (1976) Endocrinology 98. 963-974. 8 Watanabe, H.K. and Matsui. M. (1984)J. Pharmacobio-Dyn. 7, 641-647. 9 Homma, H., Sasaki, T. and Matsui, M. (1991) Chem. Pharm. Bull. 39, 1499-1503. 10 Singer. S.S. (1979) Anal. Biochem. 96, 34-38. I1 Matsui, M. and Nagai, F. (1985) J. Pharmacobio-Dyn. 8, 10481053. 12 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 13 Barnes, S., Buchina, E.S., King, RJ., McBurnett, T. and Taylor, K.B. (1989) J. Lipid. Res. 30, 529-540. 14 Laemmli, U.K. (1970) Nature 227, 680-685. 15 Homma. H., Kamakura, M., Nakagome, I. and Matsui, M. (1991) Chem. Pharm. Bull., 39, 3307-3312. 16 Kane, R.E., Chen, LJ., Herbst, J.J. and Thaler, M.M. (1988) Pediatric Res. 24, 247-253. 17 Ogura, K., Sohtome, T., Sugiyama, A., Okuda, H., Hiratsuka, A. and Watabe, T. (1990) Mol. Pharmacol. 37, 848-854. 18 Collins, R.H., Lack, L., Harman, K.M.. and Kiilenbcrg, P.G. (1986) Hepatology 6, 579-586. 19 Collins, R.H., Lack, L. and Killenberg, P.G. (1987) Am. J. Physiol. 252, G276-280. 20 Runge-Morris, M. and Wilusz, J. (1991) Biochem. Biophys. Res. Commun. 175, 1051-I056. 21 Ogura, K., Kajita, J., Narihata, H., Watabe, T., O2awa, S., Nagala, K., Yamazoe, Y. and Kato, R. (1989) Biochem. Biophys. Res. Commun, I65, 168-174. 22 Ogura, K., Kajita, J., Narihata, H., Watabe, T., Ozawa, S., Nagata, K., Yamazoe, Y. and Kato, R. (1990) Biochem. Biophys. Res. Commun. 166, 1494-1500. 23 Eliasson, E., Johasson, !. and ]ngelman-Sundberg, M. (1990) Proc. Natl. Acad. Sci. USA 87, 3225-3229. 24 Porter, T.D., Khani, S.C. and Coon, M.J. (1989) Mol. Pharmacol. 36, 61-65. 25 Song, B-J., Veech, R.L., Park, S.S.., Gelboin, H.V. and Gonzalez, FJ. (1989) J. Biol. Chem. 264, 3568-3572. 26 Singer, S.S. and Bruns, L. (1978) Exp. Geront. 13, 425-429. 27 Leakey, J.A., Cunny, H.C., Bazare, J., Jr., Webb, PJ., Lipscomb, J.C., Slikker, W., Jr., Feuers, RJ., Dully, P.H. and Hart, R.W. (1989) Mechanisms of Ageing and Development 48, 157-166. 28 Galinsky, R.E. Kane, R.E. and Franklin, M.R. (1986) J. Pharmacol. Exp. Thor. 237, 107-113. 29 Nash, A.R. and Taylor, PJ. (1990) Age 13, 13-14. 30 Song, C-S., Kim, J.M., Roy, A.K. and Chatterjee, B. (1990) Biochemistry 29, 542-551.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
69
BBAPRO 34195
lmmunochemical characterization of developmental changes in rat hepatic hydroxysteroid sulfotransferase Hiroshi Homma, Izumi Nakagome, Minoru Kamakura and Michio Matsui Kyoritsu Collegeof Pharmacy, Tokyo (Japan)
(Received 30 September 1991)
Key words: Sulfotransferase;Androsterone; Development;lsoenzyme; lsoelectricvariant; (Rat liver) A major isoenzyme of hepatic androsterone-sulfating sulfotransferase (AD-ST) was purified from adult female rats. The activity was purified ! 22-fold over that found in the cytosol and showed a single protein band with a subunit molecular mass of 30 kDa after sodium dodecyI sulfate polyacrylamide gel electrophoresis. The purified enzyme exhibited four isoelectric variants of subunits on denaturing isoelectrofocusing gels (pl = 5.8, 6.1, 6.7 and 7.2). Rabbit antiserum raised against the enzyme specifically detected AD-ST polypcptide in rat liver cytosoi, lmmunoblot analysis of liver cytosol from female and male rats at various ages showed good correlation between the levels of AD-ST activity and AD-ST polypeptide. Significant levels of AD-ST activity and polypeptide were detected in senescent male rats, though normal adult male rats have very low levels of AD-ST activity and protein. The relative content of the isoelectric variants of AD-ST were different in liver cytosol of weanling and adult females, indicating that age- and gender-related alterations of hepatic AD-ST activity are primarily determined by the levels of AD-ST polypeptide and the relative amounts of the four isoelectric variants of the enzyme.
Introduction Sulfotransferase (ST) catalyzes the transfer of sulfate group from 3'-phosphoadenoslne 5'-phosphosulfate (PAPS) to a variety of drugs, xenobiotics and endogenous compounds. Previous investigations revealed heterogeneity and multiplicity of STs in rat liver cytosol as well as in human liver and platelets [1-4]. Rat liver hydroxysteroid STs, which have high catalytic activity for steroid hormones, alcohols and bile acids were shown in purification studies to comprise several isoenzymes [2--4]. These activities are known to be under the regulation of gonadal and adrenal steroids [4] as well as pituitary growth hormone [5]. We have previously reported the following features of ST activity in rat liver cytosol using androsterone (AID) as a substrate. (1) The activity in adult female rats is 15-fold higher than in adult males [6]. Such a marked sex-related difference was not observed in ST activity with hydroxysteroids [5,7]. (2) AD-ST activity in male rats increases upon administration of estrogen [8]. Abbreviations: ST, sulfotransferase; AD-ST androsterone-sul[ating sulfotransferase; PAPS, Y-phosphoadenosine 5'-phosphosulfate; PAP, Y-phosphoadenosine5'-phosphate; SDS-PAGE, sodium dodecyi sulfate polyacrylamide gel electrophoresis. Correspondence: M. Matsui, Kyoritsu College of Pharmacy, Shibakoen, Minato-ku,Tokyo 105, Japan.
(3) The activity exhibits a characteristic alteration in male and female rats during postnatal development [6]. it increases after birth in parallel in both sexes until the weanling stage when it reaches a maximum and subsequently decreases in males. In females, on the other hand, AD-ST activity is biphasic; it declines temporarily and increases again to a high level in adult females. (4) A major isoenzyme of AD-ST was partially purified from female rat liver. It is an oligomer consisting of several subunits of the same molecular mass (30 kDa) but with distinct p l values [9]. In order to understand the molecular basis of the gender- and age-related changes in AD-ST activity, we further purified a major isoenzyme of AD-ST from adult female rats and characterized it. We also raised rabbit polyclonal antiserum against the purified enzyme and used it for immunochemical characterization of AD-ST. Materials and Methods Materials
[9,11-3H]Androsterone (60 Ci/mmol) and [1-14C]4 nitrophenol (56 mCi/mmol) were obtained from New England Nuclear (Boston, MA, USA) and Amersham International, (Buckinghamshire, UK), respectively. DEAE-eellulose DE-52 is a product of Whatman Biosystems (Maidstone, Kent, UK). Agarose adenosine 3',5'-bisphosphate type 2 (PAP-agarose)and Pharmalyte 3-10 were purchased from Pharmacia (Uppsala,
70 Sweden). 3'-Phosphoadenosine 5'-phosphate (PAP), 5'AMP, 3'-AMP, horse heart myoglobin and bovine milk /3-1actoglobulin A were obtained from Sigma (St. Louis, MO, USA). Urea and Nonidet P-40 were purchased from Wako Chemical (Osaka, Japan) and from iwai Chemical (Tokyo, Japan), respectively. Complete adjuvant H37 Ra and Freund's incomplete adjuvant are products of Difco Laboratories (Detroit, MI, USA). Peroxidase-conjugated affinipure goat anti-rabbit IgG (H + L) and Block Ace (a blocking reagent for immunoblot analysis) were obtained from Jackson Immunoresearch Laboratories (West Grove, PA, USA) and Dainippon Seiyaku (Osaka, Japan), respectively. 3'-Phosphoadenosine 5'-phosphosulfate ( P A P S ) w a s prepared by the method of Singer [10]. All other reagents were of the highest grade available.
ers used for SDS-PAGE were phosphorylase b (94 kDa), BSA (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and fl-lactoglobulin (14.4 kDa) in the Electrophoresis Calibration Kit (Pharmacia). Two-dimensional gel electrophoresis was carried out as described previously [9]. Briefly, in the first dimension, a 4% l~olyacrylamide isoelectrofocusing gel was run under denaturing conditions in the presence of 2% Nonidet P-40, 8.5 M urea and 2% Pharmalyte 3-10. In the second dimension, SDS-PAGE was performed using 12.5% polyacrylamide gels. Horse heart myoglobin (pl = 6.8 and 7.2, subunit molecular mass = 16950) and bovine milk fllactoglobulin A ( p l = 5 . 1 , subunit molecular mass = 18400) were used as internal p l markers.
Preparation of anti-AD-ST antiserum Purification of AD-ST Preparation of the cytosolic fraction of female rat liver (8 weeks of age) and determinations of AD-ST and 4-nitrophenol ST activities were carried out as previously described [9,11]. The cytosolic fractions of rat lung, kidney, brain, stomach and small intestine were prepared in a manner similar to that used for liver cytosol. Protein content was measured by the method of Bradford [ 12].
Step 1. DEAE-celkdose chromatography of AD-ST. DEAE-cellulose column chromatography of the cytosolic fraction of female rat liver was conducted as described previously [9,11] and fractions which contained the highest activity of AD-ST and little or no 4nitrophenol ST activity were pooled. These fractions were concentrated by dialysis against 10% poly(ethylene glycol) 20000 in 10 mM Tris-HCI, 250 mM sucrose, 0.1 mM EDTA and 3 mM 2-mercaptoethanol (pH 7.4), then dialyzed against 5 mM sodium phosphate (pH 8.0) (buffer A). Step 2. PAP-agarose affinity chromatography. A portion of the concentrated AD-ST fractions was subsequently subjected to a PAP-agarose affinity column chromatography following the method of Barnes et al. [131. A column of PAP-agarose (7 x 96 ram) was preequilibrated in bufier A. The sample applied to the column contained 3-6 mg protein/ml resin. After application, the column was washed at a flow rate of 46 m l / h sequentially with 50 ml of buffer A, 50 ml of 50 mM sodium phosphate (pH 8.0), 20 ml of 5 mM 5'-AMP in buffer A and 20 ml of 5 mM 3'-AMP in buffer A. The AD-ST activity was then eluted with 20 ml of 0.1 mM PAP in buffer A and 1 ml fractions were collected.
Electrophoresis SDS-PAGE was carried out by the method of Laemmli [14] and the proteins were visualized with Coomassie blue staining. The molecular weight mark-
One male New Zealand White rabbit was administered approx. 100 #g of purified AD-ST in complete adjuvant subcutaneously along the back. 4 weeks later, the rabbit received a booster injection containing the same amount of the purified enzyme in Freund's incomplete adjuvant. After 10 days, the animal was bled for preparation of the antiserum. Preimmune serum was also prepared as a control.
lmmunoblot analysis Proteins separated by SDS-PAGE were transferred from the gel to a nitrocellulose filter (Bio-Rad, Richmond, CA, USA) with a Bio-Rad Trans-Blot SD apparatus according to the procedure described by the manufacturer. The amounts of protein used in each experiment are indicated in the figure legends. When extrahepatic tissues were used, those samples contained 20 ttg of cytosolic protein. The nitrocellulose filter was blocked overnight at 4*(2 with a blocking reagent, Block Ace. A 1 : 10000 dilution of rabbit antiserum and a 1:1000 dilution of peroxidase-conjugated goat anti-rabbit IgG antibody were routinely used as primary and secondary antibody, respectively. The immunocomplex was IocaIized using a color generating reaction: the oxidation of 3,3'-diaminobenzidine tetrachloride by hydrogen peroxide. Results
Purification of androsterone-sulfating sulfotransferase
(AD-ST) The cytosolic fraction of liver cells of female rats (8 weeks of age) was subjected to DEAE-cellulose column chromatography for the initial purification of ADST as described previously [9,11]. This chromatography step yielded fractions containing the highest activity of AD-ST and lowest activity of 4-nitrophenol-ST and cortisoI-ST [11]. These fractions were pooled, concentrated and subsequently applied to a PAP-agarose
71 TABLE I
7
t~,ri]ication of A D - S T from female rat lirer cytoso!
6
C
E 0
One unit represents 1 nmoi of androsterone conjugated/rain. 5'
Protein
E
c
4.
>
3
(mg)
Cytosol 3169 DEAEcellulose 109.5
IB
g
2
PAPagarose :'
|
- l ~
1" 0
0
s
~
i
S
10
15
Fraction
20
affinity column. Affinity chromatography was then conducted and the elution pattern of the AD-ST activity from the column is shown in Fig. 1. SDS-PAGE analysis of the final preparation revealed a single protein band with a subunit molecular mass of 30 kDa (Fig. 2). The specific activity of the purified enzyme was 47.3
kDa
2
1230
0.388
1
239
2.18
5.6
15.3
47.3
122
A portion of the DEAE-cellulose fraction (22 mg protein) was applied to PAP-agarose affinity chromatography.
no.
Fig. 1. PAP-agarose affinity chromatography of AD-ST. The pooled fractions containing AD-ST activity, obtained after DEAE-cellulo~ chromatography, were concentrated and a portion of this 122 mg protein in 17 ml buffer) was applied to a PAP-agarose affinity column (7 × q6 mm). The column was washed with v~rious buffers as described in Materials and Methods and the AD-ST activity was specifically eluted with 0.1 mM PAP, The activities eluted in the various buffers were as follows: 0.07 nmol/min per ml in flow through fraction, O.! in buffer A, (I.(14 in 50 mM ,sodium phosphate buffer, 0.04 in 5'-AMP-containing buffer and 0 in 3'-AMP-containing buffer. Fractions 3-9 were pooled for the final purification step.
1
0,324
Activity Specific activity Purification (nmol/min) (nmol/min per mg) (fold)
3 "
67'
43
nmol/min per mg protein, representing a 122-fold purification over its activity in the cytosol (Table I). The final preparation of AD-ST contains PAP, since it is included in the elution medium of the affinity chromatography step. It is well known that STs have very high affinity for PAP [2]. The presence of PAP in the preparation appears to stabilize the purified enzyme, since no significant loss of the enzyme activity was observed over a month of storage at 4°C. PAP is also a competitive inhibitor of ST activity [2] (the final concentration of PAP in the assay medium is less than 10 ~tM). Thus the specific activity and purification fold should be regarded as minimal values. As shown in Fig. 3, purified AD-ST was subsequently subjected to two-dimensional gel eIectrophoresis, in which isoelectrofocusing was carried out under denaturing conditions in the first dimension, followed by SDS-PAGE in the second dimension. The purified enzyme focused mainly into four protein spots with p l values of 7.2, 6.7, 6.1 and 5.8, determined on thc basis of calculations using internal p l markers. The relative amounts of the p l 6.7 and 7.2 spots are similar, whereas the relative level of the p l 6.1 spot is low and the p l 5.8 spot is very weak (Fig. 3).
3O l
20.1 "'~r . , . ' . ~ k :k
$.1 U Fig. 2. SDS-polyacrylamide gel eleclrophoresis of AD-ST. Sample preparations of AD-ST were analyzed al each purification step by SDS-PAGE and visualized by Coomassie blue staining. Lane 1. standard markers; lane 2, cytosolic fraction (9 tLg); lane 3, pooled fractions after DEAE-cellulose chromatography {8 p,g); lane 4, purified AD-ST after PAP-agaros¢ affinity chromatography (0.3 t.tg).
Fig. 3. Two-dimensional gel electrophoresis of purified AD-ST. Purified AD-ST was analysed by two dimensional gel electrophoresis and was visualized by Coomassie blue staining. The pl value of each spot is shown and was determined using internal p l markers, as described in Materials and Methods.
72
lmmunochemical characterization of AD-ST expression in male and female rat liver We prepared rabbit polyclonal antiserum against purified AD-ST and used it for immunochemical analysis of AD-ST levels. The antiserum precipitated about 90% of the purified AD-ST enzyme activity in immunoprecipitation reactions with protein A-Sepharose (data not shown). It also recognized purified AD-ST polypeptide (Fig. 4a), however, it did not cross react significantly with purified phenol ST from male rat liver (P-STc~) [15] (data not shown). Immunoblot analysis was carried out on cytosolic fractions of rat liver taken from males a~d females at various ages. In liver preparations from female rats of 60 and 110 days of age, the antiserum recognized a polypeptide with a subunit molecular mass of 30 kDa, corresponding to the molecular mass of the purified AD-ST subunit (Fig. 4a). In contrast, very low levels of the 30 kDa polypeptide were present in the liver cy(a)
P
I, ,l 20
~ 60
I I 110
I I 550
Age (days)
(b)
- 0,0 t o.so i
[] b.-~
"'°1/
:~ 0,$01
9( 0.10] 0.0020
•
•
60
110
550
Age (days) Fig. 4. Age-related expression of AD-ST in male and female ral liver. (a) Cytosolic fractions containing 0.5 /~g protein were subjected to 12.5% SDS-PAGE and transferred to a nitrocellulose filter. This blot was probed with anti-AD-ST antiserum (1:20000 dilution) and the immunoreactive polypeptides were visualized as described in Materials and Methods. Each pair of lanes shows samples taken from male rats in the left lane and female rats in the right lane. Lane P contains purified AD-ST (0.13/~g). (b) Liver cytosolic fractions were prepared from male and female rats at 20, 60, I l0 and 550 days of age and the AD-ST activity in each fraction was determined.
2 0 days
pl
dull"
--
7.|
it.1'
-"
",-
-30kDa
ill
$.11
6.1
5.8
1 10 days
•
"
pl
T.2
8.?
~30kDa
Fig. 5. C o m p a r i ~ n of AD-ST in liver cylosolic fractions of weanling and adult female rats. The liver cytosolic fractions (0.5/zg protein) taken from female rats at 20 and i l 0 days of age were subjected to two-dimensional gel electrophoresis and transferred to nitrocellulose filters. These blots were probed with anti-AD-ST antiserum (t : 10000 dilution) and the immunoreactive proteins were visualized as described in Materials and Methods. Additional spots visible in the sample of the rat at I l0 days of age are indicated ( A, ,~ ).
tosol of male rats of the .same ages (Fig. 4a). This is consistent with the low levels of AD-ST activity observed in liver preparations from male rats of these ages (Fig. 4b). In contrast to the AD-ST levels found in older animals, the livers of male and female rats of 20 days of age contained similar amounts of AD-ST protein as well as high AD-ST activity (Fig. 4a, b). It is noteworthy that significant levels of AD-ST protein and AD-ST activity are present in the livers of male rats which are 550 days old. In addition to the AD-ST subunit, a very weak 34 kDa polypeptide was also visible throughout the immunochemically tested samples irrespective of age and sex (Fig. 4a). The identity of this protein is unknown at present, but may represent other forms of Sl'. Purified AD-ST is comprised of several isoelectric variants (Fig. 3), so we analyzed the cytosolic fraction of female rat liver by two-dimensional gel electrophoresis and followed that with immunoblot analysis using the anti-AD-ST antiserum. Four protein spots with p l values of 7.2, 6.7, 6.1 and 5.8 were present in the liver cytosol of adult female rats (Fig. 5). The relative amounts of the isoelectric variants of AD-ST were compared in weanling (20-days-old) and adult (ll0-days-old) female rats. As shown in Fig. 5, the relative amounts of the immunoreactive isoelectric variants change with age. in 20-day-old females, the amount of the pI 7.2 spot is greater than that of the p l 6.7 variant, while in ll0-day-old rat, the levels of these
73 two variants are comparable to one another. Little difference was observed in the relative levels of the weaker spots, p l 6.1 and 5.8 between liver samples taken at different ages. In the adult rat, two additional spots are visible (one is obvious, while the other is very faint, Fig. 5); the identity of these are uncertain at present. Since significant levels of AD-ST are obviously present in the liver cytosolie fraction of 550-day-old male rats (Fig. 4), we also investigated the isoelectric variant composition in these animals and found it to be similar to that seen in weanling females (data not shown). The AD-ST content was also measured in several extrahepatic tissues. Little AD-ST activity was found in the cytosolic fractions of the lung, kidney, brain, stomach or small intestine taken from adult male or female rats. Without exception, no immunoreactive polypeptide with a subunit molecular mass of 30 kDa was observed in any of these tissues (data not shown). Discussion
We previously reported age-related alterations in AD-ST activity in male and female rat liver [6]. Gender- and age-dependent changes were also observed in other ST activities in rat liver [5,7,16]. In order to clarify the molecular mechanism of these age-dependent changes of AD-ST in male and female rats, we purified a major isoenzyme of AD-ST [9,11] from the liver cytosol of adult females. PAP-agarose affinity chromatography according to the method of Barnes et at. [13], provided more effective purification than the method used in our previous studies [9,11]. Using the Barnes method, AD-ST was purified 122-fold over its activity in the liver cytosol. Several isoenzTmes of hydroxysteroid STs were isolated from female rat liver [3,4,13,17]. Purification fold of our final preparation is comparable to the results described in these reports. As described above, our final AD-ST preparation contains PAP, which is very effective in stabilizing the purified enzyme and also a competitive inhibitor of ST activity. Thus the specific activity and purification fold of the final preparation should be regarded as minimal values. The subunit molecular mass of purified AD-ST is 30 kDa and is in the range of values (28 kDa-32 kDa) reported for other hydroxysteroid STs [2-4]. The identity of this AD-ST with respect to previously isolated STs is uncertain at present. When the purified AD-ST was subjected to two dimensional gel electrophoresis, several isoelectric variants were observed, as previously described for a partially purified preparation [9]. Since these variants were present in freshly prepared liver cytosol as detected by immunoblot analysis with anti-AD-ST antiserum, they are assumed to be authentic and not to
have arisen as artifacts of purification. AD-ST is thought to be an oligomer of large molecular weight [9] and composed of several subunits of similar molecular weight, but distinct p l values. This is the first demonstration that a hydroxysteroid ST is composed of several isoelectric variants with the same subunit molecular weight. It is however, still unknown whether the different p l values are due to different primary structures or to posttranslational modification of the subunits. Immunoblot analysis showed that adult female rat liver cytosol contains much higher levels of AD-ST than does that of adult males. This is consistent with the previous observation [6] that AD-ST activity in adult females is 15-fold higher than in adult males. lmmunoblot analysis of the liver cytosol of male and female rats at various ages demonstrated good correlations between the AD-ST polypeptide content and activity levels. These results indicate that age- and gender-related differences in AD-ST activity in rats are determined primarily by the level of AD-ST polypeptide. in previous reports using monoclonai antibody against rat hepatic bile acid ST [18,19], it was demonstrated that gender differences and responses to gonadal hormones in the ST activity were correlated with the enzyme protein content. Rat hepatic ST activities towards hydroxysteroids are under endocrine control [4,5]. AD-ST activity also responds to gonadal hormone [8]. The molecular basis for control mechanism of the level of AD-ST polypeptide remains to be understood. Recently, Runge-M~;rris and Wilusz [20] measured the hepatic levels of a hydroxysteroid ST mRNA using an oligonucleotide probe of 21 base pairs, which was based on the nucleotide sequence of the eDNA encoding a rat hepatic hydroxysteroid ST (STa) [21,22]. A marked difference was observed in the mRNA levels in prepubescent male and female rats (22-30-days-old). In males, the mRNA level was 8-10-fold higher at puberty (42--45-days-old) than at prepubescent ages (22-26-days-old). These results are in marked contrast with the observations described in this study and those reported previously [6]. This contrast may indicate that the expression of these STs is controlled at the translational a n d / o r posttranslational level as reported for P-45011E1, a drug-metabolizing enzyme in phase 1 [23251. It has been reported that ST activities towards steroids and bile acids are high in the liver cytosol of senescent male rats [26-28]. Recently it was reported that the amino acid sequences of steroid STs of rat liver [21:22] and of bovine placenta [29], which were deduced from the corresponding cDNAs have considerable homology with the senescence marker protein-2 (SMP-2) [30], the function of which is unknown. The SMP-2 is significantly expressed in the livers of senescent male rats, while its level in young adult males is
74 very low [30]. On the basis of these studies, we examined AD-ST in liver cytosol of a senescent male rat and detected significant activity and protein levels. The specific expression of AD-ST in liver as described in this study and its changes during the life span of male rats appear to be regulated in a manner similar to that of SMP-2 [30]. In liver cytosol of female rats, AD-ST activity alternates biphasically from postnatal development through adulthood [6]. Similar developmental changes in the activity and isoenzyme pattern have been reported for rat hepatic steroid ST [7] and bile acid ST [16]. As described above, AD-ST is composed of subunits with distinct p/ values. We compared the isoelectric variants of AD-ST subunits in weanling (20-days-old) and adult (ll0-days-old) female rats by immunoblot analysis (Fig. 5). The differences observed in the relative amounts of the AD-ST isoelectric variants suggests that the levels of the subunits change during the temporary decline in AD-ST activity, which occurs at a ~ u t 40 days of age. It is of interest that the relative levels of these isoelectric variants in senescent male rats is similar to that in weanling females. The functional significance of the changes in the isoelectric variants during development remains to be clarified. Studies on the catalytic properties of the enzyme at different ages may reveal the significance of these various forms of AD-ST. Molecular cloning of STa cDNA using anti-STa antibody [21,22] yielded many clones from a rat liver cDNA library and nucleotide sequencing revealed that two of the clones shared significant homology. The genomic cloning of SMP-2 [30] produced two distinct clones, which had sequence homology and originated from two separate genes. It is possible that the isoelectric variants of AD-ST originate from separate genes and that their expression is regulated during aging. The multiplicity and heterogeneity of hydroxysteroid STs and regulation of their expression must be further studied at the molecular level. References I Falany, C.N. (1991) Trends Pharmacol. Sci. 12, 255-259. 2 Mulder, GJ. and Jakoby, W.B. (1990) in Conjugalion Reactions in Drug Metabolism (Mulder, G.J., ed.), pp. 107-161, Taylor & Francis. London.
3 Jakoby, W.B., Duffel, M.W., Lyon, E.S. and Ramaswamy, S. (1984) in Progre~q in Drug Metabolism, Vol. 8 (Bridges, J.W. and Chasseaud, L.F., eds.), pp. 1 !-33, Taylor & Francis, London. 4 Singer, S.S. (1985) in Biochemical Pharmacology and Toxicology, Vol. 1 (Zakim, D. and Vesscy, D.A., eds.), pp. 95-159, John Wiley & Sons, New T'ork. 5 Yamazoe, Y., Gong, D., Murayama, N., Abu-Zcid, M. and Kalo, R. (1989) Mol. Pharmacol. 35, 707-712. 6 Matsui, M. and Watanabe, H.K. (1982) Biochem. J. 204, 441-447. 7 Singer, S.S., Giera, D., Johnson, J. and Sylvester, S. (1976) Endocrinology 98. 963-974. 8 Watanabe, H.K. and Matsui. M. (1984)J. Pharmacobio-Dyn. 7, 641-647. 9 Homma, H., Sasaki, T. and Matsui, M. (1991) Chem. Pharm. Bull. 39, 1499-1503. 10 Singer. S.S. (1979) Anal. Biochem. 96, 34-38. I1 Matsui, M. and Nagai, F. (1985) J. Pharmacobio-Dyn. 8, 10481053. 12 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 13 Barnes, S., Buchina, E.S., King, RJ., McBurnett, T. and Taylor, K.B. (1989) J. Lipid. Res. 30, 529-540. 14 Laemmli, U.K. (1970) Nature 227, 680-685. 15 Homma. H., Kamakura, M., Nakagome, I. and Matsui, M. (1991) Chem. Pharm. Bull., 39, 3307-3312. 16 Kane, R.E., Chen, LJ., Herbst, J.J. and Thaler, M.M. (1988) Pediatric Res. 24, 247-253. 17 Ogura, K., Sohtome, T., Sugiyama, A., Okuda, H., Hiratsuka, A. and Watabe, T. (1990) Mol. Pharmacol. 37, 848-854. 18 Collins, R.H., Lack, L., Harman, K.M.. and Kiilenbcrg, P.G. (1986) Hepatology 6, 579-586. 19 Collins, R.H., Lack, L. and Killenberg, P.G. (1987) Am. J. Physiol. 252, G276-280. 20 Runge-Morris, M. and Wilusz, J. (1991) Biochem. Biophys. Res. Commun. 175, 1051-I056. 21 Ogura, K., Kajita, J., Narihata, H., Watabe, T., O2awa, S., Nagala, K., Yamazoe, Y. and Kato, R. (1989) Biochem. Biophys. Res. Commun, I65, 168-174. 22 Ogura, K., Kajita, J., Narihata, H., Watabe, T., Ozawa, S., Nagata, K., Yamazoe, Y. and Kato, R. (1990) Biochem. Biophys. Res. Commun. 166, 1494-1500. 23 Eliasson, E., Johasson, !. and ]ngelman-Sundberg, M. (1990) Proc. Natl. Acad. Sci. USA 87, 3225-3229. 24 Porter, T.D., Khani, S.C. and Coon, M.J. (1989) Mol. Pharmacol. 36, 61-65. 25 Song, B-J., Veech, R.L., Park, S.S.., Gelboin, H.V. and Gonzalez, FJ. (1989) J. Biol. Chem. 264, 3568-3572. 26 Singer, S.S. and Bruns, L. (1978) Exp. Geront. 13, 425-429. 27 Leakey, J.A., Cunny, H.C., Bazare, J., Jr., Webb, PJ., Lipscomb, J.C., Slikker, W., Jr., Feuers, RJ., Dully, P.H. and Hart, R.W. (1989) Mechanisms of Ageing and Development 48, 157-166. 28 Galinsky, R.E. Kane, R.E. and Franklin, M.R. (1986) J. Pharmacol. Exp. Thor. 237, 107-113. 29 Nash, A.R. and Taylor, PJ. (1990) Age 13, 13-14. 30 Song, C-S., Kim, J.M., Roy, A.K. and Chatterjee, B. (1990) Biochemistry 29, 542-551.