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Sulfation of thyroid hormones by liver of rainbow trout, Oncorhynchus mykiss
Comparative Biochemistry and Physiology Part C 120 (1998) 415 – 420
Sulfation of thyroid hormones by liver of rainbow trout, Oncorhynchus mykiss K.W. Finnson *, J.G. Eales Department of Zoology, Uni6ersity of Manitoba, Winnipeg, MB R3T 2N2, Canada Received 6 January 1998; received in revised form 1 April 1998; accepted 7 April 1998
Abstract We studied hepatic sulfation of thyroid hormones (TH) in rainbow trout, Oncorhynchus mykiss. Sulfation of thyroxine (T4) and 3,5,3%-triiodothyronine (T3) was detected in the cytosolic (63 – 67%), microsomal (12 – 16%), nuclear (12 – 14%) and mitochondrial/ lysosomal (7–8%) fractions. Using 3%-phosphoadenosine 5%-phosphosulfate (PAPS) as a sulfate donor, sulfation of T4 and T3 by the cytosolic fraction depended on protein concentration and time. The pH profiles for T4- and T3-sulfation were broad and overlapping with optimal pH values of about 6.5 and 7.0 U respectively. At pH 7.0, apparent Km (mM), Vmax (pmol/mg cytosolic protein per hour) and catalytic efficiency (Vmax/Km) values were 3,5%,3%-triiodothyronine (reverseT3, rT3) =0.7, 583 and 832; T4 = 1.7, 46 and 27; T3 =11.5, 840 and 73. Inhibitor profiles for both T4- and T3-sulfation were not significantly different with a common inhibitor preference of rT3 \pentachlorophenol\ triiodothyroacetic acid \ tetraiodothyroacetic acid T4 = T3 =3,5-diiodothyronine. T4-, T3- and rT3-sulfation activity decreased with increasing pre-incubation temperature (12, 24, 36°C); however, there were no significant differences in T4-, T3- and rT3-sulfation activity at each pre-incubation temperature. We conclude that: (i) in trout, hepatic sulfation of TH is enzymatic and obeys Michaelis – Menten kinetics; (ii) like mammalian hepatic sulfotransferases (STs), trout hepatic STs are heat-sensitive cytosolic proteins using PAPS as a sulfate donor; (iii) unlike mammalian sulfation of TH, trout hepatic sulfation of T4, T3 and rT3 may be catalyzed by a single form of ST preferring rT3 as substrate and with a catalytic efficiency of rT3 ZT3 \T4. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Oncorhynchus mykiss; Rainbow trout; Thyroid; 3%-phosphoadenosine 5%-phosphosulfate; Reverse triiodothyronine; Sulfation; Sulfotransferase; Thyroxine; Triiodothyronine
1. Introduction Deiodination and sulfation are interrelated pathways for thyroid hormone (TH) metabolism. Outer-ring deiodination (ORD) of thyroxine (T4) produces bioactive 3,5,3%-triiodothyronine (T3), while inner-ring deiodination (IRD) produces inactive 3,3%,5%-triiodothyronine (reverse T3, rT3). Further deiodination of these two types of T3 produces inactive iodothyronine metabolites [2]. These deiodinases are located in the endoplasmic reticulum of liver and other tissues, and their activities
* Corresponding author. Tel.: +1 204 4749245; fax: + 1 204 4747588; e-mail: [email protected] 0742-8413/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0742-8413(98)10015-4
can be measured in the microsomal fraction of the cell [11]. Sulfation of TH is catalyzed by phenol sulfotransferases (PSTs), a family of homologous enzymes located in the cytosolic fraction of liver and other tissues. PSTs have a broad and overlapping substrate specificity and use 3%-phosphoadenosine 5%-phosphosulfate (PAPS) as a sulfate donor [13,14]. In mammals, sulfation of the phenolic hydroxyl group of TH prevents ORD of T4, while it enhances IRD of both T4 and T3 [12–14]. Thus, since sulfation represents the initial step leading to irreversible deiodinative breakdown of TH, it is necessary to study this pathway to understand TH degradation pathways. Comparatively little is known about PSTs in nonmammalian species. However, Osborn and Simpson [8]
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have suggested the sulfation of TH in the marine plaice, Pleuronectes platessa. More recently, we have demonstrated the in vitro and in vivo sulfation of TH in the rainbow trout, Oncorhynchus mykiss [5]. In this study, our goal was to characterize hepatic sulfation of TH in trout.
2. Materials and methods
2.1. Animal maintenance Rainbow trout (250 – 500 g, 2 year old) were obtained from the Rockwood Hatchery, Balmoral, Manitoba, and held in the laboratory in flowing, aerated, dechlorinated water at 12°C, on a 12 h light:12 h dark photoperiod. Trout were fed a commercial diet (Martin Feed Mills, Elmira, Ontario, trout feed pellets, 3.2 mm diameter) 1× daily at a ration of 1–2% of body weight. Trout were used 18 – 24 h after their last meal.
2.2. Subcellular fractionation Trout were anesthetized in tricaine methanesulfonate (0.07 g/l) and killed by a blow to the head. Livers were removed and rinsed with ice-cold buffer: 0.25 M sucrose (pH 7.2) with 1 mM EDTA and 10 mM DTT. Livers were then added to 4 vol. (w/v) of buffer, minced with scissors and dispensed into a 55ml Wheaton homogenization chamber. Tissue was homogenized by two to three strokes of a motorized pestle (Tri-R Instruments, NY), filtered through two layers of cheesecloth and transferred to plastic tubes for centrifugation. The crude homogenate was centrifuged for 20 min at 730×g to separate the nuclear pellet. The post-nuclear supernatant was then centrifuged for 20 min at 25200×g to separate the crude mitochondrial/lysosomal pellet. Finally, the post-mitochondrial supernatant was centrifuged for 67 min at 110000×g to separate the microsomal pellet from the cytosol. Pellets (resuspended in 1 – 2 ml of buffer) and 1-ml aliquots of the crude homogenate and cell sap were stored at −76°C [11].
tor 3%-phosphoadenosine 5%-phosphosulfate (PAPS) (final concentration 20 mM). Control tubes received 20 ml of 0.1 M Tris buffer (pH 7.0) alone. This mixture was equilibrated in a shaking water bath for 10–15 min (12°C, 150 rpm). The reaction was started by adding 20 ml of either 125I-labeled T4, T3 or rT3 substrate (final concentration 1.0 mM; 100000 cpm). After 60 min, the reaction was stopped by adding 2 ml of chloroform/methanol (2:1 v/v), vortexed and centrifuged at 1420×g for 5 min. A volume of 250 ml was removed from the aqueous methanol layer and added to Sephadex LH-20 minicolumns containing 750 ml of 1.0 N HCl. Columns were swirled and drained, and iodide was eluted to waste with 3 ml of 0.1 N HCl. Sulfated TH conjugates were collected with 12 ml of H2O and counted in a gamma counter. Columns were washed with 3 ml of 0.1 N NH4OH in ethanol and stored with 0.1 N HCl [5]. We determined TH sulfate formation (H2O fraction) by subtracting the cpm of control tubes (− PAPS) from the cpm of experimental tubes (+PAPS). Protein concentration was determined by the Bradford method [1]. Depending on the experiment, ST activity was expressed as either (i) cpm (labeled TH sulfated)/h; (ii) nmol of TH sulfated/mg protein per hour; (iii) percentage of the control activity.
2.3. Sulfotransferase (ST) assay We based our assay on the procedure of Young et al. [16]. The various subcellular fractions were thawed on ice and diluted ( 1:25) with 0.1 M Tris buffer (pH 7.0) to achieve a final protein concentration of 0.2 – 0.8 mg/ml. We did not include BSA in our assay buffer because it decreased ST activity. A volume of 500 ml of this diluted subcellular fraction was added to siliconized test tubes containing 20 ml of the cofac-
Fig. 1. Sephadex LH-20 elution profile for synthetic 125I-labeled T3-sulfate. Profile was obtained by stepwise addition of 1-ml aliquots of 0.1 N HCl ( × 3), H2O ( ×12), and 0.1 N NH4OH in ethanol (1:1 v/v) (×3). Over 96% of the radioactivity was recovered in the H2O fraction.
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Fig. 2. Relationship between cytosolic protein concentration and amount of 125I-labeled T3 converted to T3-sulfate/h. Each point () represents the mean ( 9 SEM where it exceeds symbol size) of three separate incubations.
Fig. 3. The effect of incubation time on the amount of 125I-labeled T4 converted to T4-sulfate ( ) and 125I-labeled T3 converted to T3-sulfate () at intervals up to 1 h. Each point represents the mean of three separate incubations (SEM values did not exceed symbol size).
3. Results
3.3. Inhibitor studies
3.1. Assay conditions
Fig. 6 shows inhibition of both T4ST and T3ST activities by different inhibitors/analogs (100 nM). Inhibitor profiles for T4ST and T3ST were not signifi-
Fig. 1 shows the elution profile of synthetic 125I-labeled T3-sulfate. Over 96% of the radioactivity was recovered in the H2O fraction. Similar results were obtained using either 125I-labeled T4- or rT3-sulfate. Sulfation of T4 (T4ST) and T3 (T3ST) occurred mainly in the cytosolic fraction (63 – 67%). T4ST and T3ST activity was also detected in the microsomal (12 –16%), nuclear (12–14%) and mitochodrial/lysosomal fraction (7 – 8%) (data not shown). T3ST activity increased with cytosolic protein concentration up to 1.2 mg/ml (Fig. 2). For all remaining experiments, we used cytosolic preparations with protein concentrations ranging from 0.2–0.8 mg/ml. Both T4ST and T3ST activities increased linearly to 60 min (Fig. 3). The pH profiles for T4ST and T3ST activities were broad and overlapping with optimal pH values of about 6.5 and 7.0 U respectively (Fig. 4).
3.2. Enzyme kinetics We chose pH 7.0 U to compare kinetic properties of T4ST, T3ST and rT3ST. Fig. 5 shows Lineweaver –Burk plots for sulfation of (A) T4 (0.25 – 4.0 mM); (B) T3 (1.0 –16.0 mM); (C) rT3 (0.25 – 2.0 mM) using a PAPS concentration of 20 mM. Apparent Km (mM), Vmax (pmol/mg protein per hour) and Vmax/Km values were T4 = 1.7, 46 and 27; T3 =11.5, 840 and 73 and rT3 = 0.7, 583 and 832.
Fig. 4. The effect of pH on the sulfation of T4 ( ) and T3 (). Buffer pH was adjusted by adding 1.0 N HCl to 0.1 M Tris buffer prior to the experiments. Results are from different experiments carried out on the same cytosolic pool. Each point represents the mean ( 9SEM) of three separate incubations converted to percentage highest activity.
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Fig. 5. Lineweaver – Burk plots of hepatic cytosolic ST activities for (A) T4 (0.25 – 4.0 mM); (B) T3 (1.0 – 16.0 mM); (C) rT3 (0.25 – 2.0 mM) using a PAPS concentration of 20 mM. A different substrate range was used for (B) since higher concentrations of T3 were needed. Apparent Km (mM) and Vmax (pmol/mg protein per hour) values were T4 (1.7, 46), T3 (11.5, 840) and rT3 (0.7, 583). Each point represents the mean ( 9 SEM) of three separate incubations.
cantly different (P\0.05) with a common substrate preference of rT3 \pentachlorophenol (PCP)\ triiodothyroacetic acid (TRIAC)\tetraiodothyroacetic acid (TETRAC)\T4 =T3 =3,5,-diiodothyronine (3, 5T2). We did not carry out inhibitor studies using rT3 as substrate.
ST activities due to pre-incubation temperature (P\ 0.05)
3.4. Enzyme thermal stability
Mammalian sulfation of TH is catalyzed by multiple sulfotransferases (STs). These enzymes occur mainly in the cytosol of liver and other tissues and use PAPS as a sulfate donor [13,14]. In comparison, little is known about sulfation of TH in non-mammalian species. In this study, our main goal was to characterize hepatic sulfation of TH in trout.
Fig. 7 shows T4ST, T3ST and rT3ST activities following 15-min pre-incubation at either 12, 24 or 36°C. For each TH substrate, ST activity decreased with increasing pre-incubation temperature. However, there were no significant differences between T4ST, T3ST and rT3-
4. Discussion
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Fig. 6. Inhibition of sulfation of T4 (open bars) and T3 (hatched bars) by various analogs/inhibitors (100 nM). Differences between the inhibition of T4- and T3-sulfation by the various inhibitors/analogues were not significant (P \ 0.05). ST activity is expressed as the percentage of mean control activity (no analog/inhibitor present).
Hepatic sulfation of TH in mammals and trout share some properties. Both are catalyzed by heat-sensitive cytosolic enzymes that use PAPS as a sulfate donor, with the reaction depending on protein concentration, time and pH and obeying Michaelis – Menten kinetics [13,14]. Furthermore, apparent Km values for both
Fig. 7. ST activities for T4 (open bars), T3 (hatched bars—up) and rT3 (hatched bars — down) substrates following a 15-min pre-incubation at 12 (control), 24 or 36°C. There were no significant differences between T4ST, T3ST or rT3ST activities at any of the pre-incubation temperatures (P\ 0.05). ST activity is expressed as a percentage of control activity.
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trout (0.7–11.5 mM) and rats (1.8–4.2 mM) [6] fall in a similar range. However, a different PAPS concentration (50 mM), TH (T2) substrate, pH (7.2) and incubation temperature (37°C) were used in the rat study making a direct comparison between trout and rat Km tentative. The optimal pH values reported for human hepatic sulfation of T3 and rat hepatic sulfation of T2 are 6.6 [15] and 8.0 U [6] respectively. In comparison, the optimal pH values for trout hepatic sulfation of T4 and T3 were about 6.5 and 7.0 U respectively. Although different optimal pH values for trout hepatic sulfation of T4 and T3 suggest more than one form of ST, these pH profiles were broad and overlapping. In addition, T4 and T3 have different pKa values (T4 = 6.73; T3 = 8.45) [7], which may influence enzyme–substrate interaction. Therefore, the existence of multiple STs cannot be established in trout on the basis of pH alone. In addition to pH, differences in enzyme thermal stability can be used to distinguish multiple enzyme forms [6]. In trout, we found no differences between thermal stabilities of sulfation of either T4, T3 or rT3. Furthermore, sulfation of T4 and T3 were both inhibited to a similar extent by several analogs or inhibitors. Therefore, in contrast to the situation in mammals, trout hepatic sulfation of T4, T3 and possibly rT3 may be catalyzed by a single form of ST enzyme. One key difference between trout and mammalian sulfation of TH is iodothyronine substrate preference. Among three common TH substrates (T4, T3, rT3), rat hepatic sulfation showed a substrate preference of T3 \ rT3 \ T4 [10]. However, based on kinetic and inhibitor studies, rT3 was clearly the preferred substrate over T4 and T3 in trout. Thus, although mammalian STs may prefer iodothyronines containing less than two iodines in the outer-ring [13,14] this does not apply in trout. The different properties of trout and rat PSTs may reflect different roles of TH sulfation in these two vertebrate groups. In rats, sulfation of TH accelerates hepatic deiodination of most iodothyronines [13,14] and as a result, sulfated TH are not normally detected in rat bile [3]. The preferential sulfation of TH containing less than two outer-ring iodines favors subsequent deiodination of T3 or its derivatives, thereby salvaging iodine prior to its loss by excretion. In contrast, sulfation of TH prevents subsequent deiodination in trout liver (Finnson and Eales, unpublished data) and, probably as a consequence, sulfated forms of TH occur in bile [4] and urine [9]. Furthermore, trout hepatic sulfation prefers inactive rT3 over bioactive T3 as a substrate. Therefore, sulfation of TH in trout may not be important either in TH inactivation or in salvaging iodine by facilitating deiodination, but instead may enhance excretion by increasing water-solubility.
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Acknowledgements This study was supported by NSERC operating grant OGP1965 to JGE and by an NSERC postgraduate fellowship to KWF. The trout were kindly supplied by R. Olson and staff at the Rockwood Fish Hatchery, Department of Fisheries and Oceans, Canada.
References [1] Bradford M. A rapid and selective method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. [2] Burger A. Monodeiodinative pathways of thyroid hormone metabolism. In: Henneman G, editor. Thyroid Hormone Metabolism. New York: Marcell Dekker, 1986:255–76. [3] de Herder WW, Bonthuis F, Rutgers M, Otten MH, Hazenburg MP, Visser TJ. Effects of inhibition of type I iodothyronine deiodinase and phenol sulfotransferase on the biliary clearance of triiodothyronine in rats. Endocrinology 1988;122:153– 7. [4] Eales JG, Brown SB. Measurement and regulation of thyroidal status in teleost fish. Rev Fish Biol Fish 1993;3:299–347. [5] Finnson KW, Eales JG. Identification of thyroid hormone conjugates produced by isolated hepatocytes and excreted in bile of rainbow trout, Oncorhynchus mykiss. Gen Comp Endocrinol 1996;101:145 – 54.
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[6] Kaptein E, van Haasteren GAC, Linkels E, de Greef WJ, Visser TJ. Characterization of iodothyronine sulfotransferase activity in rat liver. Endocrinology 1997;138:5136– 43. [7] McNabb FMA. Thyroid Hormones. Englewood Cliffs, NJ: Prentice-Hall, 1992. [8] Osborn RH, Simpson TH. Thyroxine metabolism in the plaice, Pleuronectes platessa. Gen Comp Endocrinol 1969;13:524. [9] Parry JE, Zhang C, Eales JG. Urinary excretion of thyroid hormones in rainbow trout, Oncorhynchus mykiss. Gen Comp Endocrinol 1994;95:310 – 9. [10] Sekura RD, Kanji S, Cahnmann HJ, Robbins J, Jakoby W. Sulfate transfer of thyroid hormones and their analogs by hepatic aryl sulfotransferases. Endocrinology 1981;108:454–6. [11] Shields CA, Eales JG. Thyroxine 5%-monodeiodinase activity in hepatocytes of rainbow trout, Salmo gairdneri: distribution, effects of starvation, and exogenous inhibitors. Gen Comp Endocrinol 1986;63:334 – 43. [12] Visser TJ. Importance of deiodination and conjugation in the hepatic metabolism of thyroid hormone. In: Green MA, editor. The Thyroid Gland. New York: Raven Press, 1990:255–83. [13] Visser TJ. Role of sulfation in thyroid hormone metabolism. Chemico – Biol Interact 1994;92:293 – 303. [14] Visser TJ, van Buuren JCJ, Rutgers M, Eelkman Rooda SJ, de Herder WW. The role of sulfation in thyroid hormone metabolism. Trans Electron Microsc 1990;3:211 – 8. [15] Weinshilboum RM. Sulfate conjugation of neurotransmitters and drugs. Fed Proc 1986;45:2220 – 30. [16] Young WF, Gorman CM, Weinshilboum RM. Triiodothyronine: a stable substrate for the thermostable and thermolabile forms of human phenol sulfotransferases. Endocrinology 1988;122:1816– 24.
Sulfation of thyroid hormones by liver of rainbow trout, Oncorhynchus mykiss K.W. Finnson *, J.G. Eales Department of Zoology, Uni6ersity of Manitoba, Winnipeg, MB R3T 2N2, Canada Received 6 January 1998; received in revised form 1 April 1998; accepted 7 April 1998
Abstract We studied hepatic sulfation of thyroid hormones (TH) in rainbow trout, Oncorhynchus mykiss. Sulfation of thyroxine (T4) and 3,5,3%-triiodothyronine (T3) was detected in the cytosolic (63 – 67%), microsomal (12 – 16%), nuclear (12 – 14%) and mitochondrial/ lysosomal (7–8%) fractions. Using 3%-phosphoadenosine 5%-phosphosulfate (PAPS) as a sulfate donor, sulfation of T4 and T3 by the cytosolic fraction depended on protein concentration and time. The pH profiles for T4- and T3-sulfation were broad and overlapping with optimal pH values of about 6.5 and 7.0 U respectively. At pH 7.0, apparent Km (mM), Vmax (pmol/mg cytosolic protein per hour) and catalytic efficiency (Vmax/Km) values were 3,5%,3%-triiodothyronine (reverseT3, rT3) =0.7, 583 and 832; T4 = 1.7, 46 and 27; T3 =11.5, 840 and 73. Inhibitor profiles for both T4- and T3-sulfation were not significantly different with a common inhibitor preference of rT3 \pentachlorophenol\ triiodothyroacetic acid \ tetraiodothyroacetic acid T4 = T3 =3,5-diiodothyronine. T4-, T3- and rT3-sulfation activity decreased with increasing pre-incubation temperature (12, 24, 36°C); however, there were no significant differences in T4-, T3- and rT3-sulfation activity at each pre-incubation temperature. We conclude that: (i) in trout, hepatic sulfation of TH is enzymatic and obeys Michaelis – Menten kinetics; (ii) like mammalian hepatic sulfotransferases (STs), trout hepatic STs are heat-sensitive cytosolic proteins using PAPS as a sulfate donor; (iii) unlike mammalian sulfation of TH, trout hepatic sulfation of T4, T3 and rT3 may be catalyzed by a single form of ST preferring rT3 as substrate and with a catalytic efficiency of rT3 ZT3 \T4. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Oncorhynchus mykiss; Rainbow trout; Thyroid; 3%-phosphoadenosine 5%-phosphosulfate; Reverse triiodothyronine; Sulfation; Sulfotransferase; Thyroxine; Triiodothyronine
1. Introduction Deiodination and sulfation are interrelated pathways for thyroid hormone (TH) metabolism. Outer-ring deiodination (ORD) of thyroxine (T4) produces bioactive 3,5,3%-triiodothyronine (T3), while inner-ring deiodination (IRD) produces inactive 3,3%,5%-triiodothyronine (reverse T3, rT3). Further deiodination of these two types of T3 produces inactive iodothyronine metabolites [2]. These deiodinases are located in the endoplasmic reticulum of liver and other tissues, and their activities
* Corresponding author. Tel.: +1 204 4749245; fax: + 1 204 4747588; e-mail: [email protected] 0742-8413/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0742-8413(98)10015-4
can be measured in the microsomal fraction of the cell [11]. Sulfation of TH is catalyzed by phenol sulfotransferases (PSTs), a family of homologous enzymes located in the cytosolic fraction of liver and other tissues. PSTs have a broad and overlapping substrate specificity and use 3%-phosphoadenosine 5%-phosphosulfate (PAPS) as a sulfate donor [13,14]. In mammals, sulfation of the phenolic hydroxyl group of TH prevents ORD of T4, while it enhances IRD of both T4 and T3 [12–14]. Thus, since sulfation represents the initial step leading to irreversible deiodinative breakdown of TH, it is necessary to study this pathway to understand TH degradation pathways. Comparatively little is known about PSTs in nonmammalian species. However, Osborn and Simpson [8]
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have suggested the sulfation of TH in the marine plaice, Pleuronectes platessa. More recently, we have demonstrated the in vitro and in vivo sulfation of TH in the rainbow trout, Oncorhynchus mykiss [5]. In this study, our goal was to characterize hepatic sulfation of TH in trout.
2. Materials and methods
2.1. Animal maintenance Rainbow trout (250 – 500 g, 2 year old) were obtained from the Rockwood Hatchery, Balmoral, Manitoba, and held in the laboratory in flowing, aerated, dechlorinated water at 12°C, on a 12 h light:12 h dark photoperiod. Trout were fed a commercial diet (Martin Feed Mills, Elmira, Ontario, trout feed pellets, 3.2 mm diameter) 1× daily at a ration of 1–2% of body weight. Trout were used 18 – 24 h after their last meal.
2.2. Subcellular fractionation Trout were anesthetized in tricaine methanesulfonate (0.07 g/l) and killed by a blow to the head. Livers were removed and rinsed with ice-cold buffer: 0.25 M sucrose (pH 7.2) with 1 mM EDTA and 10 mM DTT. Livers were then added to 4 vol. (w/v) of buffer, minced with scissors and dispensed into a 55ml Wheaton homogenization chamber. Tissue was homogenized by two to three strokes of a motorized pestle (Tri-R Instruments, NY), filtered through two layers of cheesecloth and transferred to plastic tubes for centrifugation. The crude homogenate was centrifuged for 20 min at 730×g to separate the nuclear pellet. The post-nuclear supernatant was then centrifuged for 20 min at 25200×g to separate the crude mitochondrial/lysosomal pellet. Finally, the post-mitochondrial supernatant was centrifuged for 67 min at 110000×g to separate the microsomal pellet from the cytosol. Pellets (resuspended in 1 – 2 ml of buffer) and 1-ml aliquots of the crude homogenate and cell sap were stored at −76°C [11].
tor 3%-phosphoadenosine 5%-phosphosulfate (PAPS) (final concentration 20 mM). Control tubes received 20 ml of 0.1 M Tris buffer (pH 7.0) alone. This mixture was equilibrated in a shaking water bath for 10–15 min (12°C, 150 rpm). The reaction was started by adding 20 ml of either 125I-labeled T4, T3 or rT3 substrate (final concentration 1.0 mM; 100000 cpm). After 60 min, the reaction was stopped by adding 2 ml of chloroform/methanol (2:1 v/v), vortexed and centrifuged at 1420×g for 5 min. A volume of 250 ml was removed from the aqueous methanol layer and added to Sephadex LH-20 minicolumns containing 750 ml of 1.0 N HCl. Columns were swirled and drained, and iodide was eluted to waste with 3 ml of 0.1 N HCl. Sulfated TH conjugates were collected with 12 ml of H2O and counted in a gamma counter. Columns were washed with 3 ml of 0.1 N NH4OH in ethanol and stored with 0.1 N HCl [5]. We determined TH sulfate formation (H2O fraction) by subtracting the cpm of control tubes (− PAPS) from the cpm of experimental tubes (+PAPS). Protein concentration was determined by the Bradford method [1]. Depending on the experiment, ST activity was expressed as either (i) cpm (labeled TH sulfated)/h; (ii) nmol of TH sulfated/mg protein per hour; (iii) percentage of the control activity.
2.3. Sulfotransferase (ST) assay We based our assay on the procedure of Young et al. [16]. The various subcellular fractions were thawed on ice and diluted ( 1:25) with 0.1 M Tris buffer (pH 7.0) to achieve a final protein concentration of 0.2 – 0.8 mg/ml. We did not include BSA in our assay buffer because it decreased ST activity. A volume of 500 ml of this diluted subcellular fraction was added to siliconized test tubes containing 20 ml of the cofac-
Fig. 1. Sephadex LH-20 elution profile for synthetic 125I-labeled T3-sulfate. Profile was obtained by stepwise addition of 1-ml aliquots of 0.1 N HCl ( × 3), H2O ( ×12), and 0.1 N NH4OH in ethanol (1:1 v/v) (×3). Over 96% of the radioactivity was recovered in the H2O fraction.
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Fig. 2. Relationship between cytosolic protein concentration and amount of 125I-labeled T3 converted to T3-sulfate/h. Each point () represents the mean ( 9 SEM where it exceeds symbol size) of three separate incubations.
Fig. 3. The effect of incubation time on the amount of 125I-labeled T4 converted to T4-sulfate ( ) and 125I-labeled T3 converted to T3-sulfate () at intervals up to 1 h. Each point represents the mean of three separate incubations (SEM values did not exceed symbol size).
3. Results
3.3. Inhibitor studies
3.1. Assay conditions
Fig. 6 shows inhibition of both T4ST and T3ST activities by different inhibitors/analogs (100 nM). Inhibitor profiles for T4ST and T3ST were not signifi-
Fig. 1 shows the elution profile of synthetic 125I-labeled T3-sulfate. Over 96% of the radioactivity was recovered in the H2O fraction. Similar results were obtained using either 125I-labeled T4- or rT3-sulfate. Sulfation of T4 (T4ST) and T3 (T3ST) occurred mainly in the cytosolic fraction (63 – 67%). T4ST and T3ST activity was also detected in the microsomal (12 –16%), nuclear (12–14%) and mitochodrial/lysosomal fraction (7 – 8%) (data not shown). T3ST activity increased with cytosolic protein concentration up to 1.2 mg/ml (Fig. 2). For all remaining experiments, we used cytosolic preparations with protein concentrations ranging from 0.2–0.8 mg/ml. Both T4ST and T3ST activities increased linearly to 60 min (Fig. 3). The pH profiles for T4ST and T3ST activities were broad and overlapping with optimal pH values of about 6.5 and 7.0 U respectively (Fig. 4).
3.2. Enzyme kinetics We chose pH 7.0 U to compare kinetic properties of T4ST, T3ST and rT3ST. Fig. 5 shows Lineweaver –Burk plots for sulfation of (A) T4 (0.25 – 4.0 mM); (B) T3 (1.0 –16.0 mM); (C) rT3 (0.25 – 2.0 mM) using a PAPS concentration of 20 mM. Apparent Km (mM), Vmax (pmol/mg protein per hour) and Vmax/Km values were T4 = 1.7, 46 and 27; T3 =11.5, 840 and 73 and rT3 = 0.7, 583 and 832.
Fig. 4. The effect of pH on the sulfation of T4 ( ) and T3 (). Buffer pH was adjusted by adding 1.0 N HCl to 0.1 M Tris buffer prior to the experiments. Results are from different experiments carried out on the same cytosolic pool. Each point represents the mean ( 9SEM) of three separate incubations converted to percentage highest activity.
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Fig. 5. Lineweaver – Burk plots of hepatic cytosolic ST activities for (A) T4 (0.25 – 4.0 mM); (B) T3 (1.0 – 16.0 mM); (C) rT3 (0.25 – 2.0 mM) using a PAPS concentration of 20 mM. A different substrate range was used for (B) since higher concentrations of T3 were needed. Apparent Km (mM) and Vmax (pmol/mg protein per hour) values were T4 (1.7, 46), T3 (11.5, 840) and rT3 (0.7, 583). Each point represents the mean ( 9 SEM) of three separate incubations.
cantly different (P\0.05) with a common substrate preference of rT3 \pentachlorophenol (PCP)\ triiodothyroacetic acid (TRIAC)\tetraiodothyroacetic acid (TETRAC)\T4 =T3 =3,5,-diiodothyronine (3, 5T2). We did not carry out inhibitor studies using rT3 as substrate.
ST activities due to pre-incubation temperature (P\ 0.05)
3.4. Enzyme thermal stability
Mammalian sulfation of TH is catalyzed by multiple sulfotransferases (STs). These enzymes occur mainly in the cytosol of liver and other tissues and use PAPS as a sulfate donor [13,14]. In comparison, little is known about sulfation of TH in non-mammalian species. In this study, our main goal was to characterize hepatic sulfation of TH in trout.
Fig. 7 shows T4ST, T3ST and rT3ST activities following 15-min pre-incubation at either 12, 24 or 36°C. For each TH substrate, ST activity decreased with increasing pre-incubation temperature. However, there were no significant differences between T4ST, T3ST and rT3-
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Fig. 6. Inhibition of sulfation of T4 (open bars) and T3 (hatched bars) by various analogs/inhibitors (100 nM). Differences between the inhibition of T4- and T3-sulfation by the various inhibitors/analogues were not significant (P \ 0.05). ST activity is expressed as the percentage of mean control activity (no analog/inhibitor present).
Hepatic sulfation of TH in mammals and trout share some properties. Both are catalyzed by heat-sensitive cytosolic enzymes that use PAPS as a sulfate donor, with the reaction depending on protein concentration, time and pH and obeying Michaelis – Menten kinetics [13,14]. Furthermore, apparent Km values for both
Fig. 7. ST activities for T4 (open bars), T3 (hatched bars—up) and rT3 (hatched bars — down) substrates following a 15-min pre-incubation at 12 (control), 24 or 36°C. There were no significant differences between T4ST, T3ST or rT3ST activities at any of the pre-incubation temperatures (P\ 0.05). ST activity is expressed as a percentage of control activity.
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trout (0.7–11.5 mM) and rats (1.8–4.2 mM) [6] fall in a similar range. However, a different PAPS concentration (50 mM), TH (T2) substrate, pH (7.2) and incubation temperature (37°C) were used in the rat study making a direct comparison between trout and rat Km tentative. The optimal pH values reported for human hepatic sulfation of T3 and rat hepatic sulfation of T2 are 6.6 [15] and 8.0 U [6] respectively. In comparison, the optimal pH values for trout hepatic sulfation of T4 and T3 were about 6.5 and 7.0 U respectively. Although different optimal pH values for trout hepatic sulfation of T4 and T3 suggest more than one form of ST, these pH profiles were broad and overlapping. In addition, T4 and T3 have different pKa values (T4 = 6.73; T3 = 8.45) [7], which may influence enzyme–substrate interaction. Therefore, the existence of multiple STs cannot be established in trout on the basis of pH alone. In addition to pH, differences in enzyme thermal stability can be used to distinguish multiple enzyme forms [6]. In trout, we found no differences between thermal stabilities of sulfation of either T4, T3 or rT3. Furthermore, sulfation of T4 and T3 were both inhibited to a similar extent by several analogs or inhibitors. Therefore, in contrast to the situation in mammals, trout hepatic sulfation of T4, T3 and possibly rT3 may be catalyzed by a single form of ST enzyme. One key difference between trout and mammalian sulfation of TH is iodothyronine substrate preference. Among three common TH substrates (T4, T3, rT3), rat hepatic sulfation showed a substrate preference of T3 \ rT3 \ T4 [10]. However, based on kinetic and inhibitor studies, rT3 was clearly the preferred substrate over T4 and T3 in trout. Thus, although mammalian STs may prefer iodothyronines containing less than two iodines in the outer-ring [13,14] this does not apply in trout. The different properties of trout and rat PSTs may reflect different roles of TH sulfation in these two vertebrate groups. In rats, sulfation of TH accelerates hepatic deiodination of most iodothyronines [13,14] and as a result, sulfated TH are not normally detected in rat bile [3]. The preferential sulfation of TH containing less than two outer-ring iodines favors subsequent deiodination of T3 or its derivatives, thereby salvaging iodine prior to its loss by excretion. In contrast, sulfation of TH prevents subsequent deiodination in trout liver (Finnson and Eales, unpublished data) and, probably as a consequence, sulfated forms of TH occur in bile [4] and urine [9]. Furthermore, trout hepatic sulfation prefers inactive rT3 over bioactive T3 as a substrate. Therefore, sulfation of TH in trout may not be important either in TH inactivation or in salvaging iodine by facilitating deiodination, but instead may enhance excretion by increasing water-solubility.
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Acknowledgements This study was supported by NSERC operating grant OGP1965 to JGE and by an NSERC postgraduate fellowship to KWF. The trout were kindly supplied by R. Olson and staff at the Rockwood Fish Hatchery, Department of Fisheries and Oceans, Canada.
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