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Laboratory of Comparative Endocrinology (K.A.M., S.V.d.G., V.M.D., E.R.K.), Catholic University of Leuven, Naamsestraat 61, 3000 Leuven, Belgium; Department of Internal Medicine III (T.J.V.), Erasmus University Medical School, 3000 DR Rotterdam, The Netherlands
Address all correspondence and requests for reprints to: Prof. Dr. E. R. Kühn, Laboratory of Comparative Endocrinology, Naamsestraat 61, 3000 Leuven, Belgium. E-mail: vergendo{at}bio.kuleuven.ac.be
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In mammals, three types of enzymes catalyze the production and/or the
degradation of T3 (4). Type I iodothyronine deiodinase (D1)
possesses both ORD and IRD activity and is present in liver, kidney,
and thyroid. It is a high-Km (
10-6
M) enzyme that prefers rT3 over T4
and T3 as the substrate and is potently inhibited by
6-propyl-2-thiouracil (PTU), iodoacetic acid (IAc), and aurothioglucose
(ATG). Type II iodothyronine deiodinase (D2), which is found in
pituitary, brain, and brown adipose tissue, and in humans also in heart
and skeletal muscle (5, 6), demonstrates only ORD activity. It shows
low Km values (10-9 M), prefers
T4 over rT3 as the substrate and is not
inhibited by PTU. Type III iodothyronine deiodinase (D3) catalyzes only
IRD and prefers T3 over T4 as the substrate. It
is mainly localized in the central nervous system and the placenta and
shows intermediate Km values (10-8
nM). PTU has no effect on this enzyme, except in the
placenta at thiol concentrations below 1 mM (7). All these
enzymes are dependent on thiol cofactors such as dithiothreitol (DTT)
and are believed to be selenoenzymes (5, 6, 8, 9, 10, 11).
Knowledge of the biochemical characteristics of thyroid hormone-deiodinating enzymes in other vertebrates is limited compared with mammals. In chicken, three types of enzymes have been characterized with properties similar to the mammalian deiodinases (12, 13, 14, 15). In reptiles, scarce data are available, suggesting the presence of a D1 in liver, kidney, and pancreas (16, 17, 18). In amphibians, the existence of two deiodinases similar to mammalian D2 and D3, respectively, has been clearly demonstrated (9, 19, 20, 21, 22). In fish, deiodination has been studied extensively in salmonids by Eales and co-workers (23), but a systematic comparison with deiodinases in mammals to identify possible similarities is lacking.
Recently, preliminary data have indicated important similarities between ORD activities in the tropical fish Oreochromis niloticus and rat D1 and D2 (24). The purpose of the present study is to characterize both ORD- and IRD-catalyzing enzymes in another tilapian species, Oreochromis aureus, in comparison with the rat enzymes. Additionally we wanted to verify if the PTU insensitive D1-like enzyme is also present in other fish, besides Oreochromis niloticus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
[3',5'-125I]T4,
[3'-125I]T3 and
[3',5'-125I]rT3 were obtained from Amersham
(Amersham, UK); unlabeled T4, T3, and
rT3 from Henning Berlin GmbH (Berlin, Germany), PTU from
Janssens Pharmaceuticals (Beerse, Belgium); iodoacetic acid (IAc) and
aurothioglucose (ATG) from Sigma Chemical Co. (St. Louis, MO); and DTT
from Serva (Polylab, Antwerp, Belgium).
Preparation of microsomal fractions
Tissue was homogenized in 5 vol buffer A (0.25 M
sucrose, 10 mM HEPES, pH 7.0, 1 mM DTT) and
centrifuged for 20 min at 25,000 x g. The supernatant
was then centrifuged for 60 min at 100,000 x g. The
resulting pellet and fluffy upper layer were resuspended together in an
adequate volume of buffer B (0.1 M phosphate, pH 7.0, 2
mM EDTA, 1 mM DTT), snap-frozen in aliquots,
and stored at -80 C. All procedures were carried out on ice, whereas
centrifugation was done at 4 C. For comparison, microsomes were
prepared similarly from normal rat liver (D1), hypothyroid rat brain
(D2), and normal rat placenta (D3) (23). Protein concentrations were
determined with the BCA protein assay reagent (Bio-Rad, Nazareth,
Belgium) using BSA as a standard.
Deiodinase assays
Deiodinase activities were usually measured by incubation of an
adequate amount of microsomal protein (0.1–2 mg/ml depending on the
activity) for 1 h at 37 C with 0.1 µCi (0.25 nM)
[3',5'-125I]rT3 (plus 0.1 µM
unlabeled rT3; D1 assay),
[3',5'-125I]T4 (D2 assay) or
[3'-125I]T3 (D3 assay) and 15 (D1) or 25 (D2,
D3) mM DTT in 0.2 ml 0.1 M phosphate (pH 7.0)
and 2 mM EDTA. The radioactive iodothyronines were purified
by LH-20 chromatography (25) before each assay. Each incubation was
done in duplicate together with blanks, containing no protein, to
correct for nonenzymatic degradation of the tracer. Deiodination
products were analyzed using two methods. Release of radioiodide by ORD
of outer ring-labeled rT3 or T4 was estimated
after precipitation of protein-bound iodothyronines with
trichloroacetic acid (TCA), whereas iodothyronine products were
analyzed by HPLC (26). In the former case, the reaction was stopped by
successive addition of 100 µl 5% (wt/vol) BSA and 500 µl 10%
(wt/vol) TCA at 0 C. After centrifugation (3500 x g,
10 min), 500 µl of the supernatant was counted in a 
-counter
(Gammamaster, LKB). For HPLC analysis, the incubation was stopped by
addition of 300 µl ice-cold methanol. After centrifugation (3500
x g, 10 min), 200 µl supernatant were transferred to
vials containing 250 µl ammonium acetate buffer (0.02 M,
pH 4.0). Separation of the labeled iodothyronines and I-
was achieved by injection of 80 µl of the mixture on a C18 column
(ODS Hypersil 100 x 3.2 mm, Shandon, UK) and elution with a
45%/55% (vol/vol) mixture of methanol and ammonium acetate buffer
(0.02 M, pH 4.0) at a flow rate of 1 ml/min. Radioactivity
was assessed with an on line HPLC radioactivity monitor (LB506 C-1,
Berthold), and peaks were integrated by computer using the Winflow
program (JMBS, Grenoble, France). Deiodinase activity was expressed as
percentage substrate deiodinated or as the amount of substrate
deiodinated per minute per microgram of protein.
Experimental design
Initially all tissues of Oreochromis aureus were
screened for deiodinase activity using 0.1 µCi
[3',5'-125I]rT3 (+0.1 µM
unlabeled rT3), [3',5'-125I]T4 or
[3'-125I]T3 as substrate. The incubations
were stopped with methanol, and products were monitored by HPLC to
investigate if I- production from rT3 and
T4 was equivalent to 3,3'-T2 or T3
production, respectively. If significant deiodinase activities in the
different tissues were further characterized with respect to the
effects of substrate analogs (T4, T3,
rT3), inhibitors (PTU, IAc, and ATG), pH, DTT
concentration, and incubation temperature. In addition, the
Km and Vmax values of the different deiodinases
for their preferred substrate were determined using 5 substrate
concentrations in the range of saturation of the enzymes. All these
characterization experiments were carried out using the same microsomal
pool of each tissue, and, where appropriate, parallel incubations were
done with the corresponding rat deiodinase activities. The data
presented are the means of duplicate measurements from a representative
experiment that was repeated at least once with similar results.
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Results |
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). In these tissues, equivalent production of labeled
I- and 3,3'-T2 from
[3',5'-125I]rT3 was observed. Because the
brain represents only approximately 0.06% of the total body weight and
the activity was so low, brain rT3 ORD activity was not
further investigated. Iodide release from rT3 was also
observed in spleen and red blood cells, but this was not associated
with 3,3'-T2 formation. Similarly, formation of
I-, but not T3, from T4 was found
in these tissues (Fig. 1B), suggesting that I- release was
due to a nonenzymatic process.
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). Due to the low activity in this latter tissue,
testis T4 ORD activity could not be reliably characterized.
Significant IRD of T4 to rT3 was observed in
tilapia brain (Fig. 1B).
Active IRD of T3 to initially 3,3'-T2 and
subsequently 3'-T1 was observed in tilapia brain, whereas
some IRD of T3 to 3,3'-T2 was also seen in gill
and kidney (Fig. 1C). All other activities were close to the detection
limit of the assay and were not further examined.
No other degradation products of the various substrates than the iodothyronines mentioned above were detected after incubation with the different tissue microsomes.
Effects of substrate analogues
Figure 2A presents the effects of increasing
concentrations (0.01–10 µM) of unlabeled
rT3, T4, and T3 on the deiodination
of [125I]rT3 by rat liver and tilapia kidney
and liver microsomes. The results obtained with rat liver D1 and
tilapia kidney deiodinase were very similar, i.e. both were
only affected by micromolar concentrations of the iodothyronines, with
rT3 being more potent than T4 and
T3. Although saturation of the tilapia kidney deiodinase
required somewhat higher rT3 concentrations than rat D1,
these results suggest that rT3 ORD in tilapia kidney is
catalyzed by a high-Km, rT3-preferring, D1-like
enzyme. ORD of [125I]rT3 in tilapia liver was
already inhibited by >50% at the lowest concentration of the
unlabeled iodothyronine tested (0.01 µM); T4
was more potent than rT3, which again was much more potent
than T3. This indicates that rT3 ORD in tilapia
liver is catalyzed by a low-Km enzyme.
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). The
effects of T4 and T3 were identical in tilapia
liver and rat brain, whereas rT3 was slightly less
effective in inhibiting T4 ORD by the deiodinase in tilapia
liver than by D2 in rat brain. These results suggest that
T4 ORD in tilapia liver is catalyzed by a
low-Km, T4-preferring, D2-like enzyme.
Different dose-inhibition relationships were observed for the effects
of substrate analogs on T4 ORD in tilapia kidney.
Micromolar iodothyronine concentrations were required to inhibit
T4 ORD in tilapia kidney with potencies in the order
rT3 > T4 > T3, suggesting the
involvement of the D1-like enzyme.
IRD of [125I]T3 in tilapia brain and gill was
dose-dependently inhibited by increasing concentrations (1–1000
nM) of unlabeled iodothyronines, with potencies decreasing
in the order T3 > T4 > rT3 (Fig. 2C). Identical results were obtained in parallel incubations with rat
placenta (Fig. 2C), suggesting that IRD of T3 in both
tilapia brain and gill is catalyzed by a low-Km,
T3-preferring, D3-like enzyme. In contrast, T3
IRD in tilapia kidney is insensitive to nanomolar concentrations of the
different iodothyronines, suggesting that T3 IRD in this
tissue is probably mediated by the D1-like enzyme.
Based on these results, only rT3 ORD in kidney, T4 ORD in liver, and T3 IRD in brain and gill were subjected to further characterization.
Effects of inhibitors
Addition of 1–1000 mM PTU or IAc or 0.01–10
mM ATG-induced dose-dependent inhibitions of rat liver D1
but had much less effect on rT3 ORD by the tilapia kidney
deiodinase (Fig. 3A). The latter was completely
insensitive to 1 mM PTU, approximately 40-fold less
sensitive to IAc and approximately 10-fold less sensitive to ATG than
rat D1.
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shows the effects of increasing concentrations of PTU, IAc,
and ATG on T4 ORD in rat brain and tilapia liver. Both
enzymes showed low but similar sensitivities to inhibition by PTU and
IAc. However, T4 ORD was 
5-fold more sensitive to
inhibition by ATG in tilapia liver than in rat brain (Fig. 3B).
The effects of PTU, IAc, and ATG on T3 IRD in rat placenta
and tilapia brain and gill are demonstrated in Fig. 3C. These
deiodinase activities showed similar, low sensitivities to inhibition
by PTU and IAc, but the tilapia activities were approximately 10-fold
more sensitive to inhibition by ATG than rat placenta D3.
Optimal pH, DTT concentration, and temperature
ORD of rT3 in tilapia kidney was maximal between pH 6
and 7 (Fig. 4A) and at approximately 10 mM
DTT (Fig. 4B). Usually, substantial activity was already observed
without addition of DTT, i.e. in the presence only of the
low endogenous DTT concentration (0.025 mM) added with
the microsomes. rT3 ORD activity in tilapia kidney
gradually increased with incubation temperature from 4 to 37 C but
decreased significantly if the temperature was further increased to 45
C (Fig. 4C).
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) and reached a plateau at 30 mM DTT (Fig. 4B).
The optimal incubation temperature for the tilapia liver enzyme was 37
C (Fig. 4C).
The optimal pH for T3 IRD in tilapia brain ranged from 6 to
7, whereas the branchial enzyme showed optimal activity between pH 6.5
and 7 (Fig. 4A). Both enzyme activities reached a plateau at
approximately 20 mM DTT (Fig. 4B). The optimal incubation
temperature for T3 IRD in both tilapia brain and gill was
found to be 37 C (Fig. 4C).
Kinetic parameters
Table 1 presents the kinetic parameters for the
different deiodinase activities in rat and tilapia that were determined
in parallel. The Km values of the tilapia deiodinase
activities for their preferred substrates were in the same range as
those of the corresponding rat enzymes, although the apparent
Km for rT3 ORD by tilapia kidney was
approximately six times higher than the corresponding value in rat
liver. The Vmax value for rT3 ORD was much
higher in rat liver than in tilapia kidney, whereas the
Vmax value for T4 ORD was higher in tilapia
liver than in rat brain. The Vmax value for T3
IRD was similar in tilapia brain and rat placenta but much lower in
tilapia gill (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The tissue distribution of deiodinase activities in tilapia is clearly different from that in rats and humans, where D1 is located predominantly in liver, kidney, and thyroid, D2 in brain, pituitary, brown adipose tissue (rat), heart and skeletal muscle (human), and D3 in placenta and brain. In contrast, in tilapia the D2-like enzyme is expressed predominantly in liver and not in brain, while the D1-like enzyme is expressed predominantly in kidney and not in liver. Additionally, the present results differ in several respects from those obtained in salmonids (3, 28, 29, 30, 31). We observed no deiodination in tilapia muscle, insignificant T4 ORD in kidney, heart, brain, and gill, and negligible T3 IRD in liver.
Although the tilapia kidney enzyme and rat D1 display very similar affinities for the different iodothyronines, they exhibit strikingly different susceptibilities to inhibition by PTU, IAc, and ATG. At the time rat and human D1 were characterized as selenoproteins, it was proposed that the sensitivity to these inhibitors was related to the presence of selenocysteine (Sec) in the active site of the enzyme. Additional evidence for this hypothesis was provided by the loss of PTU and ATG sensitivity displayed by a D1 mutant in which Sec was replaced by Cys (8, 32, 33, 34). However, recently D2 and D3 have also been characterized in rat, man, and amphibians (5, 6, 9, 10, 11, 21, 22) as selenoproteins with a Sec residue in homologous domains of the proteins. Because these enzymes are much less sensitive to PTU, IAc, and ATG, these data indicate that not only the presence of Sec is responsible for the inhibitory effect of these inhibitors on D1 activity, but that they may also interact with additional residues in the proteins or that the Sec residues in these enzymes have widely different reactivities. More information on this deiodinase structure-PTU inhibition relationship was obtained by studying the dog D1. The latter is less sensitive to inhibition by PTU and gold and shows a lower affinity for rT3 and a lower turnover number for rT3 ORD than rat and human D1 (35, 36, 37). It has been hypothesized that the low sensitivity of dog D1 to PTU inhibition is related to its low turnover number, perhaps reflecting a lower nucleophilicity of Sec in this enzyme (38). Whether this also applies to the tilapia kidney deiodinase is unknown because the enzyme content in tilapia kidney microsomes and, thus, its turnover number remain to be determined. However, it should be realized that the relatively small decrease in PTU sensitivity of dog D1 is associated with a large increase in Km for rT3, whereas the PTU-insensitivity of the tilapia kidney enzyme is accompanied by only a slightly increased Km for rT3 compared with rat D1. The development of methods for the specific affinity-labeling of the tilapian kidney deiodinase and the recent cloning of its complementary DNA (39) should be very helpful in elucidating the reason for the PTU insensitivity of this enzyme. Because inhibition of D1 by PTU is competitive with DTT (4), the possible relationship between the PTU insensitivity and the low DTT requirement of tilapia D1 also needs further investigation.
The pH dependencies of the different tilapia deiodinases are only slightly different from those reported for the rat enzymes. These differences may be caused by variations in the protein structure or the dissociation constant of Sec in these enzymes and may, thus, be related to the different sensitivities to inhibitors. The optimal pH of 7 reported for trout liver T4 ORD (28, 40) agrees well with the value we obtained for the tilapia liver enzyme, but in our study there is no indication for a biphasic pH response of the kidney enzyme as demonstrated earlier for trout kidney T4 ORD activity (28, 41). Additionally, the pH optimum of T3 IRD activity in tilapia brain is slightly lower than that reported for trout brain (31).
The optimal incubation temperatures for the three tilapia deiodinases are similar to those of the mammalian enzymes, i.e. 37 C. This is surprising because tilapia is poikilothermic and does not survive temperatures above 35 C (42). The results suggest that at the optimal ambient temperature for tilapia (25–30 C) the deiodinases do not function maximally. However, our results do not exclude that on prolonged incubation denaturation of these proteins becomes more pronounced at temperatures above 30 C.
A major discrepancy between our results and those obtained by MacLatchy and Eales (28) in trout concerns their suggestion of two ORD activities in trout liver with low but slightly different Km values for T4. Based on the extensive kinetic data reported previously (24) and in this paper, we have no indications that a similar situation exists in tilapia.
There are important differences in the maximal velocities of the respective deiodinases in tilapia and rat tissues. Because of the high hepatic T4 ORD activity in tilapia and other fish as well as the weight of this tissue, the liver deiodinase may very well be the major source of circulating T3 in fish. Although T3 IRD activity is much lower in tilapia gill than in the brain, branchial T3 IRD could be more important in the regulation of plasma thyroid hormone concentrations since the gills weigh more and receive blood directly from the heart before it circulates through the rest of the body.
In conclusion, iodothyronine deiodinases in O. aureus are very similar to the corresponding enzymes in higher vertebrates, with exception of the D1-like deiodinase in tilapia kidney. This enzyme differs from rat D1 by its insensitivity for PTU and lesser susceptibility to inhibition by IAc and ATG. These observations suggest important differences in the active sites between these enzymes that only marginally affect their catalytic and substrate specificities. From the evolutionary point of view, it is interesting that at least some fish exhibit D1-like activity, whereas in the amphibians studied so far no expression of D1 could be found (18, 19, 20). These data suggest that, in amphibians, D1 activity was lost during evolution, because it is unlikely that fish and higher vertebrates independently developed D1-like enzymes with such a high grade of homology (39). Tilapia liver is an important source of D2, which makes it an excellent tissue to study the regulation of this enzyme. Also, the presence of D3 in gill is interesting, but its physiological importance remains to be determined.
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Acknowledgments |
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Received October 16, 1996.
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