Hepatic outer-ring deiodinase in a Mexican endemic lizard (Sceloporus grammicus).

Abstract

The kinetic characterization of the outer-ring deiodination pathway using rT(3) (rT(3)-ORD) in male, female, and pregnant female livers of an endemic lizard, Sceloporus grammicus, is reported. The ORD pathway does not have the characteristics of deiodinase type II; it is exclusively carried out by deiodinase type I (DI). DI enzymatic activity in lizard liver contains one of the highest activities reported in vertebrates. This activity is sexually dimorphic, with males presenting the highest activity during the reproductive season. The properties of this enzyme correspond to those described in mammals, such as specificity for rT(3), susceptibility to inhibition by 6-n-propyl-2-thiouracil and gold-thioglucose, cofactor requirement, and kinetic pattern. Unlike other vertebrates, the lizard DI exhibits conspicuous stability in the thermal range of 15 to 42 degrees C and in the pH range of 5.0 to 9.0. Male true kinetic constants exhibit a direct correlation with temperature. This is in agreement with short-term adaptation to microenvironmental changes and the feasible expression of enzymatic forms/variants which, together, endow this lizard species with a greater adaptation to natural daily ambient thermal fluctuations.

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Hepatic Outer-Ring Deiodinase in a Mexican Endemic
Lizard (Sceloporus grammicus)
Bertha Fenton and Carlos Valverde-R
Centro de Neurobiologı ´a, Campus UNAM-UAQ Juriquilla, Apartado Postal 1-1141, 76001 Quere ´taro, Qro., Me ´xico
Accepted September 15, 1999
The kinetic characterization of the outer-ring deiodin-
ation pathway using rT3(rT3-ORD) in male, female, and
pregnant female livers of an endemic lizard, Sceloporus
grammicus, is reported. The ORD pathway does not have
the characteristics of deiodinase type II; it is exclusively
carried out by deiodinase type I (DI). DI enzymatic
activity in lizard liver contains one of the highest
activities reported in vertebrates. This activity is sexu-
ally dimorphic, with males presenting the highest activ-
ity during the reproductive season. The properties of this
enzyme correspond to those described in mammals, such
as specificity for rT3, susceptibility to inhibition by
6-n-propyl-2-thiouracil and gold–thioglucose, cofactor
requirement, and kinetic pattern. Unlike other verte-
brates, the lizard DI exhibits conspicuous stability in the
thermal range of 15 to 42?C and in the pH range of 5.0 to
9.0. Male true kinetic constants exhibit a direct correla-
tion with temperature. This is in agreement with short-
term adaptation to microenvironmental changes and the
feasible expression of enzymatic forms/variants which,
together, endow this lizard species with a greater adapta-
tion to natural daily ambient thermal fluctuations.
?2000
Academic Press
Key Words: thyroid; deiodinase characterization; liver;
Mexican lizard; Sceloporus grammicus.
The fine regulation of thyroid hormone action depends
on the so-called extrathyroidal iodothyronine deiodin-
ation, which is catalyzed by a family of selenoenzymes
known as deiodinases (D). In addition to their distinct
catalytic properties, members of this enzyme family
are differentially expressed in both a tissue- and a
developmentally specific manner and are tightly regu-
lated by both pre- and posttranslational processes (St.
Germain, 1995; Ko ¨hrle et al., 1992). Iodothyronine
deiodination can occur in the outer or the inner ring of
the molecule (ORD and IRD, respectively), producing
either active or inactive iodothyronines. These two
different deiodinative routes are catalyzed by at least
three distinct types of iodothyronine deiodinases: type
I (DI) catalyzes both ORD and IRD pathways, type II
(DII) catalyzes exclusively the ORD pathway, and type
III (DIII) subserves the IRD or inactivating pathway
(Leonard and Visser, 1986). Although described in a
number of organs in different animal species, espe-
cially in endotherms (St. Germain et al., 1994; St.
Germain, 1994), information regarding thyroid hor-
mone-deiodinatingenzymesinectothermsandparticu-
larly in reptiles is scarce and incomplete. To our
knowledge, except for two reports published in ab-
stract form (Joss and John-Alder, 1989; Darras, 1994),
there are only three studies dealing with this subject in
reptiles. Kar and Chandola-Saklani (1985) analyzed the
in vivo T4-activation in lizards and Wong et al. (1993)
partially characterized total ORD activity in different
tissues in a snake species. Hugenberger and Licht
(1999) characterized the ORD pathway in liver and
kidney in a turtle (Trachemys scripta). Therefore, in the
present study we fully analyze and characterize the
hepatic rT3-ORD pathway in the endemic Mexican
lizard Sceloporus grammicus. The results show that
lizardrT3-ORDactivityiscatalyzedbyatypeIdeiodin-
ase, which exhibits conspicuous thermal stability in
General and Comparative Endocrinology 117, 77–88 (2000)
doi:10.1006/gcen.1999.7384, available online at http://www.idealibrary.com on
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broad temperature (15–42°C) and pH (5.0–9.0) ranges.
This latter finding strongly suggests that lizard DI
activity may be expressed as a family of enzymatic
forms or variants (allo- or isoenzymes), and it is
consonant with the adaptive compliance exhibited by
other enzyme systems in ectotherms (Somero, 1995;
Fields and Somero, 1997).
MATERIALS AND METHODS
Lizards
Adult male, female, and pregnant female lizards
(Sceloporus grammicus microlepidotus) were collected
from a location in Rio Frı ´o, Zoquiapan, state of Me ´xico
[Cb?(w2)(w), i.e., semi-cold climate (average annual
temperature: minimum 1.35°C, mean 9.78°C, maxi-
mum 18.23°C); Servicio Meteorolo ´gico Nacional, 1985;
Garcı ´a, 1988]. The study was approved by the Institu-
tional Animal Care Committee and was conducted
from April 1995 to December 1996. During this period,
four collections were carried out, each representative
of a different season: collection I, lateApril 1995 (males
sexually active); collection II, early September 1995
(female ovulation); collection III, early July 1996 (males
sexually active, female vitellogenesis begins); and col-
lection IV, late December 1996 (Guillette and Casas-
Andew, 1980; Me ´ndez de la Cruz and Cuellar, 1995).
Body weight and snout–vent length of each individual
were measured throughout the study [male: body
length, 4.9 ? 0.56 cm; weight, 4.29 ? 1.21 g (n ? 26);
female: body length, 4.6?0.51 cm; weight, 3.3?0.93 g
(n ? 26); pregnant female: body length, 4.76 ? 0.21 cm;
weight, 5.08 ? 0.136 g (n ? 26)]. Adult and reproduc-
tive stages were identified by body size, secondary
sexual characters, and gonad size (Me ´ndez de la Cruz
and Cuellar, 1995; Cuellar et al., 1995). Pregnancy was
identified by the presence in females of placentas and
embryos(Guillette,1982;StewartandBlackburn,1988).
Animals were cold anesthetized for 5 min at 4°C and
then decapitated. Collected livers were homogenized
in1:10(w/v)ice-coldHepesbuffer(Hepes10mmol/L,
sucrose 0.32 mmol/L, EDTA1.0 mmol/L, pH 7.0). The
homogenates were centrifuged at 1000g for 15 min.
The supernatant was separated into aliquots and
stored at ?70°C until assayed. For the enzymatic assay
(see below) and for each parameter tested, a pool of six
animals from each of the four collections was used.
Reagents
Nonradioactive iodothyronines were obtained from
Henning Co. (Berlin, Germany). Radiolabeled outer-
ring [125I]iodothyronines were purchased from New
England Nuclear (Boston, MA) (sp. act. rT31175 and T4
1200 µCi/µg). Dithiothreitol (DTT) was obtained from
Calbiochem (La Jolla, CA), 6-n-propyl-2-thiouracil
(PTU) was from U.S. Biochemical Co. (Cleveland, OH),
and gold–thioglucose (GTG) was from Sigma Chemi-
cal Co. (St. Louis, MO). Radiolabeled thyronines were
purified prior to use by means of a SEP-PACK C18
Cartridge from Millipore (Waters Chromatography,
MA, USA). Bradford reagents for protein determina-
tion were from Bio–Rad (Hercules, CA).
Deiodination Assay
Enzyme activities in pooled homogenates were mea-
sured in duplicate under varying conditions by a
modification of the radiolabeled iodide release method
(Leonard and Rosenberg, 1980). Hepatic lizard activity
was assayed by incubating (1 h at 20°C) aliquots of a
pool of six animal homogenates (15–20 µg protein).
The incubation mixture contained DTT at varying
concentrations (see below), 200 fmol of [125I]rT3 or
[125I]T4(approximately 100,000 cpm), and the corre-
sponding nonradioactive thyronine to complete a final
substrate concentration in the range of µmol/L for rT3
and nmol/L for T4. These ranges of thyronine concen-
tration were chosen to explore the presence of DI and
DII activity in lizard liver as has been reported in fish
(Orozco et al., 1997). The reaction was stopped by
adding 50 µl of a cold solution containing 50% normal
bovine serum and 10 mmol/L PTU and immediately
followed by 350 µl of 10% trichloroacetic acid. After
centrifugation (1000g for 10 min), the supernatant was
decanted onto a 1-ml Dowex-50X2 column equili-
brated in acetic acid. The acid-soluble radioactive
iodine product [125I-] of the deiodination was eluted
with 2 ml of 10% acetic acid (80% of [125I-] recovery)
and counted in a gamma spectrometer. In all cases, less
than 40% of the substrate was consumed during the
reaction. The minimum activity was at least twice that
of the released iodine in the control tubes, which was
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always less than 2%. The specific activity was calculated
using the formula 1/0.8 ? 2 ? (iodide counts?blank
iodide counts)/(total counts in assay tube) ? the final
substrate concentration used ? (1/h) ? (1/mg pro-
tein). This formula was derived on the basis of the
following considerations: (1) the assumption that all of
the iodide released, corrected for the blank, came from
enzymatic deiodination of the substrate; (2) 80% of the
final assay mixture was applied to the column; and (3)
the specific activity of the iodide released was one-half
that of the iodothyronines because the substrates rT3
and T4are randomly labeled with125I in the equivalent
3? and 5? positions of the phenolic ring (Pasos-Moura et
al., 1991). In this way, data are expressed in picomoles
125I released per milligram of protein per hour. Protein
content of homogenate serial dilutions was measured
by Bradford’s method (Bradford, 1976). Enzyme char-
acterizationincludedthefollowingparameters:homog-
enate protein concentration (0.01–0.6 mg/ml); sub-
strate (rT3) concentration for apparent Michaelis
constants (0.02–16 µmol/L) and for true Michaelis
constants (0.005–2.0 µmol/L); incubation time (0.5, 1,
2, and 3 h); pH (5.0–9.0); cofactor (DDT) concentration
(0.3–15 mmol/L); temperature (0, 4, 15, 20, 29, 37, 42,
and50°C);sensitivitytoinhibitors(PTU,0.5–5mmol/L;
GTG, 1–1000 nmol/L). Rat liver homogenates (male
Wistar 300 g) were also assessed in parallel.
Statistical Analyses
Results are presented as the mean ? SEM (n ? 6).
Statistically significant differences between male, fe-
male, and pregnant female groups and among the
assessed parameters were tested using two-way analy-
sis of variance (ANOVA) followed by the Tukey and
Duncan tests, and a P ? 0.01 was considered signifi-
cant. When comparing apparent and true Michaelis–
Menten constants, the Bonferroni test was used, and a
P ? 0.05 was considered significant.
RESULTS
Assay Validation and ORD Pathway Enzyme
Characterization
To establish valid assay conditions and to make
sound quantitative comparisons, several preliminary
experiments using either [125I]T4, to explore the pres-
ence of DII in lizard liver as has been reported in
rainbow trout (Orozco et al., 1997), or [125I]rT3were
conducted. These experiments were performed using
both lizard and rat liver homogenates, as well as brain
from 20-day-old chick embryos and rat placenta. For
the hepatic tissues, the parameters assessed included
enzyme (protein) concentration, incubation time, DTT
concentration, and temperature and pH dependency.
The deiodinative products of these preliminary ex-
periments were checked by descendent paper chroma-
tography as previously described (Fenton et al., 1997).
Averageresultsoftwoindependentexperiments(deio-
dinative percentage) using T4 (0.8 µmol/L, PTU 1
mmol/L) were the following:125I, 0%; T3, 0%; and rT3,
8.4%. The results of the absence of PTU were the
following:125I, 11.45%; T3, 3.2%; and rT3, 6.7%. When
using T3(0.3 µmol/L, PTU 1 mmol/L) the following
percentages were obtained:125I, 0; and 3,3?T2, 5.2%.
Without PTU, the results were the following:125I, 3.02;
and 3,3?T2, 4.97. These results suggest that lizard liver
expressed both the ORD and the IRD pathways.
Therefore, subsequent experiments were design to
further characterize rT3ORD in lizard liver.
Protein and Cofactor (DTT) Concentration
and Incubation Time
Using optimal assay conditions to examine T4-ORD
(DII) reported for this enzyme and confirmed at our
laboratory, lizard liver was devoid of this enzyme
activity, whereas positive controls, chick brain and rat
placenta, exhibited a significant DII activity (Fig. 1). In
contrast, when rT3-ORD was assessed, liver enzyme
activity was increased up to 30 µg of protein/tube in
both species (Fig. 2) and there was no statistical
difference (P ? 0.01) between sexes (with this protein
concentration). It should be noted that lizard liver
rT3-ORD activity is between two and four times higher
than that detected in rat. For subsequent assays, protein
concentration between 15 and 20 µg/tube was chosen for
bothspecies.Optimalratesofdeiodination(i.e.,forwhich
there is linearity in the reaction) were obtained during
1 h of incubation (data not shown), and for conve-
nience this time was fixed for the following assays.
As depicted in Fig. 3, when using rT3as substrate,
lizard and/or rat liver deiodinating activity was not
modified by DTT concentrations above 2.5 mmol/L,
Deiodination Pathway in Sceloporus grammicus
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and a typical hyperbolic saturation kinetic curve was
obtained (Leonard and Visser, 1986). Under these assay
conditions, a sexually dimorphic difference in lizards
was observed, with males presenting the highest enzy-
matic activity. To compare with data from the litera-
ture, 5 mmol/LDTT were used in subsequent assays.
Inhibitory Effects of PTU and GTG
In both lizards and rat, hepatic rT3-ORD activity was
reduced approximately 95–98%, using 1.0 mmol/L
and 1000 nmol/L of PTU (Fig. 4A) and GTG (Fig. 4B),
respectively, thus confirming the presence of DI in
lizard liver (Mol et al., 1984; Sun et al., 1997).
Effects of Temperature and pH
Lizard liver rT3-ORD activity was substantially
higher at 0°C than in rat, reached its maximum activity
at 15°C, and remained unchanged up to 42°C. In
contrast, in rat, the highest enzyme activity was ob-
served between 37 and 42°C (Fig. 5). This is congruent
FIG. 1.
T4, 50 nmol/L; DTT 20 mM; pH 7.0; 3 h at 20°C (lizards: male, female, and pregnant female) or 37°C (rat placenta and 20-day-old chick embryo); PTU 1
mmol/L; 15 to 20 µg of protein/tube. Different alphabetical superscripts indicate significant statistical differences (P ? 0.01) between the tissues.
ORDactivityinthehepatichomogenates:TypeIIORD(DII)activity.Resultsrepresentthemeans?SE.Assayconditionswere[125I]T4,200fmol;
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with the thermal adaptation type in each group, i.e.,
ectotherm and endotherm, respectively, for lizard and
rat.
In both species, hepatic rT3-ORD activity was pre-
sent at pH ranging from 5 to 9. However, lizard
enzymeactivityremainedpracticallyconstantthrough-
out the whole pH interval, males presenting a slight
increase in the range from 6.5 to 7.5. In contrast, in rat,
maximum activity was attained over a narrower pH
interval (6.5–7.5) and the enzyme was markedly inhib-
ited at both pH extremes (Fig. 6). Based on these
results, we decided to characterize hepatic lizard rT3
ORD using pH 7.0 and 20°C.
Kinetic Parameters
Apparent Michaelis–Menten constants for rT3(0.02
to 16 µmol/L) were assessed in pooled liver homoge-
nates from either sex according to the reproductive
stage of the lizard. The assay temperature was 20°C for
lizard and 37°C for rat. As summarized in Table 1,
there was a distinct sexual dimorphism in lizards, with
males presenting the highest enzymatic activity in the
reproductive season; in the nonreproductive season
there was no difference in females. Similarly, the lizard
liver rT3-ORD pathway exhibited approximately a
three- to four fold higher Kmvalue and a two- to
sevenfold higher Vmaxvalue than the rat.
FIG. 2.
different pools from adult animals (n ? 6 each collection) collected throughout 1.5 years (collections I to IV). Assay conditions for lizard and rat
liver were [125I]rT3, 200 fmol; rT3, 0.5 µmol/L; DTT 5 mmol/L; pH 7.0; 1 h at 20°C (lizards) or 37°C (rats). Different alphabetical superscripts
indicate significant statistical differences (P ? 0.01) between species and the different amounts of proteins.
Effect of protein concentration on the hepatic 5? DI activity. Results represent the means ? SE from six separate assays using four
Deiodination Pathway in Sceloporus grammicus
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Based on these results, true Michaelis–Menten con-
stants were assessed at three different temperatures
(15, 20, and 37°C), using liver homogenates from male
lizards collected during the nonreproductive season
(July to April). For these assays we used a wide range
of rT3(0.005–2.0 µmol/L), due to the fact that in fishes
two different enzyme systems catalyzing the liver
5?ORD pathway have been reported (Orozco et al.,
1997), and three different DTT concentrations (1.25, 2.5,
and 5.0 mmol/L). These results (Table 2) were calcu-
lated using replots of intercepts of the 1/Vmaxenzy-
matic activity as a function of reciprocal rT3and DTT
concentrations. While in the range of 15 to 20°C
enzyme activity remains constant, at the higher tem-
FIG. 3.
protein/tube. Different alphabetical superscripts indicate significant statistical differences (P ? 0.01) between species and the different
concentrations of the cofactor.
Effect of cofactor concentration. Assay conditions as well as tissues and data handling were as described for Fig. 2, using 15 to 20 µg of
FIG. 4.
indicated enzyme inhibitors. A 100% value corresponds to control incubations performed in the absence of inhibitors. Results are expressed as
the mean ? SE from six separate assays. Assay conditions as well as tissue and data handling were as described for Fig. 2, using 15 to 20 µg of
protein. Different alphabetical superscripts, obtained from the absolute values of 5?DI specific activity, indicate significant statistical differences
(P ? 0.01) between species and the different concentration of the inhibitors.
Sensitivity of DI activity to the inhibitory effects of (A) PTU and (B) GTG. Assays were performed in the absence or presence of the
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83
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perature tested (37°C), there is a significant increase
(twofold) of enzymatic catalytic efficiency.
DISCUSSION
This is the first study reporting the complete kinetic
characterization of the hepatic ORD pathway using rT3
in male, female, and pregnant endemic lizards (S.
grammicus). Results demonstrate that hepatic rT3-ORD
in the lizard is catalyzed by an enzyme whose opera-
tional characteristics, i.e., preferential substrate, cofac-
tor requirements, and susceptibility to inhibitors, are
similar to those reported for a typical deiodinase type I
in other vertebrates (Leonard and Visser, 1986; Galton,
1988; Kohrle, 1992; McNabb, 1992). Thus, the in vitro
substrate of lizard liver enzyme was rT3with Kmvalues
in the micromolar range. The enzyme reaction kinetics
corresponded to a typical ‘‘ping-pong’’ pattern (data
not shown), and its activity was inhibited by PTU
and/or GTG. In the presence of PTU (1 mmol/L)
lizard liver produces rT3form T4, and 3,3?T2from T3;
these results extend previous preliminary data regard-
ing the characterization of lizard skin DIII (Fenton and
Valverde, 1996).Aside from the differences in methods
FIG. 5.
handling were as described for Fig. 2, using 15 to 20 µg of protein. Different alphabetical superscripts indicate significant statistical differences
(P ? 0.01) between species and different temperatures.
Effect of temperature. Results are expressed as the mean ? SE from six separate assays. Assay conditions as well as tissue and data
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and the reptile species studied, our results are consis-
tent with the suggestion that total T4-ORD activity in
snake (Elaphe taeniura) liver (Wong et al., 1993) and
T4-ORD activity in turtle (Trachemys scripta) liver (Hu-
genberger and Licht, 1999) are catalyzed by a mamma-
lian-like type I deiodinase. Similarly, the present re-
sults demonstrating that lizard liver expresses one of
the highest DI activities documented in vertebrates
agree with those of a previous report (Darras et al.,
1994). The latter finding could be explained by the high
circulating thyronine (T4) levels in this genus, which
are twofold higher than those in rat (John-Alder and
Joos, 1991; Chopra, 1992).Altogether, these results add
further support to the notion that, excluding teleosts
(Orozco et al., 1997), the adult vertebrate hepatic
FIG. 6.
as described for Fig. 2, using 15 to 20 µg of protein. Different alphabetical superscripts indicate significant statistical differences (P ? 0.01)
between species and different pH.
Effect of pH. Results are expressed as the mean? SE from six separate assays.Assay conditions as well as tissue and data handling were
TABLE 1
Apparent Kinetic Constant of Lizard Liver 5?DI
Km
µmol/L
Vmax
pmol/mg
protein/min
Catalytic
efficiency
Vmax/Km
Malea
Male
Nonpregnant female
Pregnant female
Rats
0.79 ? 0.11 a
0.45 ? 0.03 b
0.63 ? 0.18 a
0.83 ? 0.3 a
0.17 ? 0.04 c
83.86 ? 2.64 a
37.87 ? 5.46 b
27.4 ? 7.7 b
35.4 ? 7.5 b
11.9 ? 1.5 c
106 a
84 a
43 b
42 b
71 c
Note. Results are the mean ? SE (n ? 6).Assay conditions: protein,
10–20 µg/tube; rT3, 0.02 to 1.6 µmol/L; *rT3, 200 fmol; DTT, 5
mmol/L; 1 h at 20°C for lizards and 37°C for rats. Letters represent
significant (P ? 0.05) differences between lizard sexes and between
the vertebrate species.
aMale at the reproductive season (collections I and III).
Deiodination Pathway in Sceloporus grammicus
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5?ORD pathway is exclusively catalyzed by a type I
deiodinase (McNabb, 1992; Mol et al., 1997).
The present study demonstrates a seasonal dimor-
phic increase in lizard liver DI activity (Table 1), in
which males present their highest activity during the
months of May to July, when this species is sexually
active (Guillette and Casas-Andreu, 1980). These re-
sults are in agreement with the well-known functional
interrelationship between gonadal and thyroidal gland
activities characteristic of monoestric reptiles (Lynn,
1970; Haldar-Misra and Thapliyal, 1981; John-Alder,
1984; Kar and Chandola-Saklani, 1985; Fleury et al.,
1987; Leatherland, 1987; Chandola-Saklani and Kar,
1990). Similarly, previous studies in rat have shown a
sex steroid-dependent liver DI dimorphism, attributed
to the regulation of the DI gene by testosterone (Harris
et al., 1979; Miyashita et al., 1995). Furthermore, present
results suggest a surge in lizard hepatic DI activity
during a critical time, i.e., the reproductive season.
This proposal is consistent with the idea that liver DI
plays a major role in supplying T3(Leonard and Visser,
1986) and supports the notion that the oldest or more
basic thyroid hormone functions are related to growth,
maturation, and differentiation (Johnston, 1997). The
absence of DII is not confined to liver (unpublished
data from our laboratory) and agrees with recent
reportsinturtle(HugenbergerandLicht,1999)suggest-
ing that DII may not be expressed in reptiles. The
physiological importance of this finding remains to be
assessed.
Amajor finding in the present study consisted of the
remarkable stability exhibited by hepatic lizard DI
throughout a wide pH and temperature range. These
results confirm and extend previous reports for total
T4-ORD activity in snakes (Wong et al., 1993) and they
are consistent with the natural thermal range that
reptiles encounter, as well as with their capacity to
thermoregulatebehaviorally,i.e.,basking(Avery,1976).
Moreover, these findings also agree with recent reports
regarding the thermal dependency of the deiodinase
systems in teleosts (Johnston and Eales, 1995; Fenton et
al., 1997; Orozco et al., 1997).
The pH–temperature dependence for several en-
zyme systems in ectotherms has been amply docu-
mented (Yancey and Somero, 1978; Hochachka and
Somero, 1984; Hazel, 1993; Somero, 1995). These stud-
ies have revealed the crucial role played by the proton-
ation state of the histidine (imidazole groups) within
the enzyme for its thermal activity compensation and
support the notion that environmental temperature
modulates the expression of thermally adapted enzy-
matic forms or variants (iso- or allozymes). In the
present study we assessed true Michaelis–Menten
constants at three different temperatures. Results are
congruent with the installment of an immediate ther-
mal compensation, in which Kmis directly correlated
with temperature (Hochachka and Somero, 1984) and
in which the dissociation constants of selenium–
cystein and histidine–imidazole groups are contribut-
ing to preserve the catalytic (kinetics) enzyme proper-
ties. However, these microenvironmental changes do
not fully explain the Kmdifferences observed at the
temperature extremes tested (15 and 37°C). Thus,
based on these data and the current literature regard-
ing the finely tuned regulatory mechanisms in ecto-
therms for coupling the effects of temperature changes
on in vivo biochemical processes (Hochachka and
Somero 1984; Hazel, 1993; Somero, 1995; Poly, 1997),
we propose that lizard liver DI activity may be ex-
pressed as a family of enzymatic forms or variants
(with nongenetic or genetic bases, respectively). We are
currently conducting a series of acclimation experi-
ments to further test this hypothesis.
In summary, the present results demonstrate that: (1)
this enzyme exhibits a conspicuous thermal stability in
the range of 15–42°C; (2) based on the rest of its
operational characteristics (specificity for rT3, cofactor
requirements, sensitivity to inhibitors, true and appar-
ent rT3Km), this enzyme resembles mammalian type I
TABLE 2
Male True Kinetic DI Constant Using Three Different Temperatures
°C
Km
µmol/L
Vmax
pmol/mg
protein/min
Catalytic
efficiency
Vmax/Km
rT3
15
20
37
15
20
37
0.78 ? 0.07 a
0.76 ? 0.16 a
0.29 ? 0.29 b
1.06 ? 1.15 a
1.00 ? 0.59 a
0.94 ? 1.17 a
26.7 ? 2.67 a
27.4 ? 3.03 a
18.97 ? 2.58 b
28.58 ? 2.67 a
30.41 ? 3.03 a
25.20 ? 5.76 a
33.96 a
35.99 a
65.41 b
26.96 a
30.41 a
26.80 a
DTT
Note. Results are the mean ? SE (n ? 3).Assay conditions: protein,
10–20 µg/tube; rT3, 0.005 to 2.0 µmol/L; *rT3, 200 fmol; DTT, 5
mmol/L; 1 h at 15, 20, or 37°C. Letters represent significant
(P ? 0.05) differences between temperatures.
86
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Page 11

5?D; and (3) lizard liver expresses one of the highest
rT3-ORD activities reported in vertebrates. These find-
ings strongly suggest the expression of enzymatic
forms/variants which endow this lizard species with a
greater adaptation to the daily thermal fluctuations of
its natural habitat.
ACKNOWLEDGMENTS
This work was supported in part by Grant DGAPA–PAPIIT, IN
203492, and IN 203996. B.F. received a doctoral fellowship from
DGAPA-UNAM. We thank Biol. Isabel Pe ´rez M. and Marcela
Sa ´nchez for their English revision, Dr. Carmen Aceves and Dr.
Alejandra Mainero for their critical comments, Dr. Fausto Me ´ndez
for his expert advice in lizard biology as well as for his critical
comments, M. en C. Felipe Rodrı ´guez Romero for his help in the
collection of lizards, and Dr. Luz Navarro for her expert statistical
advice.
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