Effect of coenzymes and thyroid hormones on the dual activitieswith aldehyde dehydrogenase activity Kiyoshi Yamauchi and Jun–ichiro Nakajima Department of Biology and Geoscience, Faculty
Trang 1Effect of coenzymes and thyroid hormones on the dual activities
with aldehyde dehydrogenase activity
Kiyoshi Yamauchi and Jun–ichiro Nakajima
Department of Biology and Geoscience, Faculty of Science, Shizuoka University, Shizuoka, Japan
A cytosolic thyroid-hormone-binding protein (xCTBP),
predominantly responsible for the major binding activity of
T3in the cytosol of Xenopus liver, has been shown to be
identical to aldehyde dehydrogenase class 1 (ALDH1)
[Yamauchi, K., Nakajima, J., Hayashi, H., Horiuchi, R &
Tata, J.R (1999) J Biol Chem 274, 8460–8469] Within this
paper we surveyed which signaling, and other, compounds
affect the thyroid hormone binding activity and aldehyde
dehydrogenase activity of recombinant Xenopus ALDH1
(xCTBP/xALDH1) while examining the relationship
between these two activities NAD+ and NADH (each
200 lM), and tw o steroids (20 lM), inhibit significantly the
T3-binding activity, while NADH and NADPH (each
200 lM), and iodothyronines (1 lM), inhibit the ALDH
activity Scatchard analysis and kinetic studies of xCTBP/
xALDH1 indicate that NAD+and T3are noncompetitive
inhibitors of thyroid-hormone-binding and ALDH
activit-ies, respectively These results indicate the formation of a ternary complex consisting of the protein, NAD+and thy-roid hormone Although the in vitro studies indicate that NAD+ and NADH markedly decrease T3-binding to xCTBP/xALDH1 at 10)4M, a concentration equal to the NAD content in various Xenopus tissues, photoaffinity-labeling of [125I]T3using cultured Xenopus cells demonstrates xCTBP/xALDH1 bound T3within living cells These results raise the possibility that an unknown factor(s) besides NAD+and NADH may modulate the thyroid-hormone-binding activity of xCTBP/xALDH1 In comparison, thy-roid hormone, at its physiological concentration, would poorly modulate the enzyme activity of xCTBP/xALDH1 Keywords: cytosolic thyroid-hormone-binding protein; aldehyde dehydrogenase; retinoic acid synthesis; Xenopus laevis
Hydrophobic molecules that signal via nuclear receptors,
such as thyroid and steroid hormones, retinoic acid and
vitamin D3, predominantly exist within plasma and within
intracellular compartments bound to specific proteins The
kinetics and the nature of the cellular responses to these
signaling molecules are determined by these specific binding
proteins This has been well documented for cytosolic
retinoic acid and retinol binding proteins where it has been
suggested that these binding proteins may act, not only as
buffers or reservoirs of intracellular retinoids to maintain
significant levels of free retinoids, but also as modulators
transporting retinoids to their target sites, the retinoid
responsive genes within the nucleus and the metabolic
enzymes within the cytoplasm [1–3] Although similar
functions have been assumed for cytosolic
thyroid-hor-mone-binding proteins (CTBPs), a unified viewregarding their function is yet to be decided due to their divergent molecular and hormone-binding characteristics [4–8] Recently, we purified a 59-kDa CTBP from adult Xenopus liver cytosol, xCTBP, which is responsible for most of the T3 binding activity within the Xenopus liver cytosol [9] Sequencing of the peptide, isolated after treatment of xCTBP with cyanogen bromide, revealed that xCTBP contained an amino-acid sequence similar to that of the mammalian and avian aldehyde dehydrogenases class 1 (ALDH1) [9] The possibility that xCTBP was Xenopus ALDH1 (xALDH1) was later confirmed by examining both the 3,3¢,5-triiodo-L-thyronine (T3) binding and the ALDH activities of the recombinant xALDH1 [10] The concen-trations of the 59-kDa xCTBP, investigated by photoaffin-ity-labeling with [125I]T3, in the liver and the intestinal cytosol increased gradually during the metamorphic climax stage [11] In adult Xenopus, a high level of the labeled protein was found in the cytosol from the liver and the kidney [11], although xCTBP/xALDH1 mRNA was found predominantly in the kidney and the intestine rather than in the liver [10] The restricted tissue-distribution of xCTBP/ xALDH1, particularly at the metamorphosing stages, raises the possibility that xCTBP/xALDH1 could modulate the actions of T3in a tissue-dependent manner By controlling the intracellular concentrations of free T3, xCTBP/ xALDH1 might play a critical role in regulating T3access
to its target sites within the nucleus and the cytoplasm [12] There have been several reports demonstrating interac-tions between mammalian ALDH1 and bioactive
Correspondence to K Yamauchi, Department of Biology and
Geoscience, Faculty of Science, Shizuoka University, 836 Oya,
Shizuoka 422-8529, Japan.
Fax: + 81 54 2380986, Tel.: + 81 54 2384777,
E-mail: sbkyama@ipc.shizuoka.ac.jp
Abbreviations: CTBP, cytosolic thyroid-hormone-binding protein;
xCTBP, Xenopus CTBP; ALDH1, aldehyde dehydrogenase class 1;
xALDH1, Xenopus ALDH1; T 3 , 3,3¢,5-triiodo- L -thyronine; T 4 ,
L -thyroxine; Triac, 3,3¢,5-triiodo- L -thyroacetic acid; MBC, maximum
binding capacity; IC 50 , the concentration of a chemical necessary to
inhibit an activity by 50%.
Enzymes: Xenopus aldehyde dehydrogenase class 1 (EC 1.2.1.3).
(Received 11 February 2002, accepted 20 March 2002)
Trang 2molecules, such as steroids [13–17], the polycyclic aromatic
compound benzo[a]pyrene [18,19], the anthracycline
antibi-otic daunorubicin, which has been used as one of the
effective agents for cancer chemotherapy [20], and the
synthetic flavone flavopiridol [21] Together with our
findings, it would appear that ALDH1 has acquired an
ability to bind these molecules during the evolution of
vertebrates [22] These observations have led us to suggest
that the above molecules might also bind to xCTBP/
xALDH1 as thyroid hormones do
In this report, we examine the effects of coenzymes and
several hydrophobic signaling molecules on T3-binding and
ALDH activities of xCTBP/xALDH1 We demonstrate
that NAD+, NADH and two steroids inhibit the
T3-binding activity of this protein, whereas NADH,
NADPH and iodothyronines inhibit the ALDH activity
Detailed studies revealed that NAD+and T3each act as a
noncompetitive inhibitor on the T3-binding and enzyme
activities of the protein, respectively
M A T E R I A L S A N D M E T H O D S
Materials
T3, D-T3,L-thyroxine (T4), 3,3¢,5-triiodo-L-thyroacetic acid
(Triac), all-trans-retinal, all-trans-retinoic acid,
androster-one, cortisandroster-one, 11-deoxycorticosterandroster-one,
dehydroisoandros-terone, 17-b estradiol, progesterone and testosterone were
purchased from Sigma NADP+, NADPH, NAD+,
NADH and disulfiram were obtained from Wako Pure
Chemicals Vitamin D3 (cholecalciferol) was purchased
from Nacalai Tesque [125I]T3(122 MBqÆlg)1; carrier free)
was from NEN Life Science Products AG 1-X8 resin was
from Bio-Rad Other reagents of molecular biology grade
were purchased from either Wako Pure Chemicals, Nacalai
Tesque or ICN Biomedicals
All steroids and retinal were dissolved in ethanol,
iodothyronines and the analogue Triac were dissolved in
dimethylsulfoxide, to give less < 1% (v/v) solvents Control
assays without the above compounds were performed in the
presence of the corresponding solvent at the same
concentration This dilution did not affect T3-binding and
ALDH activities in the assays described below
Expression of recombinant xCTBP/xALDH1
inEscherichia coli
E coli BL21 bearing an expression vector containing
xALDH1-I (pET15b/xALDH1-I) cDNA [10] was grown
and expression of the recombinant proteins was induced by
0.2 mM isopropyl thio-b-D-galactoside Purification of the
recombinant proteins was performed as described previously
[10] In brief, bacteria were collected by centrifugation at
1200 g for 30 min at 4C After resuspending in 0.3MNaCl,
50 mM Tris/HCl, pH 8.0, 10 mM imidazole, 1 mgÆmL)1
lysozyme, 1 mMbenzamidine hydrochloride, 1 mM
phenyl-methanesulfonyl fluoride and 50 mM 2-mercaptoethanol,
the cells were disrupted by sonication (UR200P type, Tomy,
Japan) for 10 s repeated three times The extract was
obtained by centrifugation at 105 000 g for 40 min at 4C
Recombinant proteins with a histidine tag were purified by a
nickel affinity column (ProBound Resin, Invitrogen, CA,
USA) The purified proteins were stored in 1 mMEDTA,
1 mMdithiothreitol and 10% glycerol at)85 C until further use Protein concentration was determined by the dye binding method with bovine c-globulin as the standard [23]
T3-Binding activity and photoaffinity-labeling Recombinant proteins were incubated in 250 lL of 20 mM
Tris/HCl, 1 mM dithiothreitol, pH 7.5, containing 0.1 nM
[125I]T3, in the presence or the absence of 5 lMunlabeled T3 for 30 min at 0C [125I]T3bound to proteins was separated from free [125I]T3 by the Dowex method [9] and radioac-tivity levels were measured in a c-counter (Auto Well Gamma System ARC-2000, Aloka, Japan) The amount of [125I]T3bound nonspecifically was obtained by measuring the radioactivity level within the samples incubated with
5 lM unlabeled T3 The nonspecific binding value was subtracted from the amount of total bound [125I]T3to give the values of specifically bound [125I]T3 Maximum binding capacity (MBC) and Kd values were calculated from Scatchard plots [24]
Photoaffinity-labeling with underivatized [125I]T3 was performed as described previously [9–11] Xenopus cell lines
KR and XL58, which were kindly provided by S Iwamuro (University of Toho, Japan) and R J Denver (University
of Michigan, MI, USA), respectively, were cultured according to the method of Smith & Tata [25] Xenopus cytosol was incubated with 0.5 nM[125I]T3for 0.5–1.0 h at
4C whereas the intact Xenopus cells were incubated with 0.5 nM [125I]T3 in 70% Leibovitz-L15 medium in the absence of fetal bovine serum for 0.5–1.0 h at 24C The cytosol, contained within a 0.5-mL Eppendorf tube, and the Xenopus cells, spread on a 35-mm plastic Petri dish, were placed on a UV crosslinker (CL-1000, Funakoshi Co., Japan), and exposed to UV light (254 nm, 40 W) for
3 min at 0C The resultant cytosolic proteins, and Xenopus cells, detached from the Petri dish with 0.05% trypsin, were mixed separately with an equal volume of
2· SDS-sample buffer, followed by boiling for 5 min The proteins were resolved by SDS/PAGE The affinity-labeled proteins were detected by autoradiography, exposed to X-ray XAR5 film (Kodak) on an intensifying screen at )85 C for 1–3 weeks
Aldehyde dehydrogenase activity Photometric assays were performed in triplicate in 400 lL
of 50 mM Tris/HCl, pH 8.0, 3.3 mM pyrazole, 100 mM
KCl, 1 mM dithiothreitol, 0.33 mM NAD+ and 30 lM
retinal, unless otherwise stated [10] The amount of retinoic acid formed, determined by the photometrical method, was similar to the result obtained from monitoring the absorb-ance at 340 nm by HPLC [26] Kinetic constants were determined under initial velocity conditions, which were linear with time and protein
Determination of NAD content The content of NAD (the sum of its reduced and oxidized forms) in Xenopus tissues was determined according to the method of Nisselbaum & Green [27] Rat liver cytosol was used as a control and its NAD content, determined within this report, was compared with those recorded in the literature [28] to validate this method
Trang 3Statistical analysis
Statistical significance between the control and the different
treatments was determined by Student’s t-test Differences
are considered significant at P < 0.05
R E S U L T S
Characterization of T3-binding activity of recombinant
xALDH1 protein
We obtained two, closely related cDNAs encoding ALDH1
from a Xenopus hepatic cDNA library Sequencing analysis
of the cDNAs, xALDH1-I and xALDH1-II, revealed that
xCTBP was more likely to be xALDH1-II rather than
xALDH1-I [10] Thus, we concentrated on binding studies
of xALDH1-II, termed xCTBP/xALDH1 [125I]T3binding
to recombinant xCTBP/xALDH1 was examined in the
presence of each compound listed in Table 1 Of three
iodothyronines and Triac, T3was the most potent
compet-itor of [125I]T3 binding The resulting affinity order of
T3‡ D-T3> T4> Triac, agreed with the order of their
relative binding affinity to xCTBP in the Xenopus cytosol
from adult and metamorphosing tadpole liver [9,11] At
pH 7.5, 50% inhibition of [125I]T3 binding to xCTBP/
xALDH1 was achieved with T3and D-T3at a concentration
of 18 nM, w ith T4 at 450 nM and with Triac at 15 lM
(Fig 1A)
ALDH1 catalyzes the formation of retinoic acid from retinal in the presence of NAD+ [29] We therefore examined the effects of the substrate (retinal), product (retinoic acid), coenzymes (NAD+ and NADH), related dinucleotides (NADP+and NADPH) and a typical inhib-itor of the enzyme (disulfiram) on [125I]T3 binding to xCTBP/ALDH1 NAD+and NADH, at a concentration
of 200 lM, inhibited [125I]T3 binding by more than 50% while retinal, at a concentration of 12 lM, activated [125I]T3
binding by 36%, although no significant difference was obtained The other compounds exhibited little effect on T3
binding (Table 1) The effect of NAD+is shown to be dose-dependent (Fig 1B) The concentration of NAD+ neces-sary to inhibit 50% of [125I]T3binding to xCTBP/xALDH1 (IC50) was 40 lM
As mammalian ALDH1 is known to bind steroids [13–17], we finally investigated the effects of seven steroids and cholecalciferol on T3 binding Progesterone was the most potent inhibitor of T3binding for xCTBP/xALDH1 (Table 1) Dose-dependence curves indicated that the IC50 for progesterone was 2.6 lM(Fig 1B)
To determine howNAD+and progesterone decreased the specific binding of [125I]T3 to xCTBP/xALDH1, we studied their effects in the presence of varying
concentra-Table 1 Effects of hydrophobic signaling molecules on 3,3¢,5-triiodo- L -thyronine (T 3 ) binding and retinoic acid formation (ALDH activity) of Xenopus class I aldehyde dehydrogenases (xALDH1) expressed in E coli T 3 -binding activity was examined by incubating the purified xALDH1 with 0.1 n M
[125I]T 3 for 30 min at 0 C, as described in Materials and methods Nonspecific binding was determined from the samples incubated in the presence
of 5 l M unlabeled T 3 and subtracted from the total binding The activity of the retinoic acid formation was examined by incubating the purified xALDH1 with 0.33 m M NAD + and 30 l M retinal for 1–2 min at 24 C [10] Data are mean ± SEM from at least triplicate determina-tions.*P < 0.05; **P < 0.01; ***P < 0.001.
L -3,3¢,5-Triiodothyroacetic acid 0.32 l M 95.5 ± 4.1
Trang 4tions of unlabeled T3 Scatchard plots indicated that a single
class of binding sites existed in xCTBP/xALDH1 (Fig 2)
NAD+, at a concentration of 200 lM, significantly
decreased the MBC from 338 ± 30 pmolÆmg)1 protein
(n¼ 5) to 178 ± 16 pmolÆmg)1protein (n¼ 3), although
there was no significant difference in Kdvalues between the
NAD+-treated and untreated samples, 66 ± 11 nM
(n¼ 3) vs 53 ± 5 nM (n¼ 5), respectively, as shown in
Fig 2 This result indicated that the inhibitory mode of
NAD+ was noncompetitive Progesterone, at 2 lM,
appeared to affect both the Kd (75 ± 2 nM, n¼ 3)
and MBC (310 ± 28 pmolÆmg)1 protein, n¼ 3) values,
although no significant differences were obtained for these
values when compared with the Kdand MBC values for the
untreated samples
Characterization of ALDH activity of recombinant
xCTBP/xALDH1
Formation of retinoic acid from retinal by xCTBP/
xALDH1 was examined in the presence of each compound
listed in Table 1 The reduced forms of dinucleotides, NADH and NADPH, as well as disulfiram, were powerful inhibitors for xCTBP/xALDH1, whereas retinoic acid slightly but significantly stimulated the enzyme activity Iodothyronines and Triac inhibited the enzyme activity IC50
for T3 was 700 nM (Fig 3) The narrowrange of the inhibitory concentration of T3indicates positive cooperati-vity The Hill coefficient was 2.4 (Fig 3, inset) All steroids listed in Table 1 showed little effect on the enzyme activity of xCTBP/ALDH1 at the concentrations investigated
Fig 2 Scatchard plot analysis of [125I]T 3 binding to xCTBP/xALDH1 Purified recombinant xCTBP/xALDH1 (10 lg/250 lL) was incubated with 0.1 n M [ 125 I]T 3 in the presence of various concentrations of unlabeled T 3 with (open symbols) or without (d) the effector: 200 l M
NAD + (s), 2 l M progesterone (h), for 30 min at 0 C Nonspecific binding was subtracted from total binding Each value is the mean of triplicate determinations This experiment was repeated at least three times.
Fig 3 Effect of T 3 on retinoic acid synthesis from retinal, catalyzed by xCTBP/xALDH1 ALDH activity was measured as the rate of retinoic acid synthesis The reaction was performed at 24 C w ith 5 lg of xCTBP/xALDH1 in the presence of various concentrations of T 3 The inset illustrates the Hill plot, log[v c /v i )1] vs the logarithm of T 3 molar concentration, the slope of which yields the Hill coefficient v c and v i are velocities calculated in the absence and presence of various concen-trations of T 3 The Hill coefficient, h, w as 2.4 Each value is the mean
± SEM of triplicate determinations.
Fig 1 Inhibition of [125I]T 3 binding to xCTBP/xALDH1 with various
hydrophobic signaling molecules Purified recombinant xCTBP/
xALDH1 (10 lg/250 lL) was incubated with 0.1 n M [125I]T 3 in the
presence or absence (control) of the following compounds, at various
concentrations for 30 min at 0 C In (A), T 3 (s), D-T 3 (d), T 4 (h) or
Triac (n) was added, whereas, in (B), progesterone (s) or NAD+(d)
was added Nonspecific binding was subtracted from total binding to
give values for specific binding Each value is the mean ± SEM of
triplicate determinations.
Trang 5To determine howthyroid hormones interact with
xCTBP/xALDH1, resulting in the decrease in the formation
of retinoic acid from retinal, kinetics of the inhibition of
xCTBP/xALDH1 by T3 was examined by variation of
NAD+concentration within the reaction mixture The Km
value, 9 lM, was independent of the concentration of T3,
but the Vmax value decreased from 0.18 to 0.08 lmolÆ
min)1Æmg)1with increasing concentrations of T3(Fig 4)
The Ki was 0.28 lM and 0.31 lM, calculated in tw o
independent experiments Next, kinetics of the inhibition
of xCTBP/xALDH1 by T3 were examined when retinal
concentration was varied in the reaction mixture As shown
previously [10], positive cooperativity with allosteric kinetics
was detected (Fig 5) The apparent K1/2 value did not
change in the incubations with and without T3(2.8 ± 0.3
vs 2.6 ± 0.1 lM, n¼ 6), but the Vmaxvalue decreased by
64% when 5 lMT3was added to the reaction mixture The
Hill coefficient did not change significantly in incubations
with and without 5 lMT3, 2.3 ± 0.1 vs 2.2 ± 0.1 (Fig 5,
inset) These results indicated that T3acts as a
noncompet-itive inhibitor against both NAD+and retinal upon the
enzyme activity of xCTBP/xALDH1
T3binding to xCTBP/xALDH1 in intactXenopus cells
The present studies on the dual activities of xCTBP/
xALDH1 have indicated that NAD+ is required at
concentrations of 10-5)10-4
M for expression of ALDH activity, whereas 10)4M of NAD+ or NADH
pro-foundly inhibits the T3-binding activity However, we have
no information regarding NAD+, NADH or NAD (the
sum of NAD+and NADH) content within Xenopus tissues,
although NAD content in rat liver is known to be
0.7–0.9 lmolÆ(g fresh weight))1 [27,28] As both NAD+
and NADH showed similar inhibitory effects on T3-binding
to xCTBP/xALDH1 (Table 1), we assumed that the sum
of NAD+ and NADH is important for evaluating the
inhibitory effect NAD content within rat liver was
756 ± 49 lmolÆ(kg fresh weight))1(n¼ 3), which agreed with values reported previously [27,28] On the other hand, Xenopusliver had a lowNAD content, 201 ± 23 lmolÆ(kg fresh weight))1(n¼ 6), less than one third of that in rat liver (Table 2) There were no significant differences in NAD contents among various Xenopus tissues Next, T3-binding activity of xCTBP/xALDH1 was directly examined by photoaffinity-labeling using intact Xenopus cells Analyses
of the cytosol obtained from the cell lines (KR and XL58) and the adult liver revealed the presence of single labeled 59-kDa xCTBP (lanes 1–3 in Fig 6) Photoaffinity-labeling
of [125I]T3 using intact KR and XL58 cells revealed, via autoradiography, a labeled protein band of the same size (lanes 4 and 5 in Fig 6), demonstrating that xCTBP/ xALDH1 is capable of binding T3within the Xenopus cells
Fig 4 Kinetics of the inhibition of xCTBP/xALDH1 by T 3 when
NAD+ concentration was varied within the reaction mixture The
reaction was performed at 24 C w ith 5 lg of xCTBP/xALDH1 The
concentration of retinal was 30 l M and the concentrations of T 3 were 0
(d), 0.4 (e), 0.6 (n), 0.8 (h) and 1 l M (s).The buffer used was 50 m M
Tris/HCl, pH 8.0 Each value is the mean of triplicate determinations.
This experiment was repeated twice, each with similar results.
Fig 5 Kinetics of the inhibition of xCTBP/xALDH1 by T 3 when ret-inal concentration was varied within the reaction mixture The reaction was performed at 24 C w ith 5 lg of xCTBP/xALDH1 The concen-tration of NAD + was 0.33 m M and the concentrations of T 3 was 0 (s), or 5 l M (d) The buffer used was 50 m M Tris/HCl, pH 8.0 The inset depicts the Hill plots Each value is the mean of triplicate deter-minations SEMs, which were less than the size of symbols, are not shown This experiment was repeated six times, each with similar results.
Table 2 Contents of NAD in rat liver and various Xenopus tissues Data are expressed as the mean ± SEM (number of samples) NAD content
is the sum of the oxidizaed and reduced forms.
Species/tissue NAD (lmolÆ kg wet weight)1) Rat
Xenopus
Skeletal muscle 199 ± 37 (3)
Trang 6D I S C U S S I O N
The present work was undertaken with the aim of
deter-mining which signaling molecules, and other molecules,
affected the T3-binding and ALDH activities of xCTBP/
xALDH1 We have obtained evidence that the
[125I]T3-binding activity of xCTBP/xALDH1 was markedly
inhibited by NAD+, NADH, progesterone and
11-deoxy-corticosterone, as well as iodothyronines and Triac, but not
by NADP+, NADPH, disulfiram and retinal On the other
hand, the ALDH activity was inhibited by NADH,
NADPH, disulfiram, iodothyronines and Triac, but not
by any of the steroids tested We initially expected xCTBP/
xALDH1 to be one of the target sites for endocrine
disrupting chemicals, because amphibian malformations
found in field studies were very similar to those found in
individuals experimentally treated with retinoids [30]
However, treatment with bisphenol A, nonylphenol,
octyl-phenol, and benzo[a]pyrene had little effect on ALDH
activity of xCTBP/xALDH1 (data not shown) NADH was
the only compound to affect both the thyroid hormone
binding and enzymatic activities of xCTBP/xALDH1,
suggesting that the binding of a compound to xCTBP/
xALDH1 will not necessarily inhibit both activities A
similar result was observed for flavopiridol [21] Its binding
to human ALDH1 did not affect the enzyme activity of
ALDH1 Study of the interaction of ALDH1 with bioactive
molecules revealed that the mammalian enzymes have a
significant affinity for thyroid hormone [31], progesterone,
deoxycorticosterone, diethylstilbestrol,
dehydroepiandros-terone [13,14,32], dihydroandrosterone, 17,b-estradiol,
hydrocortisone [15–17] and benzo[a]pyrene [18,19] As the
binding of the first three compounds to xALDH1 was also
witnessed in the present study (Table 1), the ability of
ALDH1 to bind the compounds appears to have occurred
at an early step during vertebrate evolution
Detailed studies revealed that NAD+noncompetitively
inhibited the T3-binding activity of xCTBP/ALDH1
whereas T3inhibited the ALDH activity in a
noncompet-itive fashion against both NAD+and retinal These results
suggested the formation of a ternary complex consisting of
xCTBP/xALDH1, NAD+and T3 For human
mitochon-drial and cytoplasmic ALDHs, T3and Triac were
compet-itive inhibitors against NAD+and uncompetitive inhibitors
against propionaldehyde [31] These distinct inhibitory
modes might reflect the differences of the iodothyronine
binding pocket within xALDH1 and mammalian ALDHs
The inhibitory interactions of NAD+upon T3binding to
xCTBP/xALDH1 and of T upon its enzyme activity must
occur in a more complex fashion Binding studies demon-strated that xCTBP/xALDH1 had a high affinity for T3, with a Kd of 53 nM(Fig 2), whereas the Kivalue for T3 against NAD+on ALDH activity was 0.3 lM(Fig 4) We can not precisely determine why there was a difference between the calculated Kdand Kivalues It may be possible that xCTBP/ALDH1 forms different conformations when bound to NAD+and/or T3, This possibility is considered due to the presence of positive cooperativity upon ALDH activity (the Hill coefficient, h¼ 2.2) when the concentra-tion of retinal was varied (Fig 5) and the presence of positive cooperativity upon the inhibition of ALDH activity (h¼ 2.4) when the concentration of T3was varied (Fig 3)
T3may be a selective, allosteric inhibitor of the xALDH1 enzyme Such an allosteric conformational change was proposed for human alcohol dehydrogenase when bound to testosterone, where testosterone acts as a noncompetitive inhibitor with respect to ethanol and NAD+[33] Alter-natively, it is possible that thyroid hormone alters the equilibrium between the tetramer and dimer conformations
or between the dimer and monomer conformations of xCTBP/ALDH1, as found in glutamate dehydrogenase, where T4 and T3induce dissociation [34] To explore the second possibility, the hepatic xCTBP/xALDH1, in the presence or absence of 5 lMT3, were subjected to centrif-ugation in a glycerol density gradient However, tetrameric xCTBP/xALDH1 was not found to dissociate into its dimer
or monomer forms (data not shown) Thus, the second possibility is unlikely to occur in xCTBP/xALDH1 There are many reports of the inhibitory effects of thyroid hormones upon the activity of several dehydrogenases: pig heart malic dehydrogenase [34], beef liver glutamic dehy-drogenase [34–36], pig heart malate dehydehy-drogenase [37], horse and human alcohol dehydrogenases [38–40] and human aldehyde dehydrogenases [31] These observations raise the possibility of the presence of a dehydrogenase-specific binding site for thyroid hormone In ALDH1, the binding sites for NAD+/NADH and retinal reside in the N-terminal region, termed the NAD-binding domain, and
in the C-terminal region, termed the catalytic domain, respectively [41] We found previously that the thyroid-hormone-binding site is located in the NAD-binding domain of xCTBP/xALDH1 [10] Zhou & Weiner [31] reached the same result by eluting human ALDHs bound to AMP-affinity column with T3 or Triac These results support the possibility of a dehydrogenase-specific binding site for thyroid hormone as the coenzyme-binding domains within dehydrogenases have a relatively conserved ternary structure [42] when compared to their catalytic domains However, Ki values for thyroid hormone binding to all dehydrogenases, including those calculated for xCTBP/ xALDH1, were in the 10-7)10-4
Mrange These are high concentrations, even if the local distribution or accumula-tion of intracellular thyroid hormones was considered The present studies demonstrate that xCTBP/xALDH1 can bind T3 in intact cells (Fig 6) However, the NAD content corresponding to 0.2 mM concentration would restrict T3-binding activity of xCTBP/xALDH1 within the Xenopuscells compared to the binding activity witnessed
in vitro It should be noted that retinal, at a concentration of
12 lM, activated the T3-binding activity by 36%, although
no significant difference was obtained In the previous studies, the affinity-labeled xCTBP/xALDH1 was found at
Fig 6 Photoaffinity-labeling of xCTBP/xALDH1 in Xenopus cells.
Xenopus cytosol from KR cells (lane 1), XL58 cells (lane 2) and adult
liver (lane 3), and the intact KR (lane 4) and XL58 (lane 5) cells were
photoaffinity-labeled with 0.5 n M [ 125 I]T 3 The resultant proteins were
analysed on a 10% SDS/PAGE, followed by autoradiography.
Trang 7a higher level in the liver cytosol than in the kidney cytosol
[11], whereas xCTBP/xALDH1 mRNA was found more
predominantly in the kidney than in the liver [10] Therefore,
it is possible that T3 binding to xCTBP/xALDH1 might
be under the control of an unknown factor(s) besides
coenzymes within the cells, while poorly influencing its
ALDH activity
A C K N O W L E D G E M E N T S
We would like to thank Mr Takashi Honda for the preparation of
recombinant xCTBP/xALDH1 We also wish to thank Drs S Iwamuro
and R J Denver for providing the Xenopus cell lines This work was
supported by Grant-in-Aid for Scientific Research (B) from the Japan
Society for the promotion of Science (no 13559001).
R E F E R E N C E S
1 Chanbon, P (1995) The molecular and genetic dissection of
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