Effect of coenzymes and thyroid hormones on the dual activities of Xenopus cytosolic thyroid-hormone-binding protein (xCTBP) 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 T 3 in 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 l M ),andtwosteroids(20l M ), inhibit significantly the T 3 -binding activity, while NADH and NADPH (each 200 l M ), and iodothyronines (1 l M ), inhibit the ALDH activity. Scatchard analysis and kinetic studies of xCTBP/ xALDH1 indicate that NAD + and T 3 are 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 T 3 -binding to xCTBP/xALDH1 at  10 )4 M , a concentration equal to the NAD content in various Xenopus tissues, photoaffinity- labeling of [ 125 I]T 3 using cultured Xenopus cells demonstrates xCTBP/xALDH1 bound T 3 within 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 D 3 , 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 view regarding 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 T 3 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 (T 3 ) binding and the ALDH activities of the recombinant xALDH1 [10]. The concen- trations of the 59-kDa xCTBP, investigated by photoaffin- ity-labeling with [ 125 I]T 3 , 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 T 3 in a tissue-dependent manner. By controlling the intracellular concentrations of free T 3 , xCTBP/ xALDH1 might play a critical role in regulating T 3 access 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) Eur. J. Biochem. 269, 2257–2264 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02891.x molecules, 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 T 3 -binding and ALDH activities of xCTBP/xALDH1. We demonstrate that NAD + , NADH and two steroids inhibit the T 3 -binding activity of this protein, whereas NADH, NADPH and iodothyronines inhibit the ALDH activity. Detailed studies revealed that NAD + and T 3 each act as a noncompetitive inhibitor on the T 3 -binding and enzyme activities of the protein, respectively. MATERIALS AND METHODS Materials T 3 ,D-T 3 , L -thyroxine (T 4 ), 3,3¢,5-triiodo- L -thyroacetic acid (Triac), all-trans-retinal, all-trans-retinoic acid, androster- one, cortisone, 11-deoxycorticosterone, 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 D 3 (cholecalciferol) was purchased from Nacalai Tesque. [ 125 I]T 3 (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 T 3 -binding and ALDH activities in the assays described below. Expression of recombinant xCTBP/xALDH1 in Escherichia 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 m M 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 at4 °C. After resuspending in 0.3 M NaCl, 50 m M Tris/HCl, pH 8.0, 10 m M imidazole, 1 mgÆmL )1 lysozyme, 1 m M benzamidine hydrochloride, 1 m M phenyl- methanesulfonyl fluoride and 50 m M 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 4 °C. 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 m M EDTA, 1m M dithiothreitol 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]. T 3 -Binding activity and photoaffinity-labeling Recombinant proteins were incubated in 250 lLof20m M Tris/HCl, 1 m M dithiothreitol, pH 7.5, containing 0.1 n M [ 125 I]T 3 , in the presence or the absence of 5 l M unlabeled T 3 for 30 min at 0 °C. [ 125 I]T 3 bound to proteins was separated from free [ 125 I]T 3 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 [ 125 I]T 3 bound nonspecifically was obtained by measuring the radioactivity level within the samples incubated with 5 l M unlabeled T 3 . The nonspecific binding value was subtracted from the amount of total bound [ 125 I]T 3 to give the values of specifically bound [ 125 I]T 3 . Maximum binding capacity (MBC) and K d values were calculated from Scatchard plots [24]. Photoaffinity-labeling with underivatized [ 125 I]T 3 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 n M [ 125 I]T 3 for 0.5–1.0 h at 4 °C whereas the intact Xenopus cells were incubated with 0.5 n M [ 125 I]T 3 in 70% Leibovitz-L15 medium in the absence of fetal bovine serum for 0.5–1.0 h at 24 °C. 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 3min at 0°C. 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 m M Tris/HCl, pH 8.0, 3.3 m M pyrazole, 100 m M KCl, 1 m M dithiothreitol, 0.33 m M NAD + and 30 l M 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. 2258 K. Yamauchi and J. Nakajima (Eur. J. Biochem. 269) Ó FEBS 2002 Statistical 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. RESULTS Characterization of T 3 -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. [ 125 I]T 3 binding to recombinant xCTBP/xALDH1 was examined in the presence of each compound listed in Table 1. Of three iodothyronines and Triac, T 3 was the most potent compet- itor of [ 125 I]T 3 binding. The resulting affinity order of T 3 ‡ D-T 3 >T 4 > 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 [ 125 I]T 3 binding to xCTBP/ xALDH1 was achieved with T 3 and D-T 3 at a concentration of 18 n M ,withT 4 at 450 n M and with Triac at 15 l M (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 [ 125 I]T 3 binding to xCTBP/ALDH1. NAD + and NADH, at a concentration of 200 l M , inhibited [ 125 I]T 3 binding by more than 50% while retinal, at a concentration of 12 l M , activated [ 125 I]T 3 binding by 36%, although no significant difference was obtained. The other compounds exhibited little effect on T 3 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 [ 125 I]T 3 binding to xCTBP/xALDH1 (IC 50 ) was 40 l M . As mammalian ALDH1 is known to bind steroids [13–17], we finally investigated the effects of seven steroids and cholecalciferol on T 3 binding. Progesterone was the most potent inhibitor of T 3 binding for xCTBP/xALDH1 (Table 1). Dose-dependence curves indicated that the IC 50 for progesterone was 2.6 l M (Fig. 1B). To determine how NAD + and progesterone decreased the specific binding of [ 125 I]T 3 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 Xen opus 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 [ 125 I]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. Effector Concentration T 3 -binding activity ALDH activity Control 100 ± 6 100 ± 2 Retinoic acid 12 l M 99.3 ± 5.4 133 ± 4** NAD + 200 l M 22.1 ± 3.0*** NADH 200 l M 18.1 ± 2.7*** 38.1 ± 1.9*** NADP + 200 l M 121 ± 10 108 ± 5 NADPH 200 l M 112 ± 7 20.7 ± 2.6*** Control 100 ± 3 100 ± 5 Retinal 12 l M 136 ± 16 Disulfiram 200 l M 87.5 ± 3.1* 41.9 ± 4.6** L -3,3¢,5-Triiodothyronine 0.32 l M 15.5 ± 1.0*** 1 l M 34.9 ± 1.6*** D -3,3¢,5-Triiodothyronine 0.32 l M 18.6 ± 2.2*** 1 l M 35.7 ± 0.6*** L -Thyroxine 0.32 l M 60.4 ± 4.4** 1 l M 36.3 ± 0.4*** L -3,3¢,5-Triiodothyroacetic acid 0.32 l M 95.5 ± 4.1 1 l M 39.5 ± 1.3*** Control 100 ± 2 100 ± 5 Testosterone 20 l M 83.7 ± 5.8 104 ± 3 Androsterone 20 l M 92.3 ± 3.0 96.4 ± 3.1 Dehydroisoandrosterone 20 l M 87.2 ± 1.9** 101 ± 1 Progesterone 20 l M 39.3 ± 2.3*** 96.2 ± 6.3 17b-Estradiol 20 l M 112 ± 3* 108 ± 1 Cortisone 20 l M 90.0 ± 2.8* 99.4 ± 1.4 11-Deoxycorticosterone 20 l M 61.3 ± 3.1*** 96.3 ± 4.4 Cholecalciferol 200 l M 117 ± 3** 136 ± 8* Ó FEBS 2002 Dual activities of xCTBP/xALDH (Eur. J. Biochem. 269) 2259 tions of unlabeled T 3 . Scatchard plots indicated that a single class of binding sites existed in xCTBP/xALDH1 (Fig. 2). NAD + ,ataconcentrationof200l M , significantly decreased the MBC from 338 ± 30 pmolÆmg )1 protein (n ¼ 5) to 178 ± 16 pmolÆmg )1 protein (n ¼ 3), although there was no significant difference in K d values between the NAD + -treated and untreated samples, 66 ± 11 n M (n ¼ 3) vs. 53 ± 5 n M (n ¼ 5), respectively, as shown in Fig. 2. This result indicated that the inhibitory mode of NAD + was noncompetitive. Progesterone, at 2 l M , appeared to affect both the K d (75 ± 2 n M , n ¼ 3) and MBC (310 ± 28 pmolÆmg )1 protein, n ¼ 3) values, although no significant differences were obtained for these values when compared with the K d and 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. IC 50 for T 3 was 700 n M (Fig. 3). The narrow range of the inhibitory concentration of T 3 indicates 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 [ 125 I]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. EffectofT 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 °Cwith5lgof 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,was 2.4. Each value is the mean ± SEM of triplicate determinations. Fig. 1. Inhibition of [ 125 I]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 [ 125 I]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)orNAD + (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. 2260 K. Yamauchi and J. Nakajima (Eur. J. Biochem. 269) Ó FEBS 2002 To determine how thyroid 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 T 3 was examined by variation of NAD + concentration within the reaction mixture. The K m value, 9 l M , was independent of the concentration of T 3 , but the V max value decreased from 0.18 to 0.08 lmolÆ min )1 Æmg )1 with increasing concentrations of T 3 (Fig. 4). The K i was 0.28 l M and 0.31 l M ,calculatedintwo independent experiments. Next, kinetics of the inhibition of xCTBP/xALDH1 by T 3 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 K 1/2 value did not change in the incubations with and without T 3 (2.8 ± 0.3 vs. 2.6 ± 0.1 l M , n ¼ 6), but the V max value decreased by 64% when 5 l M T 3 was added to the reaction mixture. The Hill coefficient did not change significantly in incubations with and without 5 l M T 3 , 2.3 ± 0.1 vs. 2.2 ± 0.1 (Fig. 5, inset). These results indicated that T 3 acts as a noncompet- itive inhibitor against both NAD + and retinal upon the enzyme activity of xCTBP/xALDH1. T 3 binding to xCTBP/xALDH1 in intact Xenopus 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 )4 M of NAD + or NADH pro- foundly inhibits the T 3 -binding activity. However, we have no information regarding NAD + ,NADHorNAD(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 T 3 -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 (n ¼ 3), which agreed with values reported previously [27,28]. On the other hand, Xenopus liver had a low NAD content, 201 ± 23 lmolÆ(kg fresh weight) )1 (n ¼ 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, T 3 -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 [ 125 I]T 3 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 T 3 within 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 °Cwith5lg 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)and1l 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 °Cwith5lg 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,pH8.0.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 Liver 756 ± 49 (3) Xenopus Liver 201 ± 23 (6) Kidney 234 ± 83 (5) Stomach 232 ± 7 (3) Intestine 291 ± 69 (3) Ovary 294 ± 94 (4) Heart 177 ± 29 (3) Skeletal muscle 199 ± 37 (3) Ó FEBS 2002 Dual activities of xCTBP/xALDH (Eur. J. Biochem. 269) 2261 DISCUSSION The present work was undertaken with the aim of deter- mining which signaling molecules, and other molecules, affected the T 3 -binding and ALDH activities of xCTBP/ xALDH1. We have obtained evidence that the [ 125 I]T 3 -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 T 3 -binding activity of xCTBP/ALDH1 whereas T 3 inhibited 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 T 3 . For human mitochon- drial and cytoplasmic ALDHs, T 3 and 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 T 3 binding to xCTBP/xALDH1 and of T 3 upon its enzyme activity must occur in a more complex fashion. Binding studies demon- strated that xCTBP/xALDH1 had a high affinity for T 3 , with a K d of 53 n M (Fig. 2), whereas the K i value for T 3 against NAD + on ALDH activity was 0.3 l M (Fig. 4). We can not precisely determine why there was a difference between the calculated K d and K i values. It may be possible that xCTBP/ALDH1 forms different conformations when bound to NAD + and/or T 3 , This possibility is considered due to the presence of positive cooperativity upon ALDH activity (the Hill coefficient, h ¼ 2.2) when the concentra- tion of retinal was varied (Fig. 5) and the presence of positive cooperativity upon the inhibition of ALDH activity (h ¼ 2.4) when the concentration of T 3 was varied (Fig. 3). T 3 may 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 T 4 and T 3 induce dissociation [34]. To explore the second possibility, the hepatic xCTBP/xALDH1, in the presence or absence of 5 l M T 3 , 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 dehydrogenase [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 T 3 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, K i values for thyroid hormone binding to all dehydrogenases, including those calculated for xCTBP/ xALDH1, were in the 10 -7 )10 -4 M range. 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 T 3 in intact cells (Fig. 6). However, the NAD content corresponding to 0.2 m M concentration would restrict T 3 -binding activity of xCTBP/xALDH1 within the Xenopus cells compared to the binding activity witnessed in vitro. It should be noted that retinal, at a concentration of 12 l M , activated the T 3 -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. 2262 K. Yamauchi and J. Nakajima (Eur. J. Biochem. 269) Ó FEBS 2002 a 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]. 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