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1 Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA
2 Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA
3 Faculté de Medecine, UMR 7561 CNRS-Université Henri Poincaré, Vandoeuvre-lès-Nancy, France
Reprint requests: to Dr. Anna Radominska-Pandya, Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W. Markham, Slot 516, Little Rock, Arkansas 72205, USA; e-mail: radominskaanna{at}exchange.uams.edufax: 501-603-1146.
(RECEIVED June 29, 2000; FINAL REVISION November 1, 2000; ACCEPTED November 1, 2000)
Article and publication are at www.proteinscience.org/cgi/doi/10.1110/ps.26501.
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Keywords: Cellular retinoic acid-binding protein; photoaffinity labeling; all-trans-retinoic acid; 9-cis-retinoic acid; retinoic acid glucuronide; 5,6-epoxy-retinoic acid
Keywords: CRABP, cellular retinoic acid–binding protein; atRA, all-trans-retinoic acid; RAR, nuclear retinoic acid receptor; RXR, nuclear retinoid receptor; 13-cis-RA, 13-cis-retinoic acid; 9-cis-RA, 9-cis-retinoic acid; 4-OH-RA, 4-hydroxy-all-trans-retinoic acid; ROH, all-trans-retinol; ROAc, all-trans-retinyl acetate; 5,6-epoxy-RA, 5,6-epoxy-all-trans-RA; KPFG, ketoprofen glucuronide; LA, lithocholic acid; LAG, lithocholic acid glucuronide; RAG, retinoic acid glucuronide; UGT, UDP-glucuronosyltransferase; UDP-GlcUA, UDP-glucuronic acid
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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The major ligand for CRABP I and II is atRA. It has been demonstrated that atRA and its metabolites are involved in diverse cellular activities including cellular growth, differentiation and morphogenesis, and the regulation of gene expression (Barua and Olson 1987; Gallup et al. 1987; Blaner and Olson 1994; Chambon 1994; Hong and Itri 1994; Mangelsdorf et al. 1994; Peck and DiGiovanna 1994; Miller 1998). Several other important ligands for both CRABPs have been identified previously. CRABPs have no binding affinity for retinyl esters; however, they do bind many retinoid analogs containing modifications to the ß-ionone ring, including such atRA metabolites as 4-OH-RA, 4-oxo-RA, 18-OH-RA, and 3,4-didehydro-RA (Fiorella et al. 1993). Evidence from mutagenesis studies and the inability of CRABPs to bind all-trans-retinol (ROH) or all-trans-retinal (Fiorella 1993) implied a preference for ligands with a terminal carboxyl group.
Glucuronidation is an important metabolic pathway for retinoids in vivo. Relatively high plasma concentrations of retinoid glucuronides have been demonstrated in experimental animals (Zile et al. 1982a, bMcCormick et al. 1983). Despite the discovery of the presence of RAG in human plasma (Barua and Olson 1986), its physiological significance has yet to be elucidated. Recently, the ability of human liver microsomes and recombinant UGT2B7 to biosynthesize carboxyl- and hydroxyl-linked glucuronides of RA and its oxidized derivatives has been demonstrated (Samokyszyn et al. 2000). It has been shown that acylglucuronides of atRA and its derivatives exhibit significant biological activity. They are less cyto- and teratotoxic than the parent compound yet retain its capacity to drive cell growth, differentiation, and proliferation (Barua and Olson 1987; Gallup et al. 1987; Janick-Buckner et al. 1991; Blaner and Olson 1994). RAG is also being considered as a possible agent for cancer chemoprevention and dermatological applications (Mehta et al. 1991; Olson et al. 1992). Although these observations indicate the underlying importance of retinoid glucuronides, the physiological significance of these compounds in the cell and their effect on cancer is not completely understood.
To elucidate the physiological functions of RAG, its interactions with CRABPs were reexamined in these studies. Photoaffinity labeling with [11,12–3H]atRA ([3H]atRA), which is covalently and specifically bound to CRABP I and II (Bernstein et al. 1995) within the atRA-binding site, was used as a tool to characterize and identify new ligands for CRABPs. Figure 1
shows the structures of the compounds used in this study. The results of these experiments indicated that the binding was specific and directed to the atRA-binding site of CRABPs and identified two novel CRABP I and II ligands, RAG and 5,6-epoxy-RA.
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20-fold lower than the affinity observed for photolabeling with atRA in experiments carried out under identical conditions. These data unambiguously demonstrate that 9-cis-RA is a ligand for both transporting proteins; however, this ligand is much less effective. |
Results |
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,ß-unsaturated carbonyl group and an isoprenyl chain, can serve as an effective photoaffinity label for atRA-binding proteins found in the cytosol and membranes from various bovine tissues. A detailed characterization of the probe with recombinant CRABP I and II was not carried out.
In these studies, binding of atRA to the RA-binding site of two recombinant, purified, homogenous CRABPs was evaluated by studying the effect of irradiation and the concentration-dependence and saturability of [3H]atRA-binding to purified CRABP I and CRABP II. As shown in Figure 2A, photoaffinity labeling of pure, recombinant CRABP I (15 kD) with [3H]atRA was light- (Fig. 2A, lane -UV) and concentration-dependent (Fig. 2A, lanes a–d). At an atRA concentration of 3.3 µM, saturable RA-binding to CRABP (3.1 µM) was reached, agreeing stoichiometrically with the model of one molecule of atRA binding to one molecule of CRABP (Kleywegt et al. 1994; Thompson et al. 1995; Li and Norris 1996).
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, lanes 1–4). Figure 2A also demonstrates that ROH failed to compete with [3H]atRA for CRABP I binding (Fig. 2A, lanes 5–8).
Similar experiments were performed with CRABP II and are shown in Figure 2B. CRABP II was photolabeled in a concentration-dependent manner with [3H]atRA and protected by unlabeled atRA. Also, ROH failed to compete for the atRA-binding site of CRABP II, as previously demonstrated for CRABP I. These data indicated that both of the CRABPs accepted or rejected the same ligands. The results presented in Figure 2, showing that photolabeling of CRABP I and II was light-dependent, concentration-dependent, and protected by unlabeled atRA in a concentration-dependent manner, indicated that [3H]atRA was bound within the RA-binding site.
Another photolabeled band at 30 kD was present in all experiments where CRABP I and CRABP II were labeled with [3H]RA. This can be explained by the fact that in buffer solution, one molecule of [3H]RA can bind to two molecules of CRABP I or II at different binding sites, forming covalently bound dimers. Whether this is a physiologically significant phenomenon remains to be determined. More extensive studies will be required for the elucidation of the dimer formation on UV irradiation, and this will be investigated separately. All data presented in Figure 2 support the use of photoaffinity labeling with [3H]atRA for characterization of both CRABP I and II.
Structure-function relationships of CRABP I investigated with different derivatives of retinoic acid
The CRABP I photoaffinity labeling system was evaluated with several known CRABP ligands and a few potential new ligands (for structures, see Fig. 1) to establish the validity of the competitive binding assay (Fig. 3). Oxidized derivatives that differ in the substituents present on the ß-ionone ring of the atRA molecule, such as 5,6-epoxy-RA, 4-OH-RA, and 4-oxo-RA, were analyzed for their ability to compete with atRA for the atRA-binding site of CRABP. Although all three oxidized derivatives inhibit the labeling of [3H]atRA (Fig. 3A,B), there appear to be differences in the Kis and, more interestingly, in the maximum degree of inhibition. Only 5,6-epoxy-RA appears to be able to completely inhibit binding of [3H]atRA (Fig. 3A). The other two derivatives inhibit only partially (40%–50%). It was evident from these experiments that all the oxidized derivatives were excellent ligands for CRABP I, especially 5,6-epoxy RA, identified here for the first time as a ligand for CRABP I.
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) established that there was no significant competition between the 13-cis-RA derivatives and [3H]atRA for the atRA-binding site of CRABP I. However, the 9-cis derivative did show significant competition at high concentrations. This work also confirmed that the carboxyl function of atRA is essential for binding to CRABPs. Neither methyl-4-OH-RA, which contains a methyl group on the terminal carboxyl function of atRA, nor 13-cis ROH or ROAc competed with [3H]RA for the RA-binding site of CRABP (data not shown). Dolichol, which lacks the typical retinoid ring but has the familiar isoprenyl chain with a substituted -OH group, was not a ligand for CRABP proteins (data not shown).
RAG as a new ligand for CRABP I
The results of additional photoaffinity labeling experiments identified RAG as a new ligand for CRABP I. Figure 4 shows that RAG could compete for the atRA-binding site of CRABP and, thus, was an effective ligand for CRABP I. From the data presented here, it is evident that RAG is an excellent ligand for CRABPs. These data support the assumption that for a retinoid derivative to be a ligand, it must contain a carboxyl group.
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).
Inhibition of CRABP I and CRABP II labeling with different derivatives of retinoic acid
Characterization of CRABP II with derivatives of retinoic acid was also carried out. Increasing concentrations of several substrates, such as 5,6-epoxy-RA, 4-OH-RA, and RAG, were tested for their ability to compete for the atRA-binding site on CRABP II (Fig. 5). The results suggested that in addition to atRA, 4-OH-RA, 5,6-epoxy-RA, and RAG also possess high affinity for CRABP II. Although photoaffinity labeling in general does not allow calculation of Kd and Ki values, it can provide very accurate comparative data in terms of IC50. Calculation of IC50 for the ligands used in this study has been performed, and the values are indicated in Table 1. The obtained values reflect very clearly the different affinities of the various retinoids in relation to atRA labeling of CRABP I and II.
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shows the comparison of photolabeling of CRABP I with atRA and 9-cis-RA. The experiments were performed under the same conditions with respect to the specific activity of labeled compounds, protein concentration, and time of irradiation. 9-cis-RA bound to CRABP I with 25% the efficiency of atRA. The labeling was effectively protected with varying concentrations of both unlabeled 9-cis-RA and atRA.
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Studies of the potential interactions of acylglucuronides with microsomal and recombinant UGTs
Studies were undertaken to determine whether the interaction between RAG and CRABP was nonspecific (i.e., not directed to the atRA active site). For this purpose, RAG, KPFG, and lithocholic acid glucuronide (LAG) were synthesized and tested for their ability to react with human liver microsomes, phenol-specific human recombinant UGT1A6, and atRA-specific human recombinant UGT2B7. As shown in Figure 7, KPFG and LAG (but not RAG) irreversibly inhibited the glucuronidation of 1-naphthol and/or androsterone catalyzed by human liver microsomes, UGT1A6, and/or UGT2B7. This indicated that RAG, an acylglucuronide of the physiological substrate atRA, does not exhibit potentially toxic interactions with cellular proteins.
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Discussion |
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The relationship between structure and function for several ligands was examined with this photoaffinity system. Three types of ligands were evaluated in these studies, as shown in Figure 1A, compounds with an oxidized ß-ionone ring: 4-OH-RA, 4-oxo-RA, and 5,6-epoxy RA; Figure 1B, isomers of the isoprenyl chain: atRA, 13-cis-RA, 9-cis-RA, and dolichol; and Figure 1C, compounds containing a modification of the carboxyl function: atRA, RA methyl ester, 4-OH-retinyl acetate, and ROH.
Our studies showed that modification to the ß-ionone ring with hydroxyl (4-OH-RA) or ketone (4-oxo-RA) functional groups did not significantly affect ligand binding to CRABP I (Fig. 3A). Also, comparison of the binding affinities of 4-OH-RA and 5,6-epoxy-RA for CRABP I demonstrated that 5,6-epoxy-RA was far superior to 4-OH-RA as a ligand. This was a novel finding, as 5,6-epoxy RA had not been identified previously as a ligand for CRABP. The ability of oxidized derivatives to serve as CRABP ligands, along with the fact that they are excellent substrates for glucuronidation (Samokyszyn et al. 2000), provides important new information on atRA biotransformation and its relation to CRABPs.
Previous reports have implied that isomeric forms of atRA, which have different configurations of isoprenyl side chains, are not, in general, good ligands for CRABPs. However, ongoing debates about this subject are presented in the literature (Fiorella et al. 1993; Norris et al. 1994; Sani et al. 1994; Horst et al. 1995). From our results, 9-cis-RA appeared to be a relatively good ligand for CRABP I at high concentrations (Fig. 3B, lane 4). However, the 13-cis stereoisomer failed to compete for the atRA-binding site of CRABP I. The lack of inhibition by 13-cis-RA has additional significance. It rules out the common concern in photoaffinity studies, which is the possibility that competitive ligands will quench the input light energy, thus preventing cross linking and providing false results. This type of quenching appears to be ruled out by the studies described in Figure 3, as 13-cis-RA, even at high concentrations, did not inhibit photoincorporation of [3H]atRA (Fig. 3B). Changing the configuration of the isoprenyl side chain, which alters the position of the carboxyl group, decreased the ability of isomeric forms of atRA to be ligands for CRABPs. Therefore, it is likely that this is one of the factors that determines CRABP specificity. An even more significant determinant of ligand specificity appeared to be the terminal carboxyl group.
Alterations to this group (acetylation or methylation) eliminated ligand binding to CRABP I (data not shown), as has been shown previously by other investigators (Fiorella et al. 1993; Norris et al. 1994; Sani et al. 1994; Horst et al. 1995). ROH, which has a terminal hydroxyl rather than a carboxyl substiutuent and has been reported in the literature not to be a ligand for CRABP I or II (Fiorella et al. 1993), did not compete with [3H]atRA for binding to CRABP (Fig. 2A,B). The photoaffinity labeling experiments presented here also indicated that the absence of either the ß-ionone ring or the carboxyl group completely abolished binding.
We also applied [3H]9-cis-RA as a novel probe for the characterization of recombinant CRABP I and II. Both proteins were photolabeled with the [3H]9-cis-RA probe; however, the affinity for the binding sites was much lower than observed for atRA. In our opinion, the ability of 9-cis-RA to bind to the active site of CRABP I and II partially resolves the controversy occurring in the literature over whether or not 9-cis-RA is a ligand for CRABPs in vitro. The significance of the 9-cis-RA as an endogenous ligand for both CRABPs in vivo is not resolved. Thus, our photolabeling approach confirmed previous results from other investigators, identified two new ligands, and, more important, for the first time allowed direct visualization of the radioactive protein/ligand complexes.
Following preincubation and irradiation of ligand and protein in solution, holo-CRABP I and II exist as monomers and dimers (shown in Figs. 2,5). Both species were formed following UV irradiation and showed concentration dependence of [3H]atRA photoincorporation. This is the first demonstration that CRABP I and II can form homodimers in solution. The nature of the dimers needs to be investigated further.
These studies have also identified RAG as one of the best ligands for both CRABP I and II. This is another novel finding, as previous experiments by other investigators using skin cytosols and sucrose density gradients have excluded RAG as a ligand for CRABPs (Sani et al. 1992). Figures 4 and 5 show that RAG is an excellent competitor for atRA-binding to CRABP I, exhibiting a binding potency similar to atRA.
Several experiments were performed to exclude the possibility of nonspecific RAG binding to CRABP I. Acylglucuronides, like RAG, are known to bind to cellular proteins. A number of drugs (diuretics, hypolipidemic agents, NSAIDs) and some endogenous compounds (bilirubin, fatty acids, bile acids, retinoids) that contain a carboxylic acid group circulate and/or are excreted in the bile or urine as acylglucuronides. These compounds can undergo spontaneous hydrolysis, releasing the parent drug, or intramolecular rearrangements leading to ß-glucuronidase-resistant 2-, 3-, and 4-O-acyl isomers. KPFG has been shown previously to irreversibly bind to plasma proteins, most notably albumin (Terrier et al. 1999b). If these metabolites are exposed to tissue macromolecules, there is the possibility of forming protein adducts, which may cause immunological side effects and hepatotoxicity. In addition, we have shown that UGTs, the enzymes responsible for biosynthesizing acylglucuronides, are irreversibly inhibited by preincubation with some of these reactive compounds (Terrier et al. 1999b).
Two types of experiments were performed to determine whether RAG binds to CRABP via covalent attachment to specific amino acids in the active site or interacts nonspecifically with cellular proteins. In the first experiment, KPFG, one of the most reactive acylglucuronides, and RAG were each preincubated with CRABP I and then photolabeled with [3H]atRA. KPFG failed to compete for the atRA-binding site of CRABP I, whereas RAG did compete effectively with [3H]atRA for binding to CRABP I (Figs. 4,5). To further confirm that RAG was a specific ligand for CRABP I, the potential reactivity of RAG, KPFG, and LAG toward microsomal and recombinant UGTs was examined. Using both KPFG as a model NSAID and the two endogenous conjugates, RAG and LAG, it was found that KPFG irreversibly inhibited the glucuronidation of phenols catalyzed by human hepatic microsomes and human recombinant UGT isoforms. This inhibition correlated with the formation of ketoprofen-protein adducts (Terrier et al. 1999b). Under the same conditions, RAG failed to exhibit inhibitory properties toward microsomal or recombinant UGTs (Fig. 7A,B,C). In contrast, incubation of LAG with the same enzymes led to an inhibition of their catalytic activity (Fig. 7A,B,C). These experiments showed that different acylglucuronides can have different reactivity toward cellular proteins. Finally, RAG does not appear to interact with intracellular proteins in a nonspecific manner.
The discovery that RAG can specifically interact with CRABPs may have important physiological implications. There are data in the literature showing that glucuronides of atRA and other atRA derivatives play a physiological role similar to that of free retinoids (Barua and Olson 1987; Gallup et al. 1987; Janick-Buckner et al. 1991; Blaner and Olson 1994).
The presence of a carboxyl group on its glucuronic acid moiety complies with the assumed structural requirement of a free carboxyl group for retinoid derivatives to be active ligands. This finding is also in agreement with holo-CRABP crystallographic studies showing direct or water-mediated interactions between the atRA carboxyl group and arginines in the binding site (Kleywegt et al. 1994; Thompson et al. 1995). Structural work on the cellular retinoic acid–binding complexes (Kleywegt et al. 1994) suggests that on binding, CRABPs can force retinoic acid derivatives into highenergy conformations because of Van der Vaals forces and hydrophobic interactions. Also, this report proposed that for ligands to have high affinity for CRABPs, the retinoid must superimpose on the atRA structure. Specifically, TTNBP (4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid; Kleywegt et al. 1994), a biologically very active retinoid that contains C13-C18 benzene ring and C19 carboxyl group may bind to CRABPs through this mechanism. Three-dimensional similarities between RAG and TTNBP with regard to the presence of ring structures containing carboxyl groups may indicate that RAG also undergoes conformational changes stabilized on binding.
Our photoaffinity-based competitive binding assay can also be applied to the characterization of other retinoid-binding proteins, such as the nuclear receptors, RAR/RXR, and enzymes involved with retinoid metabolism. Finally, it is anticipated that by using photoaffinity labeling to characterize atRA-binding proteins, such as CRABP and RARs, we will enhance our understanding of the complex interplay between the CRABPs, retinoids, and retinoid nuclear receptors.
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Materials and methods |
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Preparation of cellular retinoic acid–binding proteins
Homogenous rat CRABP I and II were provided by David E. Ong of Vanderbilt University School of Medicine. They were biosynthesized and purified by HPLC with an imidazole acetate gradient (pH 6.6) as previously described (Jamison et al. 1994). Pooled protein peaks were neutralized using Tris buffer and concentrated.
Synthesis of retinoid ligands and the acylglucuronides
The 5,6-epoxy RA and 4-OH-RA used in these assays were synthesized in our laboratory as described previously (Samokyszyn et al. 2000). RAG synthesis was carried out as previously described by Becker (Becker et al. 1996). Ketoprofen acylglucuronide (KPFG) was prepared as described by Terrier (Terrier et al. 1999b) and lithocholic acylglucuronide (LAG) was prepared using methods described by Panfil (Panfil et al. 1992).
TLC and HPLC characterization of retinoids and RAG after irradiation
All retinoids and the glucuronides, RAG, LAG, and KPFG, were analyzed by TLC and HPLC after UV irradiation. The TLC system used for the analysis of the compounds was chloroform : methanol : glacial acetic acid/water (65 : 25 : 2 : 4, v/v), and the compounds were visualized under a UV lamp. HPLC separation of retinoids and glucuronides was performed as previously described (Nowell et al. 1999). Briefly, after termination of the incubation, proteins were precipitated with isopropyl alcohol followed by centrifugation at 18,000g for 10 min. The supernatant was used for HPLC analysis. The HPLC system consisted of a Waters Alliance Model 2690 Separations Module with a photodiode array detector (Model 996) in conjunction with a Radiomatics Model 500TR Flow Scintillation Analyzer. The column was a Supelco C18 (25 x 0.46 cm, 5µm particle size), and the mobile phase was composed of 20µM diethylamine acetate, pH 5.0, and methanol. A flow rate of 1 mL/min with a gradient of 40%–70% methanol over 40 min was used to separate the metabolites.
Human liver microsomes
Human liver microsomes were prepared from transplantable livers according to the method of Dragacci et al. (1987).
Membrane fractions of recombinant cells expressing UGT1A6 or UGT2B7
The development of the recombinant V79 cell line (Chinese hamster lung fibroblasts) stably expressing the human liver UGT1A6 and of the recombinant HK293 cell line (human embryonic kidney cells) stably expressing human UGT2B7 has been described previously (Fournel-Gigleux et al. 1991; Coffman et al. 1997). Enriched membrane fractions of all recombinant cells were obtained from the cell homogenate by differential ultracentrifugation as previously described (Battaglia et al. 1994). The enriched membrane fractions were stored in aliquots at -80°C. No decrease in the enzymatic activity of the recombinant UGT1A6 and UGT2B7 was observed for up to 6 mo under these conditions.
Photoaffinity labeling with [3H]RA and [3H]-9-cis-RA
CRABP I and II were photolabeled with [3H]atRA (specific activity 52.0 Ci/mmol) using a method modified from Bernstein et al. (1995). An aliquot of [3H]atRA was dried and dissolved in methanol to a final concentration of 66 µM. CRABP I and II were diluted to 0.05 mg/mL with buffer (50 mM HEPES-NaOH, pH 7.5) and 9 µL (0.45 µg) of this solution was used per assay. Unlabeled potential ligands (retinoids and other structurally similar compounds; structures shown in Fig. 1) were added in 0.5 µL ethanol at varying concentrations (0, 5, 25, and 125 µM, final concentration). The reaction mixtures (10 µL final volume) were incubated for 2 min on ice, and 0.5 µL [3H]atRA (3.3 µM final concentration) was added and incubated on ice for an additional 5 min. The samples were irradiated with a long wavelength (366 mm) UV light source (Spectroline model ENF-260C; Spectronics) for 10 min on ice. The distance from the sample to the lamp was 2.5 cm.
Following irradiation, 4 µL of NuPAGE denaturing buffer (Novex) was added to each sample. Samples were vortexed, sonicated for 3 min, heated at 100°C for 5 min, and then applied to a 1mm NuPAGE 4%–12% Bis-Tris gel (Novex). Proteins were separated at 200 V in NuPAGE MES-SDS running buffer (Novex). Following electrophoresis, the gels were stained with Coomassie Blue, destained (10% 95% ethanol, 10% glacial acetic acid), washed with water, treated with Autofluor (National Diagnostics) for 30 min, and dried. Dried gels were subjected to autoradiography at -80°C for 2–5 d. Autoradiographs were analyzed and quantified by densitometry using IS-1000 Digital Imaging System (Alpha Innotech Corporation, software version 2.03).
CRABP I and II were also photolabeled with 9-cis-[20-methyl-3H]-retinoic acid ([3H]9-cis-RA; specific activity 52.0 Ci/mmol). Experimental conditions were exactly the same as described for the photoaffinity labeling with [3H]atRA.
Measurement of UGT activity
Androsterone and 1-naphthol were used as substrates to measure the glucuronidation catalyzed by UGT2B7 expressed in HK293 cells, UGT1A6 expressed in V79 cells, and human liver microsomes. UGT activity toward androsterone was measured with radioactive aglycon and unlabeled UDP-GlcUA as the sugar donor, as previously described (Radominska-Pyrek et al. 1986, 1987). The substrate was prepared in the form of mixed micelles with Brij 58 as described in detail in Radominska-Pyrek et al. (1986). For the assays, recombinant UGT2B7 (15 µg) and the substrate (0.1 mM final concentration) were incubated in a total volume of 60 µL with the following reaction components: 100 mM Tris-HCl buffer (pH 6), 5 mM MgCl2, 0.05% Brij 58. Reactions were started by adding 50 mM UDP-GlcUA (4.17 mM final concentration). The reactions were incubated at 37°C for 30 min and were stopped with 20 µL ethanol, vortexed, and placed on ice. Sixty microliters of the reaction mix was applied directly to the preadsorbent layer of a 19-channeled silica gel TLC plate (Baker 250Si-PA [19C]; VWR Scientific) and dried. Plates were developed in chloroform : methanol : glacial acetic acid : water (65 : 25 : 2 : 4, v/v). After development, plates were dried, sprayed with EN3HANCE (New England Nuclear), and subjected to autoradiography at -80°C for 3–7 d. Silica gel containing labeled metabolites (glucuronide bands were localized using the autoradiographs) and that from corresponding areas in control lanes was scraped into vials, and radioactivity was determined by scintillation counting (Beckman model LS 5000 TD, Beckman Instruments).
Glucuronidation of 1-naphthol was determined using a method adapted from Bock and White (1974). Human liver microsomes and membrane fractions of UGT1A6-V79 cells (10 µg protein) were incubated with 1 mM 1-naphthol in 100 µL of 100 mM Tris-HCl (pH 7.4) buffer containing 10 mM MgCl2. The reaction was initiated by the addition of UDP-GlcUA (2 mM final concentration). After 30 min incubation at 37°C, the reaction was stopped with 10 µL of 6N HCl and the unconjugated substrate was extracted into 4 mL chloroform. After centrifugation for 10 min at 2000g, 50 µL of the aqueous phase was removed and added to 1.95 mL of 0.1M NaOH. Fluorescence measurements of 1-naphthyl-ß-D-glucuronide, at excitation and emission wavelengths of 290 and 330 nm, respectively, were carried out on a LS-5 fluorescence spectrophotometer (Perkin-Elmer) with authentic 1-naphthyl-ß-D-glucuronide (0–10 nmol, Sigma) as standard.
Irreversible in vitro inhibition of glucuronidation by acylglucuronides
For the analysis of potential irreversible inhibition, RAG, LAG, and KPFG were incubated with different sources of UGTs (human liver microsomes, membrane fractions of UGT1A6-transfected V79 cells, or UGT2B7-transfected HK293 cells). Solutions of RAG and LAG were prepared just before use in the form of mixed micelles with Brij 58 (final concentration of detergent in the reaction mixture, 0.05%); KPFG was dissolved in water.
Inactivation experiments were performed at room temperature in 100 mM Tris-HCl (pH 7.4), 10 mM MgCl2, containing 5 mM D-saccharic acid 1,4-lactone, an inhibitor of ß-glucuronidases, by preincubating proteins with various concentrations (0.5–5 mM) of acylglucuronides. A control without any acylglucuronide was run simultaneously, corresponding to 100% activity. Following preincubation, the activity of the UGTs toward androsterone and/or 1-naphthol was estimated as described above.
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Acknowledgments |
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References |
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