Eur. J. Biochem.

259, 127134 (1999) q FEBS 1999

Isolation, amino acid sequence determination and binding properties of two fatty-acid-binding proteins from axolotl (Ambistoma mexicanum) liver
Evolutionary relationship
Santiago M. Di Pietro1, Jacques H. Veerkamp2 and Jose A. Santome1
1 2

Instituto de Qumica y Fisicoqumica Biologicas, Facultad de Farmacia y Bioqumica, Universidad de Buenos Aires, Argentina; Department of Biochemistry, Faculty of Medical Sciences, University of Nijmegen, the Netherlands

Up until now, the primary structure of fatty-acid-binding proteins (FABPs) from the livers of four mammalian (rat, human, cow and pig) and three nonmammalian (chicken, catfish and iguana) species has been determined. Based on amino acid sequence comparisons, it has been suggested that mammalian and nonmammalian liver FABPs may be paralogous proteins that originated by gene duplication, rather than as a consequence of mutations of the same gene. In this paper we report the isolation and amino acid sequence determination of two FABPs from axolotl (Ambistoma mexicanum) liver. One of them is similar to mammalian liver FABPs (L-FABPs) and the other to chicken, catfish and iguana liver FABPs (Lb-FABPs). The finding of both L-FABP and Lb-FABP in a single species, as reported here, indicates that they are paralogous proteins. The time of divergence of these two liver FABP types is estimated to be of < 694 million years ago. The ligand-binding properties of axolotl liver FABPs were studied by means of parinaric-acid-binding and parinaric-acid-displacement assays. L-FABP binds two fatty acids per molecule but LbFABP displays a fatty-acid-conformation-dependent binding stoichiometry; L-FABP shows a higher affinity for fatty acids, especially oleic acid, while Lb-FABP has a higher affinity for other hydrophobic ligands, especially retinoic acid. In addition, the tissue-expression pattern is different, L-FABP is present in liver and intestinal mucosa while the expression of Lb-FABP is restricted to liver. Data indicate distinct functional properties of both liver FABP types. Keywords: axolotl; Ambistoma mexicanum; fatty acid-binding protein (FABP); molecular evolution. Fatty acid-binding proteins (FABPs) belong to a multigene family of intracellular lipid-binding proteins. They have been reported for nearly all mammalian tissues and a high number of residue identities has been found between FABPs of the same tissue in different species [13]. Based on their primary structure and the first tissue from which they were isolated, FABPs have been classified into several types including heart, liver, intestinal, adipocyte, myelin, ileal and epidermal [4, 5]. The distinctive pattern of tissue expression of the different FABP types, as well as the co-expression of different FABP in a single cell type, suggests that each FABP has a specific function in cellular metabolism [6]. Several lines of evidence support the idea that the L-FABP is structurally and functionally different from the other FABP types: L-FABP binds two fatty acids per molecule [712], whereas the other FABP types have a single fatty-acid-binding site [10,13]; one of the two fatty acids bound
Correspondence to J. A. Santome, IQUIFIB, Facultad de Farmacia y Bioqumica, UBA, Junn 956, Buenos Aires 1113, Argentina. Tel.: +54-1-508-3651, Fax: +54-1-508-3652, E-mail: santome@qb.ffyb.uba.ar Abbreviations: ax L-FABP, axolotl liver fatty acid-binding protein; ax Lb-FABP, axolotl basic liver fatty acid-binding protein; DEAE, diethylaminoethyl; FABP, fatty acid-binding protein; L-FABP, liver fatty acid-binding protein; Lb-FABP, basic liver fatty acid-binding protein. Note: The novel amino acid sequence data reported here have been submitted to the SwissProt data bank and are available under accession numbers P81399 and P81400. (Received 11 August 1998, revised 6 October 1998, accepted 9 October 1998)

to the L-FABP has the carboxylate group located near the protein/solvent interface [7,8]; the L-FABP is able to bind not only fatty acids but also a wide range of hydrophobic ligands such as acyl-CoA, bilirubin, lysophosphatidylcholine, retinoic acid, bile salts, prostaglandins, heme and peroxisome proliferators [14,14]; the L-FABP, unlike other FABPs, undergoes a significant conformational change upon fatty-acid binding [15]; the transfer of a fluorescent fatty acid analogue between the L-FABP and phospholipid membranes occurs via aqueous diffusion of the unbound ligand [16], in contrast to the findings for heart FABP or adipocyte FABP, the transfer mechanism involves transient collisional protein/membrane interactions [17,18]. The L-FABP type had been reported only for mammals but, in 1994, Ceciliani et al. [19] published the amino acid sequence of a basic (pI 9.0) FABP from chicken (Gallus domesticus) liver that, when compared with other known FABPs, presents the highest amino acid identity with rat and pig ileal FABPs (42 and 43%, respectively), and with rat L-FABP (39%) while mammalian L-FABPs have 7990% identity with one another. In years that followed, other liver FABPs related to that of chicken were found in the livers of several species namely, catfish (Rhamdia sapo) [20,21], toad (Bufo arenarum) [22] and iguana (Anolis pulchellus) (GenBank accession number U28756). Because the chicken protein, the first nonmammalian liver FABP to be reported, is basic (pI = 9.0), we will refer collectively to nonmammalian liver FABPs as basic FABPs (LbFABPs). On the basis of its peculiar isoelectric point, amino acid sequence, putative fatty-acid-binding site and a binding study with 11-dansylamino-undecanoic acid, it was suggested that chicken Lb-FABP may represent a type of FABP different from

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L-FABP [19,23,24]. An evolutionary analysis by Schleicher et al. [25] placed the chicken Lb-FABP as a separate branch of a subfamily that also includes L-FABPs and ileal FABPs, although it was not clear whether it was the avian counterpart of mammalian L-FABPs or a paralogous protein, that originated by a gene duplication. The proposal that both types of liver FABPs are paralogous proteins, encoded by two different genes [25], is supported by a later evolutionary analysis and the finding of a FABP in catfish intestine that cross-reacts with anti(rat L-FABP) serum and whose four sequenced tryptic peptides average 60% identity with the corresponding fragments in mammalian L-FABPs [21]. In this paper, we report the isolation of both a L-FABP and a Lb-FABP from axolotl (Ambystoma mexicanum) liver, and their amino acid sequence, evolutionary relationship, tissue expression and ligand-binding properties, showing that they are actually encoded by two separate genes and have different biochemical properties. In addition, the presence of a L-FABP type in axolotl liver indicates that it is not exclusive to mammals. MATE R I A L S A N D ME T H O D S Materials [1-14C]oleic acid (56 Cimol21) was from DuPont NEN. Cisparinaric and trans-parinaric acids were from Molecular Probes, Inc. Horseradish-peroxidase-coupled goat anti-rabbit IgG were from Dako A/S. All other reagents were purchased from Sigma, Baker, Bio-Rad, Pharmacia Biotech or Applied Biosystems. Preparation of the cytosolic fraction from axolotl tissues Axolotls, approximately 1 year old, were from a fish hatchery. Tissues were excised, cut into small pieces, suspended in 40 mm sodium phosphate (pH 7.4), 150 mm KCl, 2 mm EDTA, 4 mm dithiothreitol and homogenized in a Teflon potter. Homogenates were centrifuged at 20 000 g for 15 min and the resulting supernatants were further centrifuged at 105 000 g for 90 min in a Beckman XL-90 ultracentrifuge with a Ti 90 rotor. Purification of FABPs The liver 105 000 g supernatant (4 mL) was incubated with 0.5 mCi of [1-14C]oleic acid at 20 8C for 5 min and loaded to a Sephadex G-75 column (2.5 cm 40 cm) equilibrated with 30 mm Tris/HCl (pH 9.0), 1 mm dithiothreitol, 1 mm EDTA. Elution was performed at 4 8C with the same buffer at a flow rate of 16 mLh21. The 1018 kDa radioactive fraction containing the FABPs was applied directly to a DEAE Sephacel column (1.1 cm 8 cm) equilibrated with 30 mm Tris/HCl (pH 9.0). The material bound to the column was subsequently eluted with 10, 20, 30, 40, 50 and 100 mm NaCl in the same buffer. Fractions eluted with 20 and 30 mm NaCl, containing the FABPs, were concentrated by using a Centriprepw concentrator (Amicon), diluted threefold with 30 mm Tris/HCl (pH 9.0) and loaded onto a Mono-Q HR 5/5 column (Pharmacia LKB) previously equilibrated with the same buffer. In both cases, the column was developed on an FPLC system (Pharmacia LKB), at a flow rate of 0.8 mLmin21, with a combination of two linear gradients of NaCl concentration (0 100 mm in 40 min followed by 100300 mm in 10 min). ax LbFABP and ax L-FABP eluted at < 30 and 50 mm NaCl, respectively. Before the ligand-binding assays, lipids were removed from the proteins by incubation at 378C for 45 min

with Lipidex beads [26], followed by a buffer change to 50 mm Tris/HCl (pH 7.4). Electrophoresis and immunoblotting SDS/PAGE and immunoblotting were carried out as described by Di Pietro and Santome [27]. Isoelectric focusing was performed on a PhastGel IEF 3-9 by using the Phast System (Pharmacia Biotech). The preparation of rabbit antiserum with specificity to rat L-FABP was as reported previously [28]. Rabbit anti-(ax Lb-FABP) serum was obtained using the method described by Harlow and Lane [29]. Enzymatic digestion and peptide purification A 150-mg sample of each of purified ax Lb-FABP and ax LFABP was digested in 0.1 m Tris/HCl (pH 8.5), 2 m urea, with 3 mg of Lys-C protease at 20 8C for 24 h. For Glu-C protease digestion, ,120 and 100 mg each of the pyridylethylated FABPs were incubated in 0.1 m Tris/HCl (pH 7.9), 2 m urea, with 2.5 mg enzyme at 20 8C for 24 h. Peptides were separated by HPLC (Pharmacia LKB) on a Vydac C18 column (4.6 mm 250 mm) equilibrated with 0.1% (vol./vol.) trifluoroacetic acid in water. Elution was performed at a flow rate of 0.8 mLmin21 with a 080% acetonitrile linear gradient in 80 min. Amino acid analysis and sequence determination Quantitative amino acid analysis and automatic amino acid sequence determination were carried out in an Applied Biosystems 420 A Amino Acid Analyzer and an Applied Biosystems 477 A Protein Sequencer, respectively, according to the manufacturer's instructions. Amino acid sequence determination of the blocked N-terminal peptide The ax L-FABP N-terminal peptide obtained from the Glu-C digestion was subjected to N-acylaminoacyl-peptide hydrolase digestion as described by Radhakrishna and Wold [30] and sequenced by Edman degradation. Sequence comparison Sequence alignment was carried out as described by Schleicher et al. [25]. Amino acid substitution rates and divergence times were calculated as described by Chan et al. [31]. Parinaric-acid-binding assay We followed the procedure described by Nemecz et al. [10,15] as modified by Schroeder et al. [32]. Samples (2 mL) of 0.67 mm delipidated ax Lb-FABP or ax L-FABP in 50 mm Tris/ HCl (pH 7.4) were titrated with the addition of small amount of cis-parinaric or trans-parinaric acid up to a final concentration of 4.6 mm. The concentration of cis-parinaric and transparinaric acid stock solutions in ethanol (350 mm) was determined spectrophotometrically by using 1308 value of 7.9 104 m21cm21 and 1306 value of 7.7 104 m21cm21, respectively [33]. Fluorescence intensity measurements (excitation 324 ^ 3 nm, emission 412 ^ 3 nm) of each sample and blank (ligand or protein only) were performed at 258C on a Jasco FP-770 spectrofluorometer (Japan Spectroscopic Co.). Data were corrected for sample dilution, the latter never exceeding 1.3%. The ethanol concentration in the cuvette was below 1.5%.

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Fatty-acid-binding proteins from axolotl liver (Eur. J. Biochem. 259) 129

band with a mobility corresponding approximately to that of FABPs (Fig. 1A I, lanes 4 and 5). Each of them eluted from a Mono-Q column as a symmetric peak that was found to be homogeneous when subjected to SDS/PAGE (Fig. 1A I, lanes 6 and 7), isoelectric focusing and HPLC using a C4 column (data not shown). Anti-(rat L-FABP) antibodies cross-reacted with the protein eluted from the DEAE column with 30 mm NaCl (Fig. 1A II, lane 5) and with the same fraction further purified on a Mono-Q column (Fig. 1A II, lane 7). Rabbit antibodies obtained against the 20 mm fraction purified on a Mono-Q column did not cross-reacted with either the protein eluted at 30 mm (Fig. 1A III, lanes 5 and 7) or with rat L-FABP (Fig. 1B, lane 6) but did cross-react with catfish Lb-FABP, toad Lb-FABP and chicken Lb-FABP (Fig. 1B, lanes 35). These results suggest that FABPs eluted from the DEAE column, with 20 and 30 mm NaCl, and then purified on a Mono-Q column are more closely related to Lb-FABPs and L-FABPs, respectively. Hereafter, the protein eluted with 20 mm NaCl and that eluted with 30 mm NaCl is called `ax Lb-FABP' and `ax L-FABP', respectively. Primary structure of ax Lb-FABP The purified protein was subjected to N-terminal sequence determination. The sequence of the first 20 residues was obtained. To complete the primary-structure determination, fragments were generated by enzymatic cleavage with proteases Glu-C and Lys-C. Peptides resulting from each digestion were separated by RPHPLC and subsequently submitted to amino acid analysis and Edman degradation. The resulting primary structure of ax Lb-FABP is shown in Fig. 2. The protein consisted of 125 residues and, on the basis of the sequence, its molecular mass was calculated to be as 13 744 Da. The calculated isoelectric point [34], pI = 7.1, agrees with the experimental value (data not shown). ax Lb-FABP is the first vertebrate FABP reported to be not blocked at its N-terminus.
Fig. 1. SDS/PAGE and Western-blot analysis. (A) SDS/PAGE and Western-blot analysis of the purification of FABPs from axolotl liver. Lane 1, molecular mass marker proteins; lane 2, 105 000 g supernatant of liver homogenate; lane 3, 1018-kDa fraction from Sephadex G-75 column; lane 4, 20 mm fraction eluted from DEAESephacel column; lane 5, 30 mm fraction from DEAESephacel column; lane 6, Mono-Q-column-purified ax Lb-FABP; lane 7, Mono-Q-column-purified ax L-FABP. I, 10% SDS/ PAGE; II, anti-(rat L-FABP) Western blot; III, anti-(axolotl Lb-FABP) Western blot. (B) anti-(axolotl Lb-FABP) Western blot of FABPs from the liver of other species. Lane 1, molecular mass marker proteins; lane 2, ax Lb-FABP; lane 3, catfish Lb-FABP; lane 4, toad Lb-FABP; lane 5, chicken Lb-FABP; lane 6, rat L-FABP. The apparent molecular mass of marker proteins (in kDa) is found on the left of each panel.

Primary structure of ax L-FABP The protein was subjected to four cycles of automatic Edman degradation. No phenylthiohydantoin derivative could be identified, thus indicating that the N-terminal amino acid was blocked. For internal sequence determination, fragments were generated by enzymatic cleavage with proteases Lys-C and GluC, separated by RPHPLC and subsequently submitted to amino acid analysis and Edman degradation. One of the peaks obtained from the Glu-C digestion was identified as the blocked Nterminal peptide. Its amino acid sequence as of the second residue was obtained following digestion with N-acyl-aminoacyl-peptidase, the first residue being established on the basis of the corresponding amino acid analysis. The resulting primary structure of ax L-FABP is shown in Fig. 2. The protein consists of 126 residues and, based on the sequence, its molecular mass, considering an N-terminal acetyl group, was calculated as 13 957 Da. The calculated isoelectric point [34], pI = 5.0, agrees with the experimental value (data not shown). Evolutionary relationship The alignment of the amino acid sequence of ax Lb-FABP and ax L-FABP with those of FABPs isolated from the liver of human, cow, pig, rat, chicken, catfish and iguana is shown in Fig. 2, which also includes the per cent identities between these two axolotl liver FABPs and those between each of them and the other liver FABPs. Taking into account the amino acid

Cis-parinaric acid displacement assay ax Lb-FABP or ax L-FABP (0.4 mm) in 50 mm Tris/HCl (pH 7.4) were incubated with cis-parinaric acid (1 mm) for 1 min to obtain maximum fluorescence. Those solutions were then titrated with the corresponding ligand from an ethanolic solution (except for palmitoyl-CoA, dissolved in 0.1 m sodium acetate, pH 6.0) up to a threefold molar excess. Measurements were performed under the same conditions as above. R E S U LT S Purification of FABPs Fractions eluted from the DEAESephacel column with 20 and 30 mm NaCl were radioactive and presented an SDS/PAGE

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substitutions between the known Lb-FABP sequences and those between L-FABPs, and assuming that fish, amphibians, reptiles and birds appeared 425, 325, 295 and 165 million years ago, respectively [35], we estimated the substitution rates of LbFABPs and L-FABPs to be (4.1 ^ 0.5) 10210 and (4.4 ^ 0.4) 10210 amino acid substitutions/residue/year, respectively. On the basis of these substitution rates and the amino acid substitutions between Lb-FABPs and L-FABPs, the divergence time between them was estimated to be 694 ^ 74 million years. The amino acid substitution rates calculated for intestinal FABPs and heart FABPs were (4.8 ^ 0.3) 10210 and (3.0 ^ 0.1) 10210 amino acid substitutions/residue/year, respectively, and the divergence time between L-FABPs and intestinal FABPs as 815 ^ 66 million years and that between liver and heart FABP types as 999 ^ 73 million years. Values represent mean ^ SE. Expression of ax Lb-FABP and ax L-FABP in other axolotl tissues In order to investigate the expression pattern of these FABPs, cytosolic fractions from liver, heart, lung, skeletal muscle, stomach mucosa, intestinal mucosa and skin were submitted to electrophoresis and immunoblotting. Lb-FABP was present only in liver (Fig. 3A) while L-FABP was detected in liver and intestine (Fig. 3B). Fluorescent fatty-acid-binding measurements Upon titration of ax Lb-FABP and ax L-FABP with cis-parinaric or trans-parinaric acids, saturation curves were observed (Fig. 4). Scatchard plots of these binding curves show that

ax L-FABP is able to bind two cis-parinaric or two transparinaric acid molecules while ax Lb-FABP also binds two cisparinaric acid but cannot accommodate two trans-parinaric acid molecules. Dissociation constants of ax L-FABP for cisparinaric acid are 30 ^ 6 nm and 250 ^ 45 nm for the primary and secondary binding site, respectively, and those for transparinaric acid are 13 ^ 4 nm and 330 ^ 48 nm, respectively. Dissociation constants of ax Lb-FABP for cis-parinaric acid are 23 ^ 7 nm and 320 ^ 41 nm for the primary and secondary binding site, respectively, and that for trans-parinaric acid is 42 ^ 9 nm. Values represent mean ^ SEM (n = 3). Displacement of cis-parinaric acid by fatty acids and other ligands In order to further investigate the relative specificities of ax LbFABP and ax L-FABP for fatty acids and other possible ligands, displacement experiments were performed (Fig. 5). The ax LbFABP affinity for fatty acids is lower than that of ax L-FABP and fatty acids can be divided into two groups of different affinity, both displacing only one cis-parinaric acid molecule (Fig. 5A). The greatest affinity is for unsaturated fatty acids of 16 and 18 carbons. ax Lb-FABP binds a variety of ligands other than fatty acids with high affinity (Fig. 5B). It is significant that the two cis-parinaric acid molecules can easily be displaced by retinoic acid indicating that its Kd value for the primary binding site is approximately in the nm range. Palmitoyl-CoA, bile salts, bilirubin and lysophosphatidylcholine seem to be able to displace one of the two cis-parinaric acid molecules and show similar affinity to that of the group of fatty acids with the higher affinity.

Fig. 2. Sequence alignment of ax L-FABP and ax Lb-FABP with FABPs from the liver of other species. The amino acid sequences of the axolotl liver FABPs were aligned with those of human (hu), cow (bo), pig (pi) and rat (ra) L-FABPs and with those of chicken (ch), catfish (ca) and iguana (ig) Lb-FABPs according to Schleicher et al. [25]. Amino acid residues identical to those in ax L-FABP and ax Lb-FABP are shown in bold type and italics, respectively. The deletion of L-FABPs Asp87 in Lb-FABPs is indicated by a dash. Identity percentages between ax L-FABP (left) and ax Lb-FABP (right), as well as those between each of them and the other liver FABPs, are also shown.

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Fatty-acid-binding proteins from axolotl liver (Eur. J. Biochem. 259) 131

for fatty acids belonging to the group with the lowest affinity. The affinity for bilirubin appears to be absent. DISCUSSION L-FABP type is also present in mammalian kidney, stomach and intestinal epithelium [16] but is the only type reported for mammalian liver and comprises up to 5% of the cytosolic proteins in rat liver [36]. To date, the L-FABP primary structure from four mammalian species has been reported: rat [37], human [31], cow [38] and pig [39]. These proteins display 79 90% amino acid identity. In our laboratory, 75% of the sequence of an FABP from the liver of armadillo, one of the ancient mammals, was determined and clearly shown to belong to the LFABP type [40]. The fact that Lb-FABPs from the liver of nonmammalian species have only < 40% amino acid identity with mammalian L-FABPs raises the question of whether they are the consequence of mutations of the same L-FABP gene or paralogous proteins that originated by a gene duplication [19, 20,23,24]. The probability that L-FABPs and Lb-FABPs are encoded by two different genes is supported by evolutionary analyses [21,25] and the possible presence of an FABP belonging to the L-FABP type in catfish intestine [21]. Nevertheless, the conclusive proof is the finding of both FABP types in a single species as reported herein for the axolotl (A. mexicanum) liver. The sequence comparison of both axolotl liver FABPs with that of the L-FABPs isolated from human, cow, pig and rat liver, and with Lb-FABPs from chicken, catfish and iguana (Fig. 2) shows that ax L-FABP is related most closely to mammalian L-FABPs and ax Lb-FABP to Lb-FABPs. ax L-FABP shows 6774% identity with mammalian L-FABPs

Fig. 3. Tissue-expression pattern of ax Lb-FABP and ax L-FABP. 105 000 g supernatant of liver (lane 1), heart (lane 2), lung (lane 3), skeletal muscle (lane 4), stomach mucosa (lane 5), intestinal mucosa (lane 6) and skin (lane 7) homogenates of axolotl were subjected to SDS/PAGE and transferred to nitrocellulose membranes. Immunodetection was carried out with anti-(ax Lb-FABP) serum (A) and with anti-(rat L-FABP) serum (B).

As shown in Fig. 5C, stearic, linoleic and especially oleic acid displace both cis-parinaric acid molecules from ax L-FABP thus implying that their Kd values should be of the same order as those of cis-parinaric acid. The nm range for the primary binding site is similar to that of rat L-FABP [12]. Fatty acids as 20:4, 16:1 and 18:3 displace only one fluorescent fatty acid. The affinity for 16:0 is intermediate between the above two groups. As with mammalian L-FABP, ax L-FABP binds, apart from fatty acids, other structurally distinct hydrophobic compounds (Fig. 5D). The affinity for bile salts, palmitoyl-CoA, retinoic acid and lysophosphatidylcholine is approximately the same as

Fig. 4. Titration curves for the binding of cis-parinaric and trans-parinaric acid to ax Lb-FABP and ax L-FABP. (A) Binding of cis-parinaric acid (04.6 mm) to ax Lb-FABP (0.67 mm) in 50 mm Tris/HCl (pH 7.4) was performed following the procedure described by Nemecz et al. [10,15] as modified by Schroeder et al. [32]. Inset, Scatchard plot of the titration. (B) Binding of trans-parinaric acid to ax Lb-FABP and the corresponding Scatchard plot (inset) were determined as in (A). (C, D) Binding of cis-parinaric and trans-parinaric acid to ax L-FABP, respectively. Experiments were carried out under the same conditions described for ax Lb-FABP. The corresponding Scatchard plots are shown in the insets.

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Fig. 5. Displacement of cis-parinaric acid from ax Lb-FABP and ax L-FABP by fatty acids and other ligands. (A, B) Cuvettes with 0.4 mm ax Lb-FABP and 1 mm cis-parinaric acid in 50 mm Tris/HCl (pH 7.4). (C, D) Cuvettes with 0.4 mm ax L-FABP and 1 mm cis-parinaric acid in 50 mm Tris/HCl (pH 7.4). After 1 min of incubation maximum cis-parinaric acid fluorescence intensity was obtained. Aliquots of different ligands were added and, after equilibration, fluorescence intensity was recorded again. Fatty acids added (A and C): palmitic acid (16:0, O), palmitoleic acid (16:1, K), stearic acid (18:0, W), oleic acid (18:1, X), linoleic acid (18:2, open rhombi), linolenic acid (18:3, closed rhombi) and arachidonic acid (20:4, A). Other ligands added (B and D): retinoic acid (P), lysophosphatidylcholine (open hexagons), bilirubin (B), bile salts (equimolar mixture of sodium cholate and sodium deoxycholate, L) and palmitoyl-CoA (closed hexagons).

and 3942% with Lb-FABPs. Conversely, the ax Lb-FABP presents 3742% and 6774% amino acid identity with the same FABP groups. Western-blot analyses further support these conclusions (Fig. 1). Axolotl is the first nonmammalian species clearly proven to express the L-FABP type in addition to the LbFABP type in liver. Our phylogenetic analyses [21, 25] suggested that the two liver FABP types diverged before the fishtetrapod split. In this paper, the divergence time between the two groups of proteins is estimated to be 694 ^ 74 million years; that is, either before the vertebrateinvertebrate split, about 600 million years ago, or at approximately the same time. Chan et al. [31] reported a divergence time of 690 million years between L-FABP and intestinal FABP. Nevertheless, we estimated it to be 815 ^ 66 million years. This difference is understandable considering that we were able to use up-to-date sequences of evolutionary distant species to estimate amino acid substitution rates for liver and intestinal FABP types, whose values were very close (4.14.8 10210 substitutions/amino acid residue/year). Chan et al. calculated a much higher value for the L-FABP amino acid substitution rate based on only two L-FABPs from two closely related species, human and rat. L-FABP is also present at high concentrations in the intestinal mucosa of mammals [14, 36] but the Lb-FABP present in the liver of chicken, toad, catfish and iguana has not been reported for the intestinal mucosa of these species. Rabbit anti-(catfish Lb-FABP) do not detect the protein in any catfish tissue other

than liver [21] and rabbit anti-(ax Lb-FABP) do not detect ax Lb-FABP in any axolotl tissue other than liver. However, anti-(rat L-FABP) do detect a 14-kDa protein both in axolotl liver and intestinal mucosa suggesting that, as reported for mammals, L-FABP is also present in the intestinal mucosa of axolotl. The co-expression of a Lb-FABP and a L-FABP in axolotl liver suggests that they should have distinctive functional properties. A number of biochemical experiments have shown that this may be the case. Cis-parinaric and trans-parinaric acid, with a kinked chain and straight chain, respectively [41], were used for a comparative study of the binding characteristics of both proteins. Our results indicate that ax L-FABP, as described for rat L-FABP [10, 15, 41], binds two cis-parinaric acid or trans-parinaric acid molecules per molecule while ax Lb-FABP binds two cis-parinaric acid molecules but can only accommodate one trans-parinaric acid molecule, thus showing binding differences between the two axolotl FABPs. However, catfish Lb-FABP appears to bind only one cis-parinaric or transparinaric acid molecule [21] and chicken Lb-FABP binds only one molecule of 11-dansylamino-undecanoic acid [24]. Both axolotl liver FABPs share the distinctive capacity of binding a wide variety of ligands with mammalian L-FABPs, but their relative affinities are different. Both axolotl and mammalian L-FABPs have more affinity for fatty acids, especially oleic acid, than for other ligands while this is not

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true for ax Lb-FABP. These results suggest that the Lb-FABP type probably displays functions in cellular metabolism other than those of L-FABP. The high affinity of ax Lb-FABP for retinoic acid may be important considering that retinoids are signalling molecules in the cell, e.g. all-trans-retinoic acid is the major bioactive retinoid required for tissue growth, epithelial differentiation, gametogenesis, embryonic morphogenesis and the reduction of mutagenesis [42, 43]. An important role of cellular-retinoicacid-binding protein (CRABP) in the elimination or inactivation of retinoic acid has been suggested [42]. The presence of a high concentration of L-FABP in rat liver (< 70 nmolg21) may be unimportant physiologically, since its affinity for retinoic acid is 23 orders of magnitude lower than that of CRABP [42]. Taking into account the high affinity of ax Lb-FABP for retinoic acid, it is possible that ax Lb-FABP may participate in the regulation of functions and metabolism of the retinoic acid. In conclusion, we have proven that L-FABP and Lb-FABP are two different FABP types that diverged at the time of the vertebrateinvertebrate split or earlier. ax L-FABP is the first LFABP sequenced for a nonmammalian species, while there has been no report of an Lb-FABP in mammals so far. However, the presence, structure and binding properties of both liver-type FABPs in axolotl liver suggest that they have different functions. ax L-FABP has the same ligand binding properties, a stoichiometry of 2:1 and specificity, as mammalian L-FABPs. In contrast, the ligand-binding characteristics of ax Lb-FABP, ligand-conformation-dependent stoichiometry and specificity, do not resemble those of other FABP types. One of these distinctive properties, the high affinity of Lb-FABP for retinoic acid deserves further investigation. A C K N OW L E D G ME NT S
We would like to thank Dr H. L. Monaco for providing chicken Lb-FABP. Amino acid analysis and sequence determination were performed at the LANAIS-PRO (National Protein Sequencing Facility, UBA-CONICET, Buenos Aires, Argentina). We gratefully acknowledge the expert collaboration of S. B. Linskens and E. V. Dacci. This work was supported by Universidad de Buenos Aires Grant FA006 and Consejo Nacional de Investigaciones Cientficas y Tecnicas de la Republica Argentina-Agencia Nacional de Promocion Cientfica y Tecnologica Grant PMT-PICT0282 Prestamo BID 802/OC-AR.

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S U P P L E M E N TA RY MAT E R I A L Supplementary material is available for this issue. Fig. S1. RP-HPLC separation of ax Lb-FABP and ax L-FABP peptides generated by digestion with Lys-C and Glu-C proteases. Fig. S2. Summary of the sequence analyses and the resulting primary structure of ax Lb-FABP. Fig. S3. Summary of the sequence analyses and the resulting primary structure of ax L-FABP. (http://www.blackwell-science.com/ejb.htm).