Isolation and characterization of fatty acid binding protein in the liver of the nurse shark, Ginglymostoma cirratum

Camp. Biorhem. Phy~iol. Vol. 98A, No. 2, pp. 355-362, 1991 Prmted in Great Britain

0300-9629191 $3.00 + 0.00 Q 1991 Pergamon Press plc

ISOLATION AND CHARACTERIZATION OF FATTY ACID BINDING PROTEIN IN THE LIVER OF THE NURSE SHARK, GINGLYMOSTOMA CIRRATUM N. M. BAss,*t J. A. MANNING* and C. A. LUER~ *Department

of Medicine and The Liver Center, University of California, San Francisco, and $Mote Marine Laboratory, Sarasota, FL 34236. USA

CA 94143, USA

(Receiwd 31 Ma), 1990) Abstract-l. A 14.5 kDa fatty acid binding protein was isolated from the liver of the nurse shark. GinglymosIoma cirmfum. 2. Purified shark liver FABP (PI = 5.4) bound oleic acid at a single site with an affinity similar to that of mammalian FABP. 3. The apparent size, pI and amino acid composition of shark liver FABP indicate a close structural relationship between this protein and mammalian heart FABP.

INTRODUCTION

and heart muscle of teleost fish (Stewart and Driedzic, 1988). Organic-anion-dye-binding proteins similar in size to the FABP have also been identified by gel filtration in the liver cytosol of the elasmobranch Platyrhinoides triseriata (Sugiyama et al.. 1982). However, binding of radiolabeled oleic acid to this low-molecular-weight dye-binding protein fraction could not be demonstrated. Proteins structurally related to the vertebrate FABP have not been reported in invertebrates or prokaryotes and the evolutionary origins of this protein family remain unknown. The purpose of this study was to identify, isolate and characterize FABP from the liver of the marine elasmobranch Ginglymostoma cirratum (nurse shark) and to compare the properties of this protein with those of rat liver FABP (L-FABP).

A diverse family of 14- to 15-kDa structurally related cytosolic fatty acid binding proteins (FABP) are abundantly expressed in mammalian tissues (for reviews, see Bass, 1988; Spener et al., 1989; Kaikaus et al., 1990). FABP, representing separate gene products, are expressed in liver (Gordon et al., 1983), intestinal epithelium (Alpers et al., 1984), heart and

skeletal muscle (Bass and Manning, 1986; Claffey et al.. 1987; Miller et al., 1988), adipose tissue (Matarese and Bernlohr, 1988) and peripheral nerve myelin (Narayanan et al., 1988). Although the precise function(s) of these proteins remains unknown, several putative FABP functions have been proposed largely on the basis of circumstantial evidence (Bass, 1988). These include roles in the intracellular transport and compartmentation of long-chain fatty acids, as well as promotion of fatty acid utilization by esterification and oxidation pathways. Also, by limiting the intracellular concentrations of unbound fatty acids and hence their potentially disruptive effects on membrane integrity and enzyme activity, the FABP may provide an important cytoprotective function in tissues exposed to a high flux of fatty acids. Differences in the regulation and fatty acid binding properties of structurally distinct FABP expressed in different tissues also have suggested that the different gene products within the FABP family may perform specialized, tissue-specific functions (Bass et al., 1985; Lowe et al., 1987; Cistola et al., 1989). Largely characterized in the rat, FABP have been identified in the tissues of a number of mammalian species (Kawashima er al., 1984; Jagschies et al., 1985; Unterberg et al.. 1986; Vincent et al., 1985; Peeters et al., 1989) as well as in several tissues of aves (Katongole and March, 1979; Collins and Hargis, 1989; Sewell ef al., 1989a; 1989b; Scapin et al., 1988),

MATERIALS AND METHODS Materials [I-‘4C]oleic acid (57 mCi/nmol), (U-‘4C]palmitic acid (613 mCi/mmol) and [U-‘Hlsqualene (24.6 Ci/mmol) were obtained from New England Nuclear. [I-‘4C]Methyloleic acid was prepared as previously described (Bass et al., 1984). Specific activities of radiolabeled lipids were adjusted as required with unlabeled lipids of analytical grade. Lipidex 1000 was obtained from Packard Instruments. Rat L-FABP was purified from the livers of Sprague-Dawley rats as previously described (Ockner et al., 1982; Bass et al., 1984) and antisera to rat L-FABP, intestinal FABP (I-FABP), and heart muscle FABP (H-FABP) were raised in rabbits as previously described (Ockner and Manning, 1974; Bass et ul., 1985; Bass and Manning, 1986). Animals Nurse shark pups captured off the Florida coast were maintained free-swimming in sea water tanks at Mote Marine Laboratories until two to three years old. Sixty-dayold male Sprague Dawley rats were allowed free access to food and water. Sharks anesthetized with tricaine methanesulfonate were killed by pithing. Rats were killed by bilateral thoractomy under ether anesthesia.

tAddress all correspondence to: Nathan M. Bass, Liver Center, HSW 1120. Box 0538, University of California, San Francisco. CA 94143, USA. 355

356 Preparation of 1iDercq’rosol and serum

Polyacryiamidr gel eiectrophoresrs

from sharks were rapidly excised and frozen at - 80°C until used. Shark liver was thawed and homogenized in 2 volumes of 0.01 M potassium phosphate buffer, pH 7.4, 0.15 M (KC1 (KCI-PO, buffer) at 4°C in a Potter-Elveheim Teflon-glass homogenizer. The homogenate was centrifuged for 20 min at 10,OOOg.The supernatant was collected and centrifuged for 1hr at 105,OOOg.The cytosolic fraction was collected and either used immediately or stored at -20°C until used. Rat liver cytosol was prepared in the identical manner after livers were perfused free of blood with ice-cold 0.15 M NaCI. Blood was obtained from sharks and rats by cardiac puncture and serum was prepared by centrifugation of the clotted blood. Liver cytosol and serum were delipidated by passage of 5ml aliquots through a 2.4 x 37cm column of Lipidex 1000 equilibrated with K&PO, buffer at 37 C (Glatz and Veerkamp, 1983). Fractions of 2 ml were collected at a flow rate of 87 ml/hr. All protein-containing fractions were pooled and concentrated to 5 ml in an Amicon apparatus with a YM-5 membrane. Following delipidation, 97-98% of protein was recovered from cytosol and serum.

Non-denaturing polyacrylamide gel electrophoresis (PAGE) using a 7% separating gel at pH 8.9 was performed as previously described (Davis, 1964). SDS-PAGE was performed according to the method of Laemmli (1970). Gels were stained with Coomassie Brilliant Blue R-250. Western blotting was performed using a Bio-Rad Transblot apparatus according to the manufacturer’s instructions.

Livers

Analyiicul gel ,filtrarion

Delipidated liver cytosol (5.5 mg protein) or serum (1 ml) were adjusted with KCI-PO, buffer to 2.5 and 2.0ml, respe&ely, and were incubate’d for 1 min at room temperature with 25 nmol of [i-‘4Cloleate (2 mCi/mmol) added in 5 ~1 of absolute alcoh~l/pro~ylene giycol (i : 3, v/b). Cytosol or serum were applied to a 1.6 x 66 cm Sephadex G-100 column equilibrated with KCI-PO, buffer at room temperature. Fractions of 3.5 ml were collected at a rate of 34.8 ml per hr. Protein in column fractions was determined from the optical density at 280 nm and radioactivity was counted in 0.5 ml aliquots. Additional studies of radiolabeled ligand binding to the low-molecular-weight fatty acid binding fraction (FABP fraction) of liver cytosoi were performed using a 1 x 48 cm column of Sephadex G-50 as previously described (Ockner et nl.. 1980). Cytosol (4. I mg of protein) adjusted to 0.5 ml with KCI-PO, buffer was incubated with radiolabeled lipids for 1 min and applied to the column. Lipids tested for binding to the shark FABP fraction were [l-‘4C]oleic acid (range: 5-200 nmoi), 15 nmol of [U-‘4C]palmitic acid and 25 nmol of MU-~H]squaiene. Fractions of 0.58 ml were collected at a rate of 39 mlihr. Isolation qj’ proteins from shark liver FABP jiraction Cytosol obtained from 40g of shark liver was concentrated to 45 ml and incubated briefly with 40nmoi [l“CJoleate. The low-molecular-weight cytosolic protein fraction exhibiting fatty acid binding (FABP fraction) was isolated by two consecutive gel filtrations through Sephadex G-50 as previously described (Ockner et ai., 1982; Bass et af., 1984). Column fractions containing the FABP fraction from the second Sephadex G-SO gel filtration were pooled, concentrated to 1ml, equilibrated with 0. I3 M glycine, and were subjected to preparative Sepadex G-75 superfine thin-layer isoelectric focusing (dimensions: 20 x 20 cm) using ampholyte pH ranges of 3.5-10 or 4.@-‘7.0, as previously described (Ockner et nl., 1982). In some experiments, [l-‘4C]methyloleate (1.8 nmol, 0.1 PCi) was added to the protein sample as an electrically neutral marker of longchain acyl binding. Contact blots (2cm wide) were taken from both edges of the electrofocusing gel and were fixed and stained as previously described (Ockner er al., 1982). Gel zones. 1cm wide, corresponding to stained protein bands on the contact blots were scraped into I ml of distilled H,O and assayed for radioactivity and pH. Proteins were eluted from the gel in 0. I M Tris/HCl, pH 8.5. Ampholytes were removed from proteins by gel filtration through a 1.6x 56 cm column of Sephadex G-50 equilibrated with KCI-PO, buffer.

Lipidex binding assa)’

Fatty acid binding by purified, delipidated proteins was analyzed by a modification (Scalien et al.. 1985) of the procedure of Glatz and Veerkamp (1983) Assays were performed in 1.5 ml polypropylene tubes containing S gg of protein in 0.45ml KC&PO, buffer. Various amounts of [I-‘“Cloleic acid in 5 1~1absolute ethanol: propylene glycol (1 : 3, v/v) were added to assay tubes to obtain a range of final oleate concentrations between 0. I I and 5.5 PM. After incubation for 10 min at 37‘C, tubes were cooled on ice, and 150 ~1 of ice-cold Lipidex-1000 suspension (I : 1, v/v) in KCI-PO, buffer was added with vortex mixing to remove unbound fatty acid. After a further 10min on ice, tubes were mixed again and centrifuged at 20,OOOg at 4°C for 2min. Radioactivity was determined in the supernatants. Protein-free blanks were included for each concentration of fatty acid and were linear with respect to fatty acid concentration. Fatty computerized,

acid binding to protein was analyzed nonlinear least-squares curve fitting.

Protein concentration

by

was estimated by the method of

et al. (1951).

Radioactivity was assayed in 10 ml Optifluor (Packard Instruments) in a Packard Tri-Carb 4530 Liquid Scintillation Spectrometer. For amino acid analysis, 60 ug of ourified shark liver FABP was hvdrolvsed in 5.7 N H&-at Ib5’C for 24 hr. Analyses were performed using a Lowry

Beckman 6300 amino acid analyser. Cysteine and methionas cysteic acid and methionine sulfone, respectively, using the technique of Hirs (1967). Amino acid compositional relatedness was assessed using the difference index of Metzger et al. (1968) and the linear regression ine were determined

correlation

coefficient.

RESULTS Fractionation of nurse shark liver cytosol incubated with radiolabeled oleate by Sephadex G-100 gel filtration revealed peaks of fatty acid binding in both the void volume and low-molecular-weight fractions (Fig. IA). The latter peak corresponded closely to the FABP peak in rat liver cytosol similarly fractionated by gel filtration (Fig. lB), but eluted earlier (fraction 26) than the rat liver FABP peak (fraction 27). Shark serum proteins fractionated on Sephadex G-100 revealed a major void volume peak of fatty acid binding and a small oleate peak corresponding in elution volume to the albumin oleate binding peak of rat serum (data not shown); no M, 12,000 oleate binding fraction was present in shark serum. These results indicated the presence of an FABP in shark liver cytosol of slightly greater apparent molecular weight than rat liver L-FABP, and that this binding activity was not attributable to low-molecular-weight fatty acid binding in serum proteins contaminating the shark liver cytosol preparation. When the binding of radioiabeled oleate, palmitate and squalene to shark liver cytosol was examined by means of Sephadex G-50 gel filtration, a distinct FABP binding peak was only obtained with oleate (Fig. 2). As shown in Fig. 3, oleate binding to the shark liver cytosolic FABP

Shark

351

liver fatty acid binding protein

0

50

100

Total Oleate

Fraction Fig. 1. Binding of radiolabeled oleic acid to proteins in liver cytosol from (A) nurse shark and (B) rat, separated by Sephadex G-100 chromatography. Cytosol (2.5 ml, 5.5 mg protein) incubated with 25 nmol [l-‘4C]oleic acid was applied to a 1.6 x IOOcm column of Sephadex G-100. Fractions of 3.7 ml were collected at a rate of 34.8 ml/hr. V, indicates the void volume.

fraction was saturable with increasing amounts of oleate. The maximum oleate binding capacity of the FABP fraction in shark liver cytosol was approximately one third of the FABP fraction in rat liver cytosol, amounting to 1.33nmol/mg total cytosol protein in the shark compared with 4.28 nmol/mg total cytosol protein in the rat liver cytosol (Fig. 3). The proteins present in the shark liver FABP fraction were isolated by two consecutive gel filtrations through Sephadex G-50 and further fractionated after incubation with [1-‘4C]methyloleate by thin-layer isolectric focusing. As shown in Fig. 4, six protein bands, which ranged in p1 from 4.2 to 8.7, were identified by contact blotting of the isoelectric focusing gel. [1-‘4C]methyloleate radioactivity was predominantly associated with the p1 5.4 band (band 5) whereas the least radioactivity was recovered with the pI 8.7 band (band 6). In the experiment illustrated in Fig. 4, the separation between bands 3 and 4 was too narrow to permit recovery of these bands individually, and they were scraped from the plate together. In subsequent experiments, isoelectric focusing was performed with an ampholyte pH range of 4&7.0.

I z sz

E

0.0 0.5

L

27

01eote I

O-0

29

31

33

35

A-A

Polmitote

a---*

Squalene

37

39

41

43

Fraction

Fig. 2. Binding of radiolabeled oleic acid, palmitic acid and squalene to the FABP fraction of shark liver cytosol on Sephadex G-50 chromatography. Cytosol (0.1 ml, 4.1 mg protein) incubated with [l-‘4C]oleic acid (10 nmol), [l-‘4C]palmitic acid (15 nmol), or [U-’ Hlsqualene (25 nmol) was applied to a 1 x 48 cm column of Sephadex G-50. Fractions of 0.5 ml were collected at a rate of 39 ml/hr.

150

200

(nmol)

Fig. 3. Binding of radiolabeled oleic acid to the FABP fraction of liver cytosol from shark (closed symbols) and rat (open symbols). Cytosol (0.2 ml, 4.1 mg protein) was incubated with the indicated amounts of [I-‘4C]oleic acid and applied to a column of Sephadex G-50. Column dimensions and flow conditions were as described in Fig. 2.

This provided sufficient separation of the pI 4.2 and 5.4 bands to allow their individual recovery. The proteins separated by isoelectric focusing from shark liver FABP fraction were further characterized by SDS-PAGE. The purification of band 5, the major methyloleate-binding protein (Fig. 4) from shark liver cytosol, is illustrated in Fig. 5. The shark liver FABP fraction isolated from Sephadex G-50 chromatography revealed two major protein bands on SDSPAGE. Band 5 from the isolectric focusing step revealed a single polypeptide with migration corresponding to the higher-molecular-weight major band in the FABP fraction. The apparent molecular weight of band 5 calculated from protein standards was 14,500 compared with 11,200 for pure rat L-FABP. The actual molecular weight of rat L-FABP is 14,184 Da and is underestimated using SDS-PAGE. Bands 1, 2, 3 and 4 all revealed a major polypeptide with identical migration to band 5 on SDS-PAGE as well as fainter bands of higher and lower apparent molecular weight (data not shown). SDS-PAGE of band 6 revealed a single band which corresponded in migration to the lower-molecular-weight major protein band present in the shark liver FABP fraction (data not shown). Nondenaturing PAGE of bands 1 through 5 and rat L-FABP is shown in Fig. 6. Rat L-FABP, which is known to contain several charge isoforms (Trulszch and Arias, 1981; Ockner et al., 1982), displayed a series of three bands. Shark FABP fraction electrofocusing bands 3, 4 and 5 displayed single bands with identical migration, while bands 1 and 2 revealed bands-two in the case of band 2-which migrated more anodally. Band 6 did not enter the nondenaturing PAGE gel; not unexpectedly in view of the basic pI of this protein. Fatty acid binding to the proteins purified from the shark liver FABP fraction was further characterized using the Lipidex binding assay. For these experiments, isoelectric focusing was performed using the FABP fraction isolated from delipidated shark liver cytosol without the addition of radiolabeled fatty acids. Binding of [I-‘4C]oleate to individual electrofocusing bands determined using the Lipidex assay is shown in Fig. 7. Band 5, the major methyloleatebinding protein, demonstrated high-affinity, saturable binding of oleate with an estimated Kd of

N. M. BASSet al.

358

Band

Blot

pl

[1-14C]Methyloleate bound (dpm x10-l)

4.2

152

4.5

135

0

1’ -_/ 2

-3

4.8 347

\

\

4

5.2

5

5.4

1953

6

8.7

26

Fig. 4. Thin-layer isoelectric focusing of shark liver FABP fraction. Shark liver FABP fraction (10 mg protein) isolated from Sephadex G-50 chromatography was incubated with 1.75 nmol (0.1 pCi) [I-‘4C]methyloleate and subjected to thin-layer isoelectric focusing as described in Methods. Gel zones, 1cm wide, corresponding to the protein bands on contact blots were scraped into distilled H,O and assayed for pH and radioactivity. 0.92pM. In contrast, bands 1. 3 and 6 showed comparatively Iittle affinity or capacity for oleate binding. The maximum binding of oleate to band 5 was estimated at 0.70mol oieate/mol protein. This calculation assumed a molecular weight of 15 kDA for band 5, and corrected the mass of protein in the assay for overestimation by the Lowry assay using a correction factor derived from the amino acid analysis. The actual aminoacyl mass of band 5, calculated on the basis of the amino acid analysis of this protein, was found to be 0.55 of the mass determined by the Lowry method. This finding is similar to that previously reported for FABP isolated from the rat (Ockner et al., 1982; Glatz ef al., 1985). Band 5 was thus identified as the low-molecularweight FABP present in shark liver on the basis of methyloleate binding during isoelectric focusing and radiolabeled oleate binding in the Lipidex assay. The degree of purification of shark liver FABP/band 5 estimated from the maximum oleate binding to the FABP fraction in cytosol (Fig 2; 1.33 nmoljmg cyto-

sol protein) and the maximum binding of oleate to band 5 in the Lipidex assay (Fig. 7; 51.2nmol~mg FABP) was approximately 39-fold. The amino acid composition of shark liver FABP/band 5 compared with the compositions of rat L-FABP, I-FABP and H-FABP is shown in Table 1. Shark liver FABP differed notably from rat L-FABP in the relative abundance of several amino acids and contained greater quantities of alanine, arginine, with comparatively less leucine and threonine, isoleucine, methionine and valine. indeed, the amino acid composition of shark liver FABP showed a closer degree of relatedness to rat H-FABP than to either L-FABP or I-FABP when compared using the difference index of Metzger et al. (1968) or simple linear regression (Table 2). The difference index value of 10 obtained for shark liver FABP versus rat H-FABP is typical for proteins sharing substantial sequence homology, whereas the higher value of 19.1 obtained in the comparison of shark liver FABP and rat L-FABP is commonly found for proteins lacking

Shark

liver fatty acid binding

Fig. 5. S~S-polyac~Iamide gel electrophoresis of proteins ~lustrating the steps of pu~~cation of shark liver FABP. Lane 1, shark liver 105,000 g supematant (170 pg protein); lane 2, shark liver FABP fraction from Sephadex G-50 column chromatography (52pg protein); lane 3, band 5 protein from isoelectric focusing of shark liver FABP fraction (8 pg protein); lane 4, pure rat L-FABP (7 pg protein); lane 5, calibration proteins (with M,): Bovine albumin (66,000), ovaIbumin (45,~0), trypsinogen (24,~), myoglobin (17,8~), cytochrome c (12,400). aprotinin (6,500).

significant structural similarity (Metzger et al., 1968). Immunological cross-reaction between shark liver FABP and the various rat FABP was sought using Western blotting. Using monospecific antisera to rat L-, I- and H-FABP, positive reactions were only obtained with rat tissues containing these proteins; no cross-reaction was observed with either shark liver cytosol or pure shark liver FABP (data not shown).

359

protein

Fig. 6. Nondenaturing polyacrylamide gel electrophoresis at pH 8.9 of proteins isolated from shark liver FABP fraction by isoelectric focusing. Each lane contains 10 to 15pg of protein. Lane I, band I; lane 2, band 2; lane 3, band 3; lane 4, band 4; lane 5, band 5; lane 6, pure rat L-FABP.

fatty acids has been commonly found for the mammalian FABP, although estimates of the number of sites for long chain fatty acid binding to L-FABP have ranged from one (Bass, 1985; Glatz et al., 1985; Wilkinson and Wilton, 1987; Peeters et al., 1989; Starch et al., 1989) to two (Haunerland et al., 1983; Lower et al., 1987; Offner et al., 1986; SchulenbergSchell et al., 1988) or more (Cistola et al., 1989; Fukai et al., 1989). Several of the other acidic pI bands isolated by isoelectric focusing of the shark liver cytosol FABP fraction contained proteins with identical migration to the pi 5.4 band on nondenaturing and SDS-PAGE. Although these polypeptides could represent charge isoforms of the p1 5.4 protein, the isoelectric focusing fractions containing them showed far less methyloleate binding than the p1 5.4 band, and also showed little affinity for oleate in the Lipidex

DISCUSSTOE

We used an approach previously employed to isolate L-FABP from rat liver (Ockner et al., 1982) to identify and purify, to our knowledge for the first time, an FABP from the liver of an elasmobranch, the nurse shark. This protein, present in the lowmolecular-weight gel-filtration fraction of shark liver cytosol, has a pI of 5.4 and migrated as a single polypeptide on both nondenaturing and SDS-PAGE, with an apparent molecular weight of 14,500 on the latter. The p1 5.4 protein was identified as shark liver FABP (sL-FABP) based on several properties similar to those of the mammalian FABP class of proteins. These include size, binding of methyloleate during isoelectric focusing (Ockner et al., 1982) and high affinity, saturable binding of oleate. In the Lipidex assay, sL-FABP bound oleate at a single site with an affinity comparable to that of rat L-FABP (Bass, 1985; Glatz et al., 1985; Takikawa and Kaplowitz, 1986; Peeters et al., 1989). A single binding site for

_.-

0.0

1.0

2.0

Total

3.0 Oleate

4.0

5.0

6.0

(/hi)

Fig 7. Lipidex assay of radiolabeled oleic acid binding to proteins isolated by isoelectric focusing from shark FABP fraction. Proteins (5 pg) were incubated with the indicated concentrations of [l-‘4C]oleic acid in 0.45 ml 0.01 M potassium phosphate buffer, pH 7.4, 0.15 M HCl as described in Methods. Unbound fatty acid was separated from proteinbound fatty acid by mixing with Lipidex 1000 at 4°C. Radioactively was determined in the supernatants of assay tubes following centrifugation.

360

N.

M.

BASS et

al.

Table I. Comparison of the amino acid compositions of FABP purified from shark liver (sL-FABP) and FABP from rat liver (L-FABP), intestinal eoithelium (I-FABP) and heart (H-FABP) Rat Shark sL-FABP Amino

1983)

acid 6 5 14 2 19 9 I

6 6 I6 0 I6 I2

II I2 2 5 x1

2 2 II I I’ I? 2 9 6 I7 7 6 6 2

I? N.D. 2

12 0 3

8

I2

5

Nearest whole number values for sL-FABP 130 amino acids. N.D.; not determmed.

Table 2. Amino acid compositional relatedness between shark liver FABP (sL-FABP) and FABP from rat tissues L-FABP

I-FABP

CornDared (R)

Values are based on amino

H-FABP (Claffey er al.. 1987)

19.1 0.80 acid compositions

H-FABP

with sL-FABP 15.5 0.85 shown

IO 0.90 in Table

6 4 I 0 I2

I

I0 3

7 IO 14 4 8 4 0

6 IO Ii 2 6 8I

IO I 5

I9 , 1

II were estimated

assay. These proteins as well as the relatively smaller pI 8.7 band present in the FABP fraction, which also failed to bind fatty acids, were not characterized further. Several properties of sL-FABP differ from rat L-FABP in a manner that suggests that the elasmobranch liver protein may be more closely related to other members of the diverse mammalian FABP family than to mammalian L-FABP. These include greater apparent size of sL-FABP compared with rat L-FABP on gel filtration and SDS-PAGE, a more acidic pI [5.4 for sL-FABP compared with the neutral to basic p1 reported for rat L-FABP (Ockner et al., 1982; Glatz et al., 1985; Offner et al., 1986)] and a substantial difference in amino acid compositional relatedness between the shark and rat liver proteins. On the other hand, a higher degree of amino acid compositional relatedness was evident between sLFABP and mammalian H-FABP, the member of the mammalian FABP multigene family that is expressed in a wide variety of tissues and most abundantly in heart and skeletal muscle (Bass et al., 1986; Claffey er al.. 1987; Miller et al., 1988). The similarity between sL-FABP and mammalian H-FABP also extends to the larger size and more acidic pI of H-FABP compared with L-FABP in the rat (Glatz et al., 1985; Offner et al., 1986). Mammalian L-FABP, in addition to binding long chain fatty acids, shows a broad affinity for numerous organic anions including heme, bilirubin and dyes such as bromosulphophthalein (Bass, 1988). Using bromosulphophthalein or bilirubin as ligands,

Difference index Correlation coefficient

I-FABP (Alpers er al.. 1984)

Residues/Molecule

Ala Arg A% CYS Glx Gl> Hia lieu Le11 LYS Met Phe PKI SU Thre TW Tyr Val

L-FABP (Gordon PI al,

I.

assuming

13 a total

residue content of

previous studies failed to identify a low-molecularweight organic-anion-binding protein in the livers of several elasmobranch species including Squalus acanthias (Levine et al., 197 1; Boyer et al., 1976). Ruju erinacea (Boyer et al., 1976) and Platyrhinoides triseriata (Sugiyama et d., 1982). Sugiyama et al. (1982) however, were successful in identifying a lowmolecular-weight organic-anion-binding peak on gel filtration of the liver cytosol of P. triseriuta with the dyes Rose Bengal and 8-aminonaphthalene-lsulphonate. However, this protein fraction did not bind oleic acid, which contrasts with the findings of the present study. The reason for the difference in oleate binding in the liver cytosol low-molecularweight fraction between P. triseriata (Sugiyama et al.. 1982) and the nurse shark is unclear, but may reflect differences in methodology. Thus, we routinely delipidated liver cytosol prior to determining ligand binding by gel filtration, whereas this was not done by Sugiyama et al. (1982). It is also conceivable that FABP-like organic-anion-binding low-molecular-weight liver cytosol proteins differ in their affinity for fatty acids among different species of elasmobranchs. Fatty acid binding to this class of proteins may represent a more recent evolutionary specialization in their function. By analogy, serum albuminlike proteins from teleost fish bind fatty acids (Fellows and Hird, 1981: Davidson et al., 1988) as well as bilirubin (Fellows and Hird, 1982) whereas similar proteins in elasmobranchs bind bilirubin but not fatty acids (Fellows and Hird, 1981; 1982). Teleost fish also appear to have FABP-like proteins with high affinity for long chain fatty acids. Thus. Stewart and Driedzic (1987) recently identified a 12,800 Mr protein in the heart muscle of both Macrozoarces americanus (ocean pout) and Hemitripteru americanus (sea raven). This protein was isolated, and bound palmitate at two binding sites with a Kd of 0.7 PM. Of note, palmitate was poorly bound by the FABP fraction of nurse shark liver cylosol. in contrast to the binding of oleate to this fraction.

Shark liver fatty acid binding protein Differences in binding affinity for saturated and unsaturated fatty acids are well recognized in the mammalian FABP (Bass, 1988) and also clearly exist in the elasmobranch. The FABP in mammals represent a diverse group of proteins which, along with the cellular retinoid binding proteins, comprise a family of at least 10 separate gene products (Kaikaus et al., 1990). In non-mammalian vertebrates, evidence for tissuespecific expression of structurally different FABP has been sought and found to date only in the chicken, Galfus domesticus (Sewell et al., 1989a, b). The tissuespecific expression of structurally different FABP may point to a specialization of function among the different members of this protein family. Convincing evidence for such structure- and tissue-dependent functional specialization is, however, lacking. The present study has isolated and characterized a liver FABP from the most primitive vertebrate species studied to date. Of considerable interest, a sL-FABP appears to be more closely related to mammalian H-FABP than L-FABP. H-FABP is also closely structurally related to A-FABP, the mammalian FABP expressed in adipose tissue (Matarese and Bernlohr, 1988). Indeed, shark liver differs from the liver of mammais in that it behaves in some respects more like adipose tissue. Thus, shark liver contains considerable quantities of lipid, which serves as an energy store and provides buoyancy (Van Vleet et al., 1984; Rossouw, 1987). In addition to the uptake and incorporation of fatty acids into triglyceride (Malins and Robisch, 1973) shark liver is also capable of releasing fatty acids into the circulation (Sargent PI al., 1972; Lipshaw ef nf., 1972). More detailed understanding of the structural relationship between sLFABP and mammalian FABP, and characterization of FABP that may be present in other tissues of the shark, will help to address questions pertaining to the evolution of the FABP family of proteins as well as the functions served lipid metabolism.

by these abundant

proteins

in

Acknowledgements-The authors thank Diana Fedorchak and Michael Karasik for help in preparinn the manuscript. This work was supported in-part by research grant DK32926 from the National Institutes of Health.

361

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