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Isolation and characterization of a fatty acid binding protein in adipose tissue of Gallus domesticus
Comp. Biochem. PhysioL Vol. 96B,No. 3, pp. 585-590, 1990
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ISOLATION A N D CHARACTERIZATION OF A FATTY ACID B I N D I N G PROTEIN IN ADIPOSE TISSUE OF GALLUS D O M E S T I C U S G. H. SAMS,* B. M. HARGIS*~"and P. S. HARGIS*J/ Departments of *Poultry Science and tVeterinary Microbiology and Parasitology, Texas Agricultural Experiment Station, Texas A & M University System, College Station, TX 77843-2472, USA (Tel: 409 845 7537) (Received 11 December 1989)
Almtraet--1. Fatty acid binding protein (A-FABP) was isolated from chicken adipose cytosol. 2. Relative tool. wt of chicken A-FABP was determined to be 14,400 from SDS-polyaerylamide electrophoresis; the pI was 5.1; and amino acid composition data indicated structural homology with mammalian heart and adipose FABPs. 3. Polyclonal antisera prepared against A-FABP exhibited monospecificity for chicken A-FABP and no cross-reactivity with chicken liver proteins was observed. 4. Determination of relative ligand binding characteristics indicated A-FABP exhibited greatest binding activity in response to linoleate, followed by oleate, palmityl CoA and palmitate; no binding attinity for cholesterol was detected.
INTRODUCTION Several tissue-specific fatty acid binding proteins (FABPs) have been identified in a number of species (Ockner et al., 1972; Smith et al., 1985; Sweetser et al., 1987; Sewell et al., 1989a,b). Sequence analysis of mammalian FABPs has shown that these proteins are each structurally unique yet each contain regions of homology suggesting they have descended from a common ancestral sequence (Chan et al., 1985). These low tool. wt proteins demonstrate an affinity for fatty acids and each appear to function in the metabolism and intracellular transport of lipids (Bass, 1985; Dempsey et al., 1985; Glatz et al., 1985; Sweetser et al., 1987). Of these low mol. wt (12,000-15,000) proteins, hepatic FABP (L-FABP) and intestinal FABP from rat tissues are best characterized (Sweetser et al., 1987). Only limited investigations of FABP in adipose tissue exist in current literature (Haq et al., 1982; Smith et al., 1985; Bernlohr et al., 1985). Adipose FABP (A-FABP) isolated from the rat has been shown to be immunochemically similar to rat L-FABP (Haq et al., 1982), but to be immunochemically distinct from A-FABP isolated from human adipose. Haq et al. (1982) suggested that the apparent differences in antigenic epitopes may be due to species specific differences in lipid metabolic pathways and consequent differences in the evolutionary differentiation of the proteins in the two species. Proteins exhibiting similar activities to FABPs have also been identified in adipose tissue of the rat by Potter et al. (1987), but these are large mol. wt proteins (40,000) associated with the plasma membrane. Bernlohr et al. (1985) identified a low mol. wt protein in murine 3T3-L1 adipocytes ~/Author to whom correspondence should be addressed.
that exhibits 20-30% amino acid sequence homology to FABPs of rat liver and intestine. This protein was originally called p422 and is now designated as adipocyte lipid binding, protein (ALBP) (Bernlohr, 1988). Through the use of immunoblotting and cDNA hybridization techniques, Bernlohr et al. (1985) concluded that ALBP was Specific to adipose tissue in the mouse. Findings that ALBP mRNA increased in abundance during differentiation of 3T3-L1 fibroblasts to mature adipocytes lead Bernlohr et al. (1985) to suggest a role for this protein in intracellular fatty acid transfer and recent evidence indicates murine ALBP binds fatty acids and retinoids in vitro (Matarese et al., 1988). The objective of this study was to isolate and characterize A-FABP in the chicken, to assess relative binding characteristics of this protein for various ligands and to develop a monospecific polyclonal antisera. The chicken provides a unique model for the study of mechanisms involved in lipid metabolism due to the fact that in this species, nearly all de novo synthesis of fatty acids occurs in the liver, as it does in man, and adipose tissue functions mainly for lipid storage. Adipose tissue contains the largest overall energy stores in the body, primarily in the form of triacylglycerol fatty acids. Previous work has resulted in the purification of L-FABP from chicken liver cytosol (Sewell et al., 1989a) and provided evidence for the presence of a low tool. wt fatty acid binding protein in adipose tissue (Collins and Hargis, 1989). MATERIALS AND
METHODS
Tissue homogenization
Abdominal fat pads from 9-week-old broilers were excised and frozen at -70°C for subsequent analysis. Tissues were homogenized with a Virtis (Model 23) stainless steel blade homogenizer in 3.3 vols of phosphate-buffered saline, pH 7.4, at room temperature. The homogenate was 585
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centrifuged at 800g for I0 min at 4°C and the supernatant collected and centrifuged at 100,000g for 60 min. In preparation for gel filtration, the cytosolic fraction was concentrated /0-fold with an Amicon 8200 ultrafiltration unit containing a Diaflo ultrafiltration membrane (10 YM2 62 mm) (Amicon, Beverly, MA, USA) with a mol. wt cut off at 1000Da and the concentrate was filtered through a 0.22 # m membrane to remove particulate matter.
Support Laboratory (College Station, TX, USA). The A-FABP sample was lyophilized and rehydrated in 10 mM ammonium bicarbonate buffer. Three nanomoles of this sample was dot-blotted onto an Imobilon (Millipore Corporation, Bedford, MA, USA) membrane and subjected to HCI vapor-phase hydrolysis for 60min at 150°C. Free amino acids were then derivatized with phenylisothiocyanate (PITC) and the PITC-amino acid derivatives were analyzed by high performance liquid chromatography (HPLC).
Gel filtration Filtered adipose concentrate was applied to a column (2.6 x 100cm) of Sephadex G-75 (bead size 40-120#m) equilibrated with 30 mM Tris-HCl, pH 9.0, at 4°C. Fractions were collected at a flow rate of 35 ml/hr. Optical densities were read at 280 nm using a Beckman spectrophotometer (Model DU-6). Fractions from the Sephadex G-75 column containing fatty acid binding activity were identified using a modification of the fatty acid binding assay of Glatz and Veerkamp (1983) and the presence of low mol. wt proteins were verified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970).
Fatty acid binding assay Relative fatty acid binding activities were determined according to a modification of the method of Glatz and Veerkamp (1983). A constant concentration of protein from G-75 fractions of purified A-FABP was incubated with 250pmol of t4C-labeled fatty acid (spec. act. of ~ 55 mCi/mmol) (Amersham, Arlington Heights, IL, USA) in 200 pl of 10 mM K+PO4, 0. 1% gelatin, 0.01% thimerasol buffer for 3 hr at room temperature. Following incubation, 500#1 of hydroxyalkoxypropyl dextran VI (60:40) (Glatz and Veerkamp, 1983) was added, and the tubes vortexed. Assay tubes were then centrifuged at 14,000g for 2min, 250 pl of supernatant collected, and DPM determined using a Beckman (Model LS 5800) liquid scintillation counter. Non-specific binding (NSB) was determined by incubation of 250pmol 14C-labeled fatty acid with 300#1 of buffer only. Binding of fatty acid by A-FABP was adjusted for NSB.
Electrophoresis and isoelectric focusing Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed by the method of Laemmli (1970). A dissociating, discontinuous buffer system was used in 1.0 mm thick slab gels with a 10-20% gradient (pH 8.8) resolving gel and a 4% (pH 6.8) stacking gel. Isoelectric focusing was performed by a modification of the method of Saravis and Zamcheck (1979) at 4°C in a horizontal isoelectric focusing chamber (LKB Inst. Gaithersburg, MD, USA). Pre-poured Isogel agarose gels (FMC Bioproducts, Rockland, ME, USA) pH range 3-10 were used. Acetic acid (0.5 N) and NaOH (I .0 N) served as the anode and cathode solutions, respectively. Samples (15-45/~1) of A-FABP and pI markers were loaded into wells of the sample mask. Electrofocusing was performed for 10 min at 2.0 W, the sample mask was then removed, and focusing was continued for 35-40 min at 17 W (1500 V max.). Gels were silver stained according to the method of Black (1985).
Cholesterol binding assay Relative cholesterol binding activity was determined according to a modification of the fatty acid binding assay used in this study. Constant concentrations of purified A-FABP were incubated with 0.144pmol of 3H-labeled cholesterol (spec. act. of 93 Ci/mmol) (Amersham, Arlington Heights, IL, USA) in 10mM K+PO4, 0.1% gelatin, 0.01% thimerasol buffer to a total assay volume of 700 ttl for 3 hr at room temperature. Following incubation, the contents of each assay tube was applied to a column of Sephadex G-25M (Pharmacia LKB Biotechnology, Piscataway, N J, USA) to separate protein-bound 3H-cholesterol and free 3H-cholesterol. Columns were eluted with assay buffer and fractions of 0.25 ml were collected and DPM determined using a Beckman (Model LS 5800) liquid scintillation counter.
Amino acid composition Amino acid composition analysis was performed by the Texas Agricultural Experiment Station Biotechnology
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Fig. 1. Separation of chicken adipose cytosolic proteins on a column of Sephadex G-75. Chicken adipose soluble proteins were applied to a I00 x 2.6 cm column of Sephadex G-75 equilibrated with 30 mM Tris-HC1, pH 9.0. Fractions were collected at a rate of 35 ml/hr. Optical densities were read at 280 nm and relative fatty acid binding activity was determined by a modification of the method of Glatz and Veerkamp (1983).
Adipose fatty acid binding protein
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Fig. 2. SDS-PAGE at pH 8.8 of FABP containing column fractions from chicken adipose at various steps of purification. Fractions exhibiting fatty acid binding activity were examined by SDS-PAGE (10-20% gradient, 1.0 mm slab gels). Lane 1, calibration proteins (with mol. wt): bovine albumin (66,000), ovalbumin (45,000), glyceraldehyde-3-phosphate dehydrogenase (36,000), carbonic anhydrase (29,000), trypsinogen (24,000), trypsin inhibitor (20,100), alpha-lactalbumin (14,200); lane 2, A-FABP from Sephadex G-75 column fraction 26; lane 3, adipose soluble proteins after concentration with Amicon 8200 ultrafiltration; lane 4, pooled adipose soluble proteins prior to concentration; lane 5, calibration proteins as in lane 1. Preparation of polyclonal antisera Domestic goats (Capra hircus) were injected intradermally with approximately 150#g of A-FABP in an adjuvated, emulsified solution. Two weeks later, goats were given a second injection of 75/~g of A-FABP in a saline-oil emulsion. Ten days later, sera was titered and monospecificity determined using Western Blot analysis. Animals were immunized and bled according to federal laws and guidelines by a state licensed and federally accredited veterinarian. Western blot analysis Proteins in chicken liver, heart and adipose cytosol were separated using SDS-PAGE (Laemmli, 1970) and then electrophoretically transferred to nitrocellulose. Following blocking of the nitrocellulose membrane with 3% bovine serum albumin (BSA), the membrane was incubated with goat anti-chicken A-FABP sera, 1: 5,000. Rabbit antigoat IgG alkaline phosphatase conjugate (Bio-Rad, Richmond, CA, USA), 1:2,000 was used as the secondary antibody. Alkaline phosphatase conjugate substrate kit (Bio-Rad, Richmond, CA, USA) was used for color development.
RESULTS AND DISCUSSION A concentrated preparation of chicken adipose cytosolic protein was fractionated by gel filtration chromatography and column fractions were analyzed for FABP activity. Fatty acid binding activity was present in both high and low mol. wt fractions (Fig. 1). These samples were subjected to S D S - P A G E (10-20% gradient, 1.0mm slab gels) (Laemmli, 1970). The high mol. wt fractions contained albumin (data not shown) and the low mol. wt fractions (fractions 30-40) contained a single protein band at a mol. wt of ca 14,400 (Fig. 2). Adipose samples from the low mol. wt fractions (fractions 30--40) were pooled and subjected to isoelectric focusing. The A-FABP sample focused as a single band at pI 5.1, verifying purification (Fig. 3). A number of investigators have provided evidence for the participation of fatty acid binding proteins in the uptake, transport and esterification of longchain fatty acids in a variety of mammalian tissues
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G.H. SAMSet al. Table I. Amino acid composition of fatty acid binding proteins Chicken Rat Mouse A-FABP H-FABP* A-FABP* Amino probable probable probable acid residues residues residues Asx 15 15 14 GIx 16 12 10 Ser 10 9 9 Gly 14 I1 10 His 2 4 (1 Arg 6 5 7 Thr 10 18 10 Ala 9 7 6 Pro 5 I I Tyr 3 2 2 Val I0 10 [ Met 1 2 4 Ile 6 4 Leu I0 10 6 Phe 6 6 5 Lys 12 12 13 Trp n.d. 2 I *Data from Offner et al., 1986.
Fig. 3. isoelectric focusing of fractions containing chicken adipose FABP isolated in this study, lsoelectric focusing was performed at 4'C in a horizontal isoelectric focusing chamber (LKB Inst., Gaithersburg, MD, USA). Prepoured Isogel agarose gels (FMC Bioproducts, Rockland, ME, USA) pH range 3 10 were used. The gel was silver stained. Lane A, calibration proteins (with pI): horse myoglobin major (7.4), horse myoglobin minor (7.0), carbonic anhydrase (6.1), fl-lactoglobulin B (5.5), fl-lactoglobulin A (5.4), ovalbumin (4.8); lane B, A-FABP from chicken adipose. including liver, intestine and heart (Ockner et al., 1972; Ockner and Manning, 1974; Fournier et al., 1978; Bass, 1985; Dempsey et al., 1985; Glatz et al., 1985). The present study provides evidence for the existence of a low molecular weight F A B P ( ~ 14,400) in chicken adipose tissue. Low mol. wt proteins exhibiting fatty acid binding activity have previously been purified from avian liver and intestinal duodenum (Sewell et al., 1989a,b). Unlike the procedures to purify FABPs from intestinal and hepatic tissues which resulted in relatively low yields (Sewell et al., 1989a,b), avian adipose F A B P was isolated in relatively high concentrations using a simple two-step procedure involving molecular sieve ultrafiltration and molecular sieve chromatography. Due to the primary function of adipose tissue in lipid storage, the large energy stores present in this tissue, and the proposed function of these low mol. wt proteins in lipid transport, an abundance of A - F A B P , as observed, might be expected.
The isoelectric point of 5.1 determined for chicken A - F A B P is similar to that reported for rat heart (pl 5.0) and bovine heart (pi4.9 to 5.0) FABPs (Fournier et al., 1978; Jagschies, 1985). There have been no reports in the literature of a pl value for mammalian A - F A B P , but Sacchettini et al. (1986) reported heart FABPs to be highly homologous structurally to adipose FABP. An F A B P isolated from rat m a m m a r y tissue was reported to have a pl value of 4.8-4.9 (Jones et al., 1988). Jones et al. (1988) also demonstrated immunochemical homology between the rat m a m m a r y protein and rat heart FABP. The acidic pl of chicken A - F A B P may be due in part to the presence of associated fatty acids. Several forms of L - F A B P with different isoelectric points ranging from 5.2-6.9 have been reported (Bass, 1985). The multiple forms of L - F A B P have been proposed to be due to various ligands bound to the protein (Bass, 1985; Takahashi et al., 1983). The amino acid composition of chicken A - F A B P is shown in Table 1. Comparison with amino acid composition of rat heart F A B P and murine 3T3 adipocyte F A B P indicate some compositional homology of chicken A - F A B P with mammalian FABPs (Offner et al., 1986). Correlation coefficients of amino acid composition data from both avian and mammalian species suggests A - F A B P is relatively more homologous with rat heart F A B P than with murine 3T3 adipocyte F A B P (Table 2). The murine adipocyte F A B P is high in valine, lysine and aspartare, while rat heart F A B P is highest in threonine, aspartate, glutamate, glycine and lysine. Chicken A - F A B P is also high in relative content of aspartate. glutamate, glycine and lysine. The determination of amino acid composition did not distinguish between the acid or amide forms of the Glx and Asx residues. It may be speculated, however, that the Asx and Glx Table 2. Correlation coefficientsof FABP amino acid composition with chicken A-FABP Chicken L-FABP 0.81 Rat H-FABP 0.83 Murine A-FABP 0.75 Rat L-FABP 0.74
Adipose fatty acid binding protein Table 3. In vitro bindingof fatty acids to A-FABP pmole FA/ Labeled ligand nag A-FABP '4C-palmitate(CI6: 0) 225 + 7* 14C-oleate(C18:1) 348 ! 91"?:~ 14C-linoleate(CI8:2) 453 + 10t 14C-palmitylCoA 318 _+51~ *?:~Means+ SEM significantlydiffer (P ~<0.06) if superscriptsdiffer. residues were mainly in the carboxyl form as reflected in the acidic pI of chicken A-FABP. As an indice of functionality, relative binding capacity of chicken A-FABP for various ligands was assessed. Relative binding activity of A-FABP was greatest for linoleate followed by oleate, palmityl CoA and palmitate (Table 3); no binding affinity ( < 0.4 pmol cholesterol/mg A-FABP) for cholesterol was detected. Few studies examining the binding affinity of mammalian A-FABP for various ligands have been conducted. Haq et al. (1982) reported that flavaspidic acid, a compound which competes with long-chain fatty acids for binding to FABPs, inhibited the uptake and esterification of [14C]-palmitic acid in rat adipocytes. Matarese et aL (1988) utilized a liposome
589
assay to determine binding properties of murine ALBP. Both oleic and retinoic acid were saturably bound by ALBP. These results led these authors to suggest that a role for retinoic acid in lipid metabolism of 3T3-L1 adipocytes may be mediated by ALBP. Few investigations have examined binding activity of cholesterol to FABPs. Bass (1985) reported L-FABP bound cholesterol and its derivatives only weakly, if at all. This finding is in contrast to investigations by Dempsey et al. (1985) which demonstrated that rat L-FABP was required for cholesterol biosynthesis. This discrepancy in L-FABP affinity for sterols may be due to an artifact of assay procedures used by Dempsey et al. (1985) and Vahouny et al. (1987). The endogenous ligands associated with A-FABPs have not been defined. Polyclonal antisera developed against A-FABP (Fig. 4) reacted monospecifically with a single low mol. wt protein in adipose cytosol. Additionally, at the concentration of 1 : 5000 used, anti-A-FABP sera exhibited a slight immunorecognition for a low mol. wt protein in chicken heart soluble proteins (not visible in photograph), but did not cross-react with chicken liver soluble proteins (Fig. 4). Analysis of anti-chicken A-FABP affinity for bovine skeletal
Fig. 4. Western blot analysis of Sephadex G-75 column fractions of chicken adipose, liver and heart tissues. Chicken liver, heart, and adipose cytosols were subjected to SDS-PAGE (10-20% gradient, 1.0 mm slab gels). Lane 1, calibration proteins (with tool. wt): bovine albumin (66,000), ovalbumin (45,000), glyceraldehyde-3-phosphate dehydrogenase (36,000), carbonic anhydrase (29,000), trypsinogen (24,000), trypsin inhibitor (20,000), alpha-lactalbumin (14,200); lane 2, chicken liver soluble proteins, lane 3, chicken heart soluble proteins; lane 4, chicken adipose soluble proteins. The proteins were electrophoretically transferred to nitrocellulose. A variation of the Bio-Rad immunoblot assay system (Bio-Rad, Richmond, CA, USA) was then used to assess homology of FABPs to chicken adipose FABP. Primary antibody (goat anti-chicken A-FABP) was used in a I: 5000 dilution and secondary antibody (rabbit anti-goat Ig-G, Bio-Rad, Richmond, CA, USA was used at a 1: 2000 dilution. Antisera prepared against chicken A-FABP exhibited affinity for 14,400mol. wt adipose protein (lane C), slight immunorecognition for the heart protein (lane B), and no immunorecognition for the liver protein (lane A).
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muscle, liver, or adipose soluble proteins indicated chicken A - F A B P sera exhibited immunorecognition for a low mol. wt protein in bovine skeletal muscle and to a lesser extent in bovine adipose but not for bovine liver soluble proteins (data not shown). F r o m this investigation, it appears that a low mol. wt protein exhibiting fatty acid binding properties exists in chicken adipose tissue. A m i n o acid composition and Western Blot data suggest this protein is structurally distinct from L - F A B P previously isolated from chicken liver (Sewell et al., 1989a). A family of these low mol. wt proteins which bind fatty acids, their metabolic products and sterols have been identified previously in tissues possessing active lipid metabolism pathways in mammals. Structural analysis of mammalian FABPs indicate regions of sequence homology between several tissue-specific FABPs and comparison with amino acid composition and functionality of avian FABPs suggests these proteins have been highly conserved throughout evolution. The abundance and distinctive pattern of tissue expression of F A B P s suggests a specialized role for these proteins in fatty acid metabolism. Further study is necessary to elucidate the functional importance of these abundant and apparently teleologically conserved lipid transport proteins. Acknowledgements--The authors would like to thank Dr T. Hayes and the Texas Agricultural Experiment Station Biochemistry Support Laboratory for determination of amino acid composition of A-FABP. We wish to thank Charles R. Young for his advice concerning isoelectric focusing and we thank Stella Wiese for photographing the electrophoresis gels.
REFERENCES Bass N. M. (1985) Function and regulation of hepatic and intestinal fatty acid binding proteins. Chem. Phys. Lipids 38, 95-114. Bernlohr D. A. (1988) Cloning, expression and characterization of a murine adipocyte lipid binding protein (ALBP) in E. coli. FASEB J., A1037. Bernlohr D. A., Doering T. L., Kelly T. J., Jr. and Lane M. D. (1985) Tissue specific expression of p422 protein, a putative lipid carrier, in mouse adipocytes. Biochem. biophys. Res. Commun. 132, 850-855. Black J. A. (1985) A silver stain for isoelectric focusing in agarose gel and its application for analyzing unconcentrated cerebrospinal fluid. Electorphoresis 6, 27-29. Chan L., Wei C-F., Li W-H., Yang C-Y., Ratner P., Pownall H., Gotto A. M., Jr. and Smith L. C. (1985) Human liver fatty acid binding cDNA and amino acid sequence. J. biol. Chem. 260, 2629-2632. Collins D. M. and Hargis P. S. (1989) Distribution of fatty acid binding proteins in tissues and plasma of Gallus Domesticus. Comp. Biochem. Physiol. 92B, 283-289. Dempsey M. E., Hargis P. S., McGuire D. M., McMahon A., Olson C. D., Salati L. M., Clarke S. D. and Towle H. C. (1985) Role of sterol carrier protein in cholesterol metabolism. Chem. Phys. Lipids 38, 223-237. Fournier N. C., Geoffroy M. and Deshusses J. (1978) Purification and characterization of long chain fatty acid binding proteins supplying the mitochondrial fl-oxidative system in the heart. Biochim. biophys. Acta 533, 457-464. Glatz J. F. C., Janssen A. M., Baerwaldt C. C. T. and Veerkamp J. H. (1985) Purification and characterization
of fatty acid binding proteins from rat heart and liver. Biochim. biophys. Acta 837, 57456. Glatz J. F. C. and Veerkamp J. H. (1983) Removal of fatty acids from serum albumin by Lipidex 1000 chromatography. J. biochem, biophys. Meth. 8, 57451. Haq R-U., Christodoulides L., Ketterer B. and Shrago E. (1982) Characterization and purification of fatty acid binding protein in rat and human adipose tissue. Biochim. biophys. Acta 713, 193-198. Jagschies G., Reers M., Unterberg C. and Spener F. (1985) Bovine fatty acid binding proteins. Isolation and characterization of two cardiac fatty acid binding proteins that are distinct from corresponding hepatic proteins. Eur. J. Biochem. 152, 537-545. Jones P. D., Carne A., Bass N. M. and Grigor M. R. (1988) Isolation and characterization of fatty acid binding proteins from mammary tissue of lactating rats. Biochem. J. 251,919-925. Laemmli U. K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage-T4. Nature 227, 680-683. Matarese V., Stone R. L. and Bernlohr D. A. (1988) Adipocyte lipid-binding protein, formerly p422, binds oleate retinoate in liposome assay. FASEB J., A1036. Ockner R. K. and Manning J. A. (1974) Fatty acid binding protein in small intestine. Identification, isolation and evidence for its role in cellular fatty acid transport. J. clin. Invest. 54, 326-338. Ockner R. K., Manning J. A., Poppenhausen R. B. and Ho W. K. L. (1972) A binding protein for fatty acids in cytosol of intestinal mucosa, liver, myocardium and other tissues. Science 177, 56-58. Offner G. D., Troxler R. F. and Brecher P. (1986) Characterization of a fatty acid binding protein from rat heart. J. biol. Chem. 261, 5584-5589. Potter B. J., Stump D., Schwieterman W., Sorrentino D., Jacobs L. N., Kiang C-L., Rand J. H. and Berk P. D. (1987) Isolation and partial characterization of plasma membrane fatty acid binding proteins from myocardium and adipose tissue and their relationship to analogous proteins in liver and gut. Biochem. biophys. Res. Commun. 148, 1370-1376. Sacchettini J. C., Said B., Schulz H. and Gordon J. 1. (1986) Rat heart fatty acid binding protein is highly homologous to the murine adipocyte 422 protein and the P2 protein of peripheral nerve myelin. J. biol. Chem. 261, 8218-8223. Saravis C. A. and Zamcheck N. (1979) Isoelectric focusing in agarose. J. immunol. Meths. 29, 91-96. Sewell J. E., Davis S. K. and Hargis P. S. (1989a) Isolation, characterization, and expression of fatty acid binding protein in the liver of Gallus domesticus. Comp. Biochem. Physiol. 92B, 509-516. Sewell J. E., Young C. R., Davis S. K. and Hargis P. S. (1989b) Isolation and characterization of two fatty acid binding proteins from intestine of Gallus domesticus. Comp. Biochem. Physiol. 92B, 6234529. Smith S. B., Ekeren P. A. and Sanders J. O. (1985) Fatty acid binding protein activities in bovine muscle, liver and adipose tissue. J. Nutr. 115, 1535-1539. Sweetser, D. A., Heuckerotb R. O. and Gordon J. 1. (1987) The metabolic significance of mammalian fatty acid binding proteins: Abundant proteins in search of a function. A. Rev. Nutr. 7, 337-359. Takahashi K., Odani S. and Ono T. (1983) Primary structure of rat-liver Z-protein. A low MR cytosol protein that binds sterols, fatty acids, and other small molecules. Eur. J. Biochem. 135, 589-601. Vahouny G. V., Chanderbhan R. and Kharroubi A. (1987) Sterol carrier and lipid transfer proteins. Adv. Lipid Res. 22, 83 113.
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ISOLATION A N D CHARACTERIZATION OF A FATTY ACID B I N D I N G PROTEIN IN ADIPOSE TISSUE OF GALLUS D O M E S T I C U S G. H. SAMS,* B. M. HARGIS*~"and P. S. HARGIS*J/ Departments of *Poultry Science and tVeterinary Microbiology and Parasitology, Texas Agricultural Experiment Station, Texas A & M University System, College Station, TX 77843-2472, USA (Tel: 409 845 7537) (Received 11 December 1989)
Almtraet--1. Fatty acid binding protein (A-FABP) was isolated from chicken adipose cytosol. 2. Relative tool. wt of chicken A-FABP was determined to be 14,400 from SDS-polyaerylamide electrophoresis; the pI was 5.1; and amino acid composition data indicated structural homology with mammalian heart and adipose FABPs. 3. Polyclonal antisera prepared against A-FABP exhibited monospecificity for chicken A-FABP and no cross-reactivity with chicken liver proteins was observed. 4. Determination of relative ligand binding characteristics indicated A-FABP exhibited greatest binding activity in response to linoleate, followed by oleate, palmityl CoA and palmitate; no binding attinity for cholesterol was detected.
INTRODUCTION Several tissue-specific fatty acid binding proteins (FABPs) have been identified in a number of species (Ockner et al., 1972; Smith et al., 1985; Sweetser et al., 1987; Sewell et al., 1989a,b). Sequence analysis of mammalian FABPs has shown that these proteins are each structurally unique yet each contain regions of homology suggesting they have descended from a common ancestral sequence (Chan et al., 1985). These low tool. wt proteins demonstrate an affinity for fatty acids and each appear to function in the metabolism and intracellular transport of lipids (Bass, 1985; Dempsey et al., 1985; Glatz et al., 1985; Sweetser et al., 1987). Of these low mol. wt (12,000-15,000) proteins, hepatic FABP (L-FABP) and intestinal FABP from rat tissues are best characterized (Sweetser et al., 1987). Only limited investigations of FABP in adipose tissue exist in current literature (Haq et al., 1982; Smith et al., 1985; Bernlohr et al., 1985). Adipose FABP (A-FABP) isolated from the rat has been shown to be immunochemically similar to rat L-FABP (Haq et al., 1982), but to be immunochemically distinct from A-FABP isolated from human adipose. Haq et al. (1982) suggested that the apparent differences in antigenic epitopes may be due to species specific differences in lipid metabolic pathways and consequent differences in the evolutionary differentiation of the proteins in the two species. Proteins exhibiting similar activities to FABPs have also been identified in adipose tissue of the rat by Potter et al. (1987), but these are large mol. wt proteins (40,000) associated with the plasma membrane. Bernlohr et al. (1985) identified a low mol. wt protein in murine 3T3-L1 adipocytes ~/Author to whom correspondence should be addressed.
that exhibits 20-30% amino acid sequence homology to FABPs of rat liver and intestine. This protein was originally called p422 and is now designated as adipocyte lipid binding, protein (ALBP) (Bernlohr, 1988). Through the use of immunoblotting and cDNA hybridization techniques, Bernlohr et al. (1985) concluded that ALBP was Specific to adipose tissue in the mouse. Findings that ALBP mRNA increased in abundance during differentiation of 3T3-L1 fibroblasts to mature adipocytes lead Bernlohr et al. (1985) to suggest a role for this protein in intracellular fatty acid transfer and recent evidence indicates murine ALBP binds fatty acids and retinoids in vitro (Matarese et al., 1988). The objective of this study was to isolate and characterize A-FABP in the chicken, to assess relative binding characteristics of this protein for various ligands and to develop a monospecific polyclonal antisera. The chicken provides a unique model for the study of mechanisms involved in lipid metabolism due to the fact that in this species, nearly all de novo synthesis of fatty acids occurs in the liver, as it does in man, and adipose tissue functions mainly for lipid storage. Adipose tissue contains the largest overall energy stores in the body, primarily in the form of triacylglycerol fatty acids. Previous work has resulted in the purification of L-FABP from chicken liver cytosol (Sewell et al., 1989a) and provided evidence for the presence of a low tool. wt fatty acid binding protein in adipose tissue (Collins and Hargis, 1989). MATERIALS AND
METHODS
Tissue homogenization
Abdominal fat pads from 9-week-old broilers were excised and frozen at -70°C for subsequent analysis. Tissues were homogenized with a Virtis (Model 23) stainless steel blade homogenizer in 3.3 vols of phosphate-buffered saline, pH 7.4, at room temperature. The homogenate was 585
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centrifuged at 800g for I0 min at 4°C and the supernatant collected and centrifuged at 100,000g for 60 min. In preparation for gel filtration, the cytosolic fraction was concentrated /0-fold with an Amicon 8200 ultrafiltration unit containing a Diaflo ultrafiltration membrane (10 YM2 62 mm) (Amicon, Beverly, MA, USA) with a mol. wt cut off at 1000Da and the concentrate was filtered through a 0.22 # m membrane to remove particulate matter.
Support Laboratory (College Station, TX, USA). The A-FABP sample was lyophilized and rehydrated in 10 mM ammonium bicarbonate buffer. Three nanomoles of this sample was dot-blotted onto an Imobilon (Millipore Corporation, Bedford, MA, USA) membrane and subjected to HCI vapor-phase hydrolysis for 60min at 150°C. Free amino acids were then derivatized with phenylisothiocyanate (PITC) and the PITC-amino acid derivatives were analyzed by high performance liquid chromatography (HPLC).
Gel filtration Filtered adipose concentrate was applied to a column (2.6 x 100cm) of Sephadex G-75 (bead size 40-120#m) equilibrated with 30 mM Tris-HCl, pH 9.0, at 4°C. Fractions were collected at a flow rate of 35 ml/hr. Optical densities were read at 280 nm using a Beckman spectrophotometer (Model DU-6). Fractions from the Sephadex G-75 column containing fatty acid binding activity were identified using a modification of the fatty acid binding assay of Glatz and Veerkamp (1983) and the presence of low mol. wt proteins were verified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970).
Fatty acid binding assay Relative fatty acid binding activities were determined according to a modification of the method of Glatz and Veerkamp (1983). A constant concentration of protein from G-75 fractions of purified A-FABP was incubated with 250pmol of t4C-labeled fatty acid (spec. act. of ~ 55 mCi/mmol) (Amersham, Arlington Heights, IL, USA) in 200 pl of 10 mM K+PO4, 0. 1% gelatin, 0.01% thimerasol buffer for 3 hr at room temperature. Following incubation, 500#1 of hydroxyalkoxypropyl dextran VI (60:40) (Glatz and Veerkamp, 1983) was added, and the tubes vortexed. Assay tubes were then centrifuged at 14,000g for 2min, 250 pl of supernatant collected, and DPM determined using a Beckman (Model LS 5800) liquid scintillation counter. Non-specific binding (NSB) was determined by incubation of 250pmol 14C-labeled fatty acid with 300#1 of buffer only. Binding of fatty acid by A-FABP was adjusted for NSB.
Electrophoresis and isoelectric focusing Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed by the method of Laemmli (1970). A dissociating, discontinuous buffer system was used in 1.0 mm thick slab gels with a 10-20% gradient (pH 8.8) resolving gel and a 4% (pH 6.8) stacking gel. Isoelectric focusing was performed by a modification of the method of Saravis and Zamcheck (1979) at 4°C in a horizontal isoelectric focusing chamber (LKB Inst. Gaithersburg, MD, USA). Pre-poured Isogel agarose gels (FMC Bioproducts, Rockland, ME, USA) pH range 3-10 were used. Acetic acid (0.5 N) and NaOH (I .0 N) served as the anode and cathode solutions, respectively. Samples (15-45/~1) of A-FABP and pI markers were loaded into wells of the sample mask. Electrofocusing was performed for 10 min at 2.0 W, the sample mask was then removed, and focusing was continued for 35-40 min at 17 W (1500 V max.). Gels were silver stained according to the method of Black (1985).
Cholesterol binding assay Relative cholesterol binding activity was determined according to a modification of the fatty acid binding assay used in this study. Constant concentrations of purified A-FABP were incubated with 0.144pmol of 3H-labeled cholesterol (spec. act. of 93 Ci/mmol) (Amersham, Arlington Heights, IL, USA) in 10mM K+PO4, 0.1% gelatin, 0.01% thimerasol buffer to a total assay volume of 700 ttl for 3 hr at room temperature. Following incubation, the contents of each assay tube was applied to a column of Sephadex G-25M (Pharmacia LKB Biotechnology, Piscataway, N J, USA) to separate protein-bound 3H-cholesterol and free 3H-cholesterol. Columns were eluted with assay buffer and fractions of 0.25 ml were collected and DPM determined using a Beckman (Model LS 5800) liquid scintillation counter.
Amino acid composition Amino acid composition analysis was performed by the Texas Agricultural Experiment Station Biotechnology
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Fig. 1. Separation of chicken adipose cytosolic proteins on a column of Sephadex G-75. Chicken adipose soluble proteins were applied to a I00 x 2.6 cm column of Sephadex G-75 equilibrated with 30 mM Tris-HC1, pH 9.0. Fractions were collected at a rate of 35 ml/hr. Optical densities were read at 280 nm and relative fatty acid binding activity was determined by a modification of the method of Glatz and Veerkamp (1983).
Adipose fatty acid binding protein
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Fig. 2. SDS-PAGE at pH 8.8 of FABP containing column fractions from chicken adipose at various steps of purification. Fractions exhibiting fatty acid binding activity were examined by SDS-PAGE (10-20% gradient, 1.0 mm slab gels). Lane 1, calibration proteins (with mol. wt): bovine albumin (66,000), ovalbumin (45,000), glyceraldehyde-3-phosphate dehydrogenase (36,000), carbonic anhydrase (29,000), trypsinogen (24,000), trypsin inhibitor (20,100), alpha-lactalbumin (14,200); lane 2, A-FABP from Sephadex G-75 column fraction 26; lane 3, adipose soluble proteins after concentration with Amicon 8200 ultrafiltration; lane 4, pooled adipose soluble proteins prior to concentration; lane 5, calibration proteins as in lane 1. Preparation of polyclonal antisera Domestic goats (Capra hircus) were injected intradermally with approximately 150#g of A-FABP in an adjuvated, emulsified solution. Two weeks later, goats were given a second injection of 75/~g of A-FABP in a saline-oil emulsion. Ten days later, sera was titered and monospecificity determined using Western Blot analysis. Animals were immunized and bled according to federal laws and guidelines by a state licensed and federally accredited veterinarian. Western blot analysis Proteins in chicken liver, heart and adipose cytosol were separated using SDS-PAGE (Laemmli, 1970) and then electrophoretically transferred to nitrocellulose. Following blocking of the nitrocellulose membrane with 3% bovine serum albumin (BSA), the membrane was incubated with goat anti-chicken A-FABP sera, 1: 5,000. Rabbit antigoat IgG alkaline phosphatase conjugate (Bio-Rad, Richmond, CA, USA), 1:2,000 was used as the secondary antibody. Alkaline phosphatase conjugate substrate kit (Bio-Rad, Richmond, CA, USA) was used for color development.
RESULTS AND DISCUSSION A concentrated preparation of chicken adipose cytosolic protein was fractionated by gel filtration chromatography and column fractions were analyzed for FABP activity. Fatty acid binding activity was present in both high and low mol. wt fractions (Fig. 1). These samples were subjected to S D S - P A G E (10-20% gradient, 1.0mm slab gels) (Laemmli, 1970). The high mol. wt fractions contained albumin (data not shown) and the low mol. wt fractions (fractions 30-40) contained a single protein band at a mol. wt of ca 14,400 (Fig. 2). Adipose samples from the low mol. wt fractions (fractions 30--40) were pooled and subjected to isoelectric focusing. The A-FABP sample focused as a single band at pI 5.1, verifying purification (Fig. 3). A number of investigators have provided evidence for the participation of fatty acid binding proteins in the uptake, transport and esterification of longchain fatty acids in a variety of mammalian tissues
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G.H. SAMSet al. Table I. Amino acid composition of fatty acid binding proteins Chicken Rat Mouse A-FABP H-FABP* A-FABP* Amino probable probable probable acid residues residues residues Asx 15 15 14 GIx 16 12 10 Ser 10 9 9 Gly 14 I1 10 His 2 4 (1 Arg 6 5 7 Thr 10 18 10 Ala 9 7 6 Pro 5 I I Tyr 3 2 2 Val I0 10 [ Met 1 2 4 Ile 6 4 Leu I0 10 6 Phe 6 6 5 Lys 12 12 13 Trp n.d. 2 I *Data from Offner et al., 1986.
Fig. 3. isoelectric focusing of fractions containing chicken adipose FABP isolated in this study, lsoelectric focusing was performed at 4'C in a horizontal isoelectric focusing chamber (LKB Inst., Gaithersburg, MD, USA). Prepoured Isogel agarose gels (FMC Bioproducts, Rockland, ME, USA) pH range 3 10 were used. The gel was silver stained. Lane A, calibration proteins (with pI): horse myoglobin major (7.4), horse myoglobin minor (7.0), carbonic anhydrase (6.1), fl-lactoglobulin B (5.5), fl-lactoglobulin A (5.4), ovalbumin (4.8); lane B, A-FABP from chicken adipose. including liver, intestine and heart (Ockner et al., 1972; Ockner and Manning, 1974; Fournier et al., 1978; Bass, 1985; Dempsey et al., 1985; Glatz et al., 1985). The present study provides evidence for the existence of a low molecular weight F A B P ( ~ 14,400) in chicken adipose tissue. Low mol. wt proteins exhibiting fatty acid binding activity have previously been purified from avian liver and intestinal duodenum (Sewell et al., 1989a,b). Unlike the procedures to purify FABPs from intestinal and hepatic tissues which resulted in relatively low yields (Sewell et al., 1989a,b), avian adipose F A B P was isolated in relatively high concentrations using a simple two-step procedure involving molecular sieve ultrafiltration and molecular sieve chromatography. Due to the primary function of adipose tissue in lipid storage, the large energy stores present in this tissue, and the proposed function of these low mol. wt proteins in lipid transport, an abundance of A - F A B P , as observed, might be expected.
The isoelectric point of 5.1 determined for chicken A - F A B P is similar to that reported for rat heart (pl 5.0) and bovine heart (pi4.9 to 5.0) FABPs (Fournier et al., 1978; Jagschies, 1985). There have been no reports in the literature of a pl value for mammalian A - F A B P , but Sacchettini et al. (1986) reported heart FABPs to be highly homologous structurally to adipose FABP. An F A B P isolated from rat m a m m a r y tissue was reported to have a pl value of 4.8-4.9 (Jones et al., 1988). Jones et al. (1988) also demonstrated immunochemical homology between the rat m a m m a r y protein and rat heart FABP. The acidic pl of chicken A - F A B P may be due in part to the presence of associated fatty acids. Several forms of L - F A B P with different isoelectric points ranging from 5.2-6.9 have been reported (Bass, 1985). The multiple forms of L - F A B P have been proposed to be due to various ligands bound to the protein (Bass, 1985; Takahashi et al., 1983). The amino acid composition of chicken A - F A B P is shown in Table 1. Comparison with amino acid composition of rat heart F A B P and murine 3T3 adipocyte F A B P indicate some compositional homology of chicken A - F A B P with mammalian FABPs (Offner et al., 1986). Correlation coefficients of amino acid composition data from both avian and mammalian species suggests A - F A B P is relatively more homologous with rat heart F A B P than with murine 3T3 adipocyte F A B P (Table 2). The murine adipocyte F A B P is high in valine, lysine and aspartare, while rat heart F A B P is highest in threonine, aspartate, glutamate, glycine and lysine. Chicken A - F A B P is also high in relative content of aspartate. glutamate, glycine and lysine. The determination of amino acid composition did not distinguish between the acid or amide forms of the Glx and Asx residues. It may be speculated, however, that the Asx and Glx Table 2. Correlation coefficientsof FABP amino acid composition with chicken A-FABP Chicken L-FABP 0.81 Rat H-FABP 0.83 Murine A-FABP 0.75 Rat L-FABP 0.74
Adipose fatty acid binding protein Table 3. In vitro bindingof fatty acids to A-FABP pmole FA/ Labeled ligand nag A-FABP '4C-palmitate(CI6: 0) 225 + 7* 14C-oleate(C18:1) 348 ! 91"?:~ 14C-linoleate(CI8:2) 453 + 10t 14C-palmitylCoA 318 _+51~ *?:~Means+ SEM significantlydiffer (P ~<0.06) if superscriptsdiffer. residues were mainly in the carboxyl form as reflected in the acidic pI of chicken A-FABP. As an indice of functionality, relative binding capacity of chicken A-FABP for various ligands was assessed. Relative binding activity of A-FABP was greatest for linoleate followed by oleate, palmityl CoA and palmitate (Table 3); no binding affinity ( < 0.4 pmol cholesterol/mg A-FABP) for cholesterol was detected. Few studies examining the binding affinity of mammalian A-FABP for various ligands have been conducted. Haq et al. (1982) reported that flavaspidic acid, a compound which competes with long-chain fatty acids for binding to FABPs, inhibited the uptake and esterification of [14C]-palmitic acid in rat adipocytes. Matarese et aL (1988) utilized a liposome
589
assay to determine binding properties of murine ALBP. Both oleic and retinoic acid were saturably bound by ALBP. These results led these authors to suggest that a role for retinoic acid in lipid metabolism of 3T3-L1 adipocytes may be mediated by ALBP. Few investigations have examined binding activity of cholesterol to FABPs. Bass (1985) reported L-FABP bound cholesterol and its derivatives only weakly, if at all. This finding is in contrast to investigations by Dempsey et al. (1985) which demonstrated that rat L-FABP was required for cholesterol biosynthesis. This discrepancy in L-FABP affinity for sterols may be due to an artifact of assay procedures used by Dempsey et al. (1985) and Vahouny et al. (1987). The endogenous ligands associated with A-FABPs have not been defined. Polyclonal antisera developed against A-FABP (Fig. 4) reacted monospecifically with a single low mol. wt protein in adipose cytosol. Additionally, at the concentration of 1 : 5000 used, anti-A-FABP sera exhibited a slight immunorecognition for a low mol. wt protein in chicken heart soluble proteins (not visible in photograph), but did not cross-react with chicken liver soluble proteins (Fig. 4). Analysis of anti-chicken A-FABP affinity for bovine skeletal
Fig. 4. Western blot analysis of Sephadex G-75 column fractions of chicken adipose, liver and heart tissues. Chicken liver, heart, and adipose cytosols were subjected to SDS-PAGE (10-20% gradient, 1.0 mm slab gels). Lane 1, calibration proteins (with tool. wt): bovine albumin (66,000), ovalbumin (45,000), glyceraldehyde-3-phosphate dehydrogenase (36,000), carbonic anhydrase (29,000), trypsinogen (24,000), trypsin inhibitor (20,000), alpha-lactalbumin (14,200); lane 2, chicken liver soluble proteins, lane 3, chicken heart soluble proteins; lane 4, chicken adipose soluble proteins. The proteins were electrophoretically transferred to nitrocellulose. A variation of the Bio-Rad immunoblot assay system (Bio-Rad, Richmond, CA, USA) was then used to assess homology of FABPs to chicken adipose FABP. Primary antibody (goat anti-chicken A-FABP) was used in a I: 5000 dilution and secondary antibody (rabbit anti-goat Ig-G, Bio-Rad, Richmond, CA, USA was used at a 1: 2000 dilution. Antisera prepared against chicken A-FABP exhibited affinity for 14,400mol. wt adipose protein (lane C), slight immunorecognition for the heart protein (lane B), and no immunorecognition for the liver protein (lane A).
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muscle, liver, or adipose soluble proteins indicated chicken A - F A B P sera exhibited immunorecognition for a low mol. wt protein in bovine skeletal muscle and to a lesser extent in bovine adipose but not for bovine liver soluble proteins (data not shown). F r o m this investigation, it appears that a low mol. wt protein exhibiting fatty acid binding properties exists in chicken adipose tissue. A m i n o acid composition and Western Blot data suggest this protein is structurally distinct from L - F A B P previously isolated from chicken liver (Sewell et al., 1989a). A family of these low mol. wt proteins which bind fatty acids, their metabolic products and sterols have been identified previously in tissues possessing active lipid metabolism pathways in mammals. Structural analysis of mammalian FABPs indicate regions of sequence homology between several tissue-specific FABPs and comparison with amino acid composition and functionality of avian FABPs suggests these proteins have been highly conserved throughout evolution. The abundance and distinctive pattern of tissue expression of F A B P s suggests a specialized role for these proteins in fatty acid metabolism. Further study is necessary to elucidate the functional importance of these abundant and apparently teleologically conserved lipid transport proteins. Acknowledgements--The authors would like to thank Dr T. Hayes and the Texas Agricultural Experiment Station Biochemistry Support Laboratory for determination of amino acid composition of A-FABP. We wish to thank Charles R. Young for his advice concerning isoelectric focusing and we thank Stella Wiese for photographing the electrophoresis gels.
REFERENCES Bass N. M. (1985) Function and regulation of hepatic and intestinal fatty acid binding proteins. Chem. Phys. Lipids 38, 95-114. Bernlohr D. A. (1988) Cloning, expression and characterization of a murine adipocyte lipid binding protein (ALBP) in E. coli. FASEB J., A1037. Bernlohr D. A., Doering T. L., Kelly T. J., Jr. and Lane M. D. (1985) Tissue specific expression of p422 protein, a putative lipid carrier, in mouse adipocytes. Biochem. biophys. Res. Commun. 132, 850-855. Black J. A. (1985) A silver stain for isoelectric focusing in agarose gel and its application for analyzing unconcentrated cerebrospinal fluid. Electorphoresis 6, 27-29. Chan L., Wei C-F., Li W-H., Yang C-Y., Ratner P., Pownall H., Gotto A. M., Jr. and Smith L. C. (1985) Human liver fatty acid binding cDNA and amino acid sequence. J. biol. Chem. 260, 2629-2632. Collins D. M. and Hargis P. S. (1989) Distribution of fatty acid binding proteins in tissues and plasma of Gallus Domesticus. Comp. Biochem. Physiol. 92B, 283-289. Dempsey M. E., Hargis P. S., McGuire D. M., McMahon A., Olson C. D., Salati L. M., Clarke S. D. and Towle H. C. (1985) Role of sterol carrier protein in cholesterol metabolism. Chem. Phys. Lipids 38, 223-237. Fournier N. C., Geoffroy M. and Deshusses J. (1978) Purification and characterization of long chain fatty acid binding proteins supplying the mitochondrial fl-oxidative system in the heart. Biochim. biophys. Acta 533, 457-464. Glatz J. F. C., Janssen A. M., Baerwaldt C. C. T. and Veerkamp J. H. (1985) Purification and characterization
of fatty acid binding proteins from rat heart and liver. Biochim. biophys. Acta 837, 57456. Glatz J. F. C. and Veerkamp J. H. (1983) Removal of fatty acids from serum albumin by Lipidex 1000 chromatography. J. biochem, biophys. Meth. 8, 57451. Haq R-U., Christodoulides L., Ketterer B. and Shrago E. (1982) Characterization and purification of fatty acid binding protein in rat and human adipose tissue. Biochim. biophys. Acta 713, 193-198. Jagschies G., Reers M., Unterberg C. and Spener F. (1985) Bovine fatty acid binding proteins. Isolation and characterization of two cardiac fatty acid binding proteins that are distinct from corresponding hepatic proteins. Eur. J. Biochem. 152, 537-545. Jones P. D., Carne A., Bass N. M. and Grigor M. R. (1988) Isolation and characterization of fatty acid binding proteins from mammary tissue of lactating rats. Biochem. J. 251,919-925. Laemmli U. K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage-T4. Nature 227, 680-683. Matarese V., Stone R. L. and Bernlohr D. A. (1988) Adipocyte lipid-binding protein, formerly p422, binds oleate retinoate in liposome assay. FASEB J., A1036. Ockner R. K. and Manning J. A. (1974) Fatty acid binding protein in small intestine. Identification, isolation and evidence for its role in cellular fatty acid transport. J. clin. Invest. 54, 326-338. Ockner R. K., Manning J. A., Poppenhausen R. B. and Ho W. K. L. (1972) A binding protein for fatty acids in cytosol of intestinal mucosa, liver, myocardium and other tissues. Science 177, 56-58. Offner G. D., Troxler R. F. and Brecher P. (1986) Characterization of a fatty acid binding protein from rat heart. J. biol. Chem. 261, 5584-5589. Potter B. J., Stump D., Schwieterman W., Sorrentino D., Jacobs L. N., Kiang C-L., Rand J. H. and Berk P. D. (1987) Isolation and partial characterization of plasma membrane fatty acid binding proteins from myocardium and adipose tissue and their relationship to analogous proteins in liver and gut. Biochem. biophys. Res. Commun. 148, 1370-1376. Sacchettini J. C., Said B., Schulz H. and Gordon J. 1. (1986) Rat heart fatty acid binding protein is highly homologous to the murine adipocyte 422 protein and the P2 protein of peripheral nerve myelin. J. biol. Chem. 261, 8218-8223. Saravis C. A. and Zamcheck N. (1979) Isoelectric focusing in agarose. J. immunol. Meths. 29, 91-96. Sewell J. E., Davis S. K. and Hargis P. S. (1989a) Isolation, characterization, and expression of fatty acid binding protein in the liver of Gallus domesticus. Comp. Biochem. Physiol. 92B, 509-516. Sewell J. E., Young C. R., Davis S. K. and Hargis P. S. (1989b) Isolation and characterization of two fatty acid binding proteins from intestine of Gallus domesticus. Comp. Biochem. Physiol. 92B, 6234529. Smith S. B., Ekeren P. A. and Sanders J. O. (1985) Fatty acid binding protein activities in bovine muscle, liver and adipose tissue. J. Nutr. 115, 1535-1539. Sweetser, D. A., Heuckerotb R. O. and Gordon J. 1. (1987) The metabolic significance of mammalian fatty acid binding proteins: Abundant proteins in search of a function. A. Rev. Nutr. 7, 337-359. Takahashi K., Odani S. and Ono T. (1983) Primary structure of rat-liver Z-protein. A low MR cytosol protein that binds sterols, fatty acids, and other small molecules. Eur. J. Biochem. 135, 589-601. Vahouny G. V., Chanderbhan R. and Kharroubi A. (1987) Sterol carrier and lipid transfer proteins. Adv. Lipid Res. 22, 83 113.