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Identification and characterization of a fatty acid binding protein in bovine mammary gland
Comp. Biochem. Physiol. Vol. 85B, No. 3, pp. 687~92, 1986
0305-0491/86 $3.00 +0.00 Pergamon Journals Ltd
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I D E N T I F I C A T I O N A N D C H A R A C T E R I Z A T I O N OF A F A T T Y A C I D B I N D I N G P R O T E I N IN B O V I N E MAMMARY GLAND* H. D. WHETSTONE,t W. L. HURLEY3~ and C. L. DAVIS Department of Animal Sciences, University of Illinois, Urbana, IL 61801, U.S.A (Tel: 217-333-1327) (Received 20 February 1986)
A fatty acid binding protein (FABP) was isolated from bovine mammary cytosol by gel filtration and ion exchange chromatography. 2. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a mol. wt. of 12,000. Isoelectric focusing showed two bands at pH 5.6 and 5.8. 3. FABP bound long chain fatty acids and their CoA thioesters, but not medium or short chain fatty acids. Affinity constant (Ka) for 18:1 was about 2 micromolar. 4. Endogenously bound fatty acids included 16:0, 18:0 and 18:1, in both covalent and noncovalent association with FABP. 5. Activities of microsomal phosphatidic acid phosphatase, fatty acid:CoA ligase or diacylglycerol acyltransferase were not affected by purified FABP in vitro. Abstract--1.
INTRODUCTION
Properties of fatty acid binding proteins have recently been reviewed (Glatz and Veerkamp, 1985). Fatty acid binding protein (FABP) is a low mol. wt (about 12,000) cytosolic protein which binds fatty acids and acyl CoA thioesters with high affinity. Its presence has been documented in a number of tissues which metabolize fatty acids including rat liver, myocardium, lung, intestinal mucosa, adipose, kidney, adrenal, and other tissues, bovine liver (Glatz and Veerkamp, 1985), and recently in bovine muscle and adipose (Smith et al., 1985). These FABP are thought to participate in intracellular fatty acid transport and utilization. Inhibitors of fatty acid binding to FABP result in marked decrease in fatty acid esterification into triglycerides in a number of in vitro systems including hepatocytes, prefused liver, adipocytes, and everted jejunal sacs. Activities of aeetyl CoA carboxylase, fatty acid:CoA ligase, acyl CoA:glycerol3-phosphate acyltransferase, and diacylglycerol acyltransferase are enhanced in vitro by FABP. However, the exact role of FABP in intracellular lipid metabolism remains unclear. Large quantities of fatty acids are used in synthesis of milk fat by the mammary gland. Milk fat is 98% triglyceride (Jenness, 1974) and in the case of the dairy cow, as much as 1.5 kg of triglyceride may be synthesized daily. Fatty acids used for triglyceride synthesis in ruminants arise from two sources, the de novo synthesis of short-chain saturated fatty acids
*This research was supported in part by USDA Hatch projects 35-0364 and 35-0363 of the Illinois Agricultural Experiment Station. tCurrent address: Eastman Chemicals Division, Eastman Kodak Company, Rochester, NY 14603, USA. ~;To whom correspondence should be addressed.
(C4-C16) from blood-borne volatile fatty acids derived from rumen fermentation, and from uptake of long chain fatty acids (C~6, C~8, C18:1, C~8:2) derived from in situ lipoprotein lipase activity on blood lipoproteins (Dils, 1983). While FABP might be expected to have a major role in mammary function and milk fat synthesis, its presence has not been noted previously in the literature. Here we report the isolation, identification, and characterization of a fatty acid binding protein from bovine mammary gland. MATERIALS AND METHODS
Materials
Sephadex G-25, G-75, and DEAE Sephadex A-25 were products of Pharmacia, Inc., Piscataway, NJ, USA. Aquasol liquid scintillation cocktail, EN3HANCE, [l-t4C] octanoic and oleic acids were obtained from New England Nuclear, Boston, MA, USA. [1-L4C] butyric, laurie, and palmitic acids were purchased from ICN Radiochemicals, Irvine, CA, USA. [l-14C] Palmitoyl-CoA was from Amersham Corp., Arlington Heights, IL, USA. Dithiothreitol (reduced), gelatin (Type I from swine skin, 300 bloom), dextran (clinicalgrade), bovine serum albumin (Fraction V), ovalbumin, trypsinogen, cytochrome c, pentadecanoic acid, methyl pentadecanoate, phosphatidic acid, sodium adenosine triphosphate, and coenzyme A were obtained from Sigma Chemical Co., St. Louis, MO, USA. Activated charcoal (carbon decolorizing neutral Norit) was purchased from Fisher Scientific,Pittsburg, PA, USA. Acrylamide, bisacrylamide, TEMED, and ammonium persulfate were products of Bio-Rad, Richmond, CA, USA. Ampholine ampholytes (pH3.5 10) were products of LKB Instruments, Inc., Gaithersburg, MD, USA. Fatty acids, mono-, di- and triglycerides and thin layer plates (Adsorbosil Plus 1 preparative soft layer Prekotes) were obtained from Applied Science Laboratories, State College, PA, USA. Fat and Oil Reference Mixture # 6 was a product of Supelco, Inc., Bellefonte, PA, USA. All other chemicals were reagent grade. Chloroform and methanol were redistilled prior to
687
use.
688
H . D . WHETSTONE et al.
Preparation o f cytosol Mammary tissue was obtained at slaughter from lactating Holstein cows from the University dairy herd. Production ranged from 5-25 kg of milk per day prior to slaughter. The tissue was minced and homogenized in ice cold Tris-sucrose buffer (30mM Tris, pH 7.2, 0.3 M sucrose, 1 mM DTT, l mM EDTA) (1:2, w/v) in a Sorvall Omnimixer. Whole cells, nuclei, and mitochondria were removed by centrifugation at 10,000 g for 15 min. The resulting supernatant was centrifuged at 100,000g for 2 hr in a Beckman L5-50B ultracentrifuge at 5°C to obtain cytosol and microsomal (pellet) fractions. For experiments requiring lipid-free cytosol, noncovalently bound and free fatty acids were removed from cytosol by extraction with diisopropyl ether (Ockner et al., 1982). Protein concentrations of the microsomes, cytosol, and FABP fractions were determined by the method of Lowry et al. (1951) using BSA as the standard. Preparation o f F A B P Cytosol (15ml, 250-350mg protein) was applied to a Sephadex G-75 column (90 x 2.6 cm) at 5°C. Protein was eluted at 2(~30 ml/hr in 20 mM Tris, pH 7.9. Fractions of 8-9 ml were collected and absorbance at 280 nm was determined. The assay procedure of Morrow and Martin (1983) for fatty acid binding activity was used to locate FABP. The assay was performed as described, except that [14C] oleic acid was used in place of [14C] palmitic acid. Fractions containing FABP from several chromatographic runs were pooled and concentrated (Amicon ultrafiltration cell with YM5 membrane-mol, wt cutoff 5000). Four to 5 ml of this G-75 fractionated FABP were applied to a DEAE Sephadex A-25 column (20 × 1.6 cm) at 25°C. Unbound protein was eluted with 20mM Tris, pH 7.9. The addition of 0.1 M NaCI to the buffer removed the bulk of bound protein. Residual protein was eluted by 0.5 M NaC1. Fractions containing FABP were combined, concentrated and stored at -20°C. Gel electrophoresis and isoelectric focusing Polyacrylamide gel electrophoresis using sodium dodecyl sulfate (SDS) as a denaturant, was as described by Laemmli (1970). Samples were run on 15% acrylamide slab gels, 10.5 x 0.08 cm, with a 5% acrylamide stacking gel. Ten to 15/al (1 #g protein) were electrophoresed per lane. Gels were fixed and silver-stained (Merrill et al., 1981). Isoelectric focusing was carried out in tube gels by procedures of Pharmacia (1982) and Wrigley (1971). A 5% acrylamide gel was prepared containing ampholytes (1/30 Ampholine) pH 3.5-10. Gels were prefocused at 1 mA/gel for 30 min. The anode solution was 0.2% phosphoric acid and Cathode solution was 0.4% ethanolamine. After the sample was applied, the gels were focused for 7 hr at 500 V. A focused gel without sample was cut into 5 mm pieces, each piece placed in one ml distilled water and the pH determined. Proteins were stained with Coomassie blue. Binding o f f a t t y acids and F A B P f a t t y acid composition Binding affinity (Ka) and capacity were determined for oleic acid using the assay procedure of Morrow and Martin (1983). Substrate concentrations used were 2.9, 2.1, 1.1, 0.21, and 0.11 micromolar. Assays were run in triplicate with 45/~g of FABP per tube. Binding constants were estimated by the direct linear plotting procedure of Eisenthal and Cornish-Bowden (1974). Binding of [t4C] labeled fatty acids to FABP was determined by the method of Oekner et al. (1972). One milligram of FABP (G-75 fraction) was incubated with 25 nmol [ ~4C]fatty acid. Bound fatty acids were separated from unbound by gel filtration on Sephadex G-25. Endogenous fatty acid composition of FABP (G-75 or DEAE fractions) was determined using the lipid extraction method of Kates (1972). Extracted fatty acids were isolated by thin layer chromatography and then converted to their
methyl esters by incubation for 2 hr at 90°C with 4% sulfuric acid in methanol. The methyl esters were analyzed by gas-liquid chromatography on a Vista 44 gas chromatography system (Varian Associates, Walnut Creek, CA) employing a stainless steel column (150 × 0.3 cm) packed with 20% diethylene glycol succinate on Chromosorb W. Separation was performed at 172°C. Pentadecanoic acid was added as an internal standard. For determination of covalently bound fatty acids, FABP was extracted as before. The protein pellet was suspended in 6 N HC1, sealed in an ampule and incubated at 60°C for 24 hr. The hydrolysate was extracted 3 times with 4 ml of petroleum ether. Derivatization and analysis of fatty acids were carried out as described for noncovalent fatty acids. Enzyme assays Phosphatidic acid phosphatase (EC 3.1.3.4) activity was measured in bovine mammary microsomes as the release of inorganic phosphate (Mavis et al., 1978). Fatty acid:CoA ligase (EC 6.2.1.3) activity was measured in bovine mammary microsomes as the formation of the hydroxamates by the method of Ockner and Manning (1976). Diacylglycerol acyltransferase (EC 2.3.1.20) activity was measured in bovine liver microsomes (because of insufficient activity present in mammary microsomes; Whetstone, 1985) using the method of O'Doherty and Kuksis (1975). Statistical analyses of FABP or BSA effects on enzyme assays were done using a paired t-test. Linear regression analyses were performed for the levels of FABP and BSA addition. Analysis o f milk and colostrum Fresh milk and colostral samples were centrifuged for 30 min at 15,000g at 25°C to remove particulate matter and the fat layer. The aqueous fraction was treated by filtration through a sintered glass disc. The filtered solutions were subjected to gel filtration chromatography, FABP assay, and electrophoretic analysis. RESULTS AND DISCUSSION T h e elution profile of fatty acid binding in bovine m a m m a r y cytosol after c h r o m a t o g r a p h y o n Sephadex G-75 is s h o w n in Fig. 1. The smaller binding peak represents F A B P which accounts for 3 % of total cytosolic protein a n d has a s p . act. of 7.7 n m o l oleate b o u n d / u n i t A280. The large binding peak includes serum a l b u m i n a n d / ~ - l a c t o g l o b u l i n as determined by polyacrylamide gel electrophoresis (data not shown). F a t t y acid binding to serum a l b u m i n has been observed previously in efforts to purify F A B P (Ockner a n d M a n n i n g , 1976; Smith et al., 1985). The milk protein fl-lactoglobulin, which a c c o u n t s for 3 - 5 % of bovine milk proteins (Jenness, 1974), binds fatty acids at multiple sites (Spector and Fletcher, 1970), a n d is a fatty acid binding c o m p o n e n t unique to the m a m mary gland. F u r t h e r purification o f F A B P material from gel filtration was achieved by D E A E ion exchange c h r o m a t o g r a p h y (Fig. 2). The m a j o r peak o f protein a n d of oleate binding, which eluted with 0 . 1 M N a C I , h a d a sp. act. of 1 2 . 8 n m o l oleate bound/unit A280, a n d accounted for 1% o f the total cytoplasmic protein. Polyacrylamide gel electrophoresis (with SDS) of protein in the m a j o r peak from the D E A E c o l u m n showed one protein with a mol. wt. of a b o u t 12,000 (data n o t shown). This is similar to the size of F A B P identified in o t h e r tissues including rat liver, rat intestine a n d adipose, h u m a n adipose, bovine liver, a n d others (Glatz a n d Veerkamp, 1985). Isoelectric
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Fig. 1. Bovine mammary cytosol chromatographed on Sephadex G-75 column. Protein elution is shown as the solid line and expressed as absorbance at 280 nm. Binding of [14C] oleate to fractions is shown as the dashed line.
focusing of bovine mammary FABP material after DEAE chromatography showed two closely associated bands focusing at about pH 5.6 and 5.8 (data not shown). This is similar to the pK found for bovine liver FABP when bound to oleic acid (Haunerland et al., 1984), while delipidated FABP in that study separated into two forms focusing at pH 6 and pH 7. Multiple forms of FABP from rat liver cytosol also have been identified (Glatz and Veerkamp, 1985). Differences between some of these rat liver FABP forms were due to bound fatty acids (Takahashi et aL, 1983).
Long chain fatty acids and their CoA derivatives were bound by FABP (Table 1) but short and medium chain fatty acids were not. A binding capacity of 33.6 nmol/mg FABP was determined representing a ratio of 0.5 fatty acid bound per molecule FABP. The affinity constant for oleic acid was determined to be 2.14 x 10 -6 M, similar to that found for rat liver FABP (Kin = 2.8 x 10 -6 M; Mishkin et al., 1972). Selective binding to long chain fatty acids also had been observed for other isolated FABP (Glatz and Veerkamp, 1985). In this study, palmitate was the most extensively bound (Table 1). This could be
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Fig. 2. Sephadex G-75 purified FABP chromatographed on DEAE Sephadex A-25 column. Protein elution is shown as the solid line and expressed as absorbance at 280 nm. Binding of [ 14C] oleate to fractions is shown as the dashed line. Arrows indicate change in buffer salt concentration. FABP elutes in the presence of 0.1 M NaCI.
690
H, D. WHETSTONEet al. Table 1, Binding of fatty acids to FABW ~om bovine mammarycytosol Percent of added f a t t y acid bounde
Fatty acid b
nmoles fatty acid/mg protein o
Fatty acidb
4:0 (3)
0.35
8:0 (2)
0.92 ±
0.53
16:1
3.02 ±
12:0 (2)
1.82 ±
0.58
18:0
13.77 ±
1.37
56.28 ± 8.46
18:1
1.02 ±
0.89
16:0 (2)
± 0.10
Table 3, Composition of covalently attached fatty acid of F A B W ~ o m b o v i n e m a m m a r y cytosol
18:1 (4)
41.42 ±
1.37
16:0-CoA (2)
29.66 ±
1,64
~One mg FABP from Sephadex G-75 per assay,
hFatty acid carbons:double bonds. Number of determinations in parentheses. "Mean ± SEM of label eluting with protein.
a reflection of the diminished proportion of unsaturated fatty acids available to the ruminant due to the microbial fermentation in the rumen. The affinity constant for oleate determined here for the mammary FABP is about one order of magnitude greater than that for free fatty acid binding to the primary site of fl-lactoglobulin (Spector and Fletcher, 1970). It has not been established whether fl-lactoglobulin has a role in binding or transport of fatty acids intracellularly. Similarity between fl-lactoglobulin structure and the structure of human serum retinol binding protein recently has been demonstrated (Pervaiz and Brew, 1985). Bovine FABP isolated from mammary cytosol had noncovalently associated fatty acids (Table 2), with the predominant fatty acids being oleate and palmirate, while no arachidonate was detected. The ratio of fatty acids nonconvalently bound per FABP molecule was 0.11 for the DEAE preparation. The relative binding affinities shown in Table 1 reflect the relative
Table 2. Composition of endogenous, noncovalently associated fatty acids of FABP a from bovine mammary cytosol Fatty acidb
G-75e
DEAEd
nmo'l'es fatty acid/mg protein 14:0
0.39 ± 0.20
ND
16:0
0,65 ± 0.12
2.08
16:1
0.21 ± 0.03
0.46
18:0
0.27 ± 0.13
1.00
18:1
0.39 ± 0.10
5.29
18:2
0.12 ± 0.04
0.06
18:3
0.05 ± 0.02
0.02
2,07
8.91
Total fatty acids
~FABP from Sephadex G-75 fraction or DEAE
fraction. ~Fatty acid carbons:double bonds. ~Mean _+ SEM of 3 determinations, dOne determination, N D = not detected,
16:0
32.54 ± 15.21
18:2
0.52
15.93 ± 15,94
Total fatty acid
66,28
~FABP from D E A E fraction. hFatty acid carbons:double bonds. CMean + SEM of 3 determinations.
Table 4. Effect of bovine mammary FABP ° and bovine serum albumin on activity of phosphatidic acid phosphatase in mammary microsomes
Incubation
Inorganic phosphate formed nmoles/minb
Microsomes
50 ~g
0.63
+ 0.05
Microsomes
100 ~g
1.30
+ 0.06
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50 vg 50 ~g
0.58
+ 0.02
+ FABP
20 ~g
0.70
+ 0.07
Microsomes + albumin
50 u9 25 ~g
0,68
+ 0.04
aFABP from Sephadex G-75 fraction. hMean + SEM of 3 determinations.
proportions of fatty acids found associated with purified FABP (Table 2). The amount of noncovalently associated fatty acids were quite low, possibly due to losses during the purification procedure or the physiological state of the animal at slaughter, which was unknown. Covalent or tightly associated fatty acids also were found on this mammary FABP (Table 3). The majority of these fatty acids were palmitate, stearate, and linoleate. The ratio of fatty acids bound per FABP molecule was 0.8. Covalently attached fatty acids were found in rat liver FABP preparations (Dempsey et aL, 1981) but not in bovine liver preparations (Haunerland et al., 1984). These fatty acids may increase the hydrophobicity of the molecule and aid in interactions with other lipid components, although the exact role of covalently bound fatty acids is not known. The effects of bovine mammary FABP (DEAE fraction) on activity of microsomal phosphatidic acid phosphatase (Table 4), fatty acid:CoA tigase (Table 5) and diacylglycerol acyltransferase (Table 6) were examined. In contrast to other studies, bovine mammary FABP (or albumin) did not affect these enzymes of lipid metabolism. Ockner and Manning (1976) observed a significant stimulation of fatty acid:CoA ligase activity by FABP using the same
Bovine mammary gland FABP Table 5. Effect of bovine mammary FABPa and bovine serum albumin on the activity of fatty acid:CoA ligase in mammary microsomes Hydroxamate formed nmoles( min/mg microsomal p r o t e i n ~
Incubation
Microsomes
I
m9
21.50 +- 0.75
Microsomes + FABP
I 0.5
mg mg
22.00 ± 0.50
0.75 mg
22.50 + 1.00
+ FABP + FABP
i
mg
20.60 ± 1.12
+ FABP
2
mg
22.40 ± 0.62
Microsomes + albumin
i 1
mg mg
22.10 ± 1.87
+ albumin
2
mg
19.50 ± 0.25
+ albumin
4
mg
17.40 ± 1.60
aFABP from Sepadex G-75 fraction. hMean + SEM of duplicate determinations. Table 6. Effect of bovine mammary FABPa and bovine serum albumin on the activity of diacylglycerol acyltransferase in bovine liver microsomes T r i g l y c e r i d e formed nmoles/15 min~
Incubation
Microsomes
2.0 mg
0.47
± 0.03
Microsomes + FABP
2.0 mg 0.3 mg
0.50
± 0.04
+ FABP
0.6 mg
0.489 ± 0.02
Microsomes 2.0 mg + albumin 1.3 m9
0.32
+ 0.06
aFABP from Sephadex G-75 fraction. hMean _+SEM of 3 determinations. assay (with rat intestinal microsomes) and quantities of F A B P used in this study. O ' D o h e r t y and Kuksis (1975) used the same assay (with rat liver and intestinal microsomes) for triacylglycerol synthesis as described here and obtained large increases in enzyme activity with addition of FABP. The reason for the discrepancy is not known, but difference in tissue is a probable explanation. In the case of diacylglycerol acyltransferase, bovine liver microsomes were used due to insufficient activity with bovine m a m m a r y microsomes (Whetstone, 1985). In incubations of bovine m a m m a r y microsomes and cytosol Bennink (1973) found that of the lipid synthesized, at most 10% was triglyceride, while in other species it accounted for up to 86%. A linear decrease (not statistically significant) in activity of fatty acid : C o A ligase was observed with increasing amounts of albumin, suggesting that fatty acids bound to F A B P may have been more readily available to those enzymes than fatty acids bound to albumin. Examination of skim milk or colostral proteins fractionated by gel filtration chromatography showed considerable fatty acid binding activity (data not shown). Polyacrylamide gel electrophoresis of fractions with binding activity indicated that this primarily was due to the presence of #-lactoglobulin. C.B,P 8 5 , ' 3 ~ M
691
By these methods, F A B P was not detected in either the skim milk or colostral preparations. Bovine m a m m a r y F A B P has many properties in c o m m o n with other m a m m a l i a n FABP; however its role in m a m m a r y gland lipid metabolism and milk fat synthesis remains to be determined. Determination of the fatty acid composition and levels of F A B P in m a m m a r y tissue from cows producing different a m o u n t of milk fat and at different stages of lactation may provide an insight into this role. M a m m a r y F A B P also might be involved in positioning of fatty acids on the milk fat triglyceride molecule, where there is a substantial preference of position by different fatty acids (Parodi, 1982). For example, blood derived palmitate has a different pattern of esterification in milk fat triglycerides than palmitate synthesized in the gland (Dimick et al., 1966), and F A B P may function specifically in the intracellular transport and positioning of fatty acids arising from breakdown of lipoprotein triglycerides.
REFERENCES
Bennink M. R. (1973) Factors affecting fatty acid esterification by rabbit and bovine mammary gland preparations. PhD Thesis, University of Illinois, Urbana-Champaign. Dempsey M. E., McCoy K. E., Baker H. N., DimitriadouVafiadou A., Lorsbach T. and Howard J. B. (1981) Large scale purification and structural characterization of squalene and sterol carrier protein. J. biol. Chem. 256, 1867-1873. Dils R. R. (1983) Milk fat synthesis. In Biochemistry of Lactation (Edited by Mepham T. B.), pp. 141-157. Elsevier, Amsterdam. Dimick P. S., McCarthy R. D. and Patton S. (1966) Paths of palmitic acid incorporation into milk fat triglycerides. Biochim. biophys. Acta 116, 159-162. Eisenthal R. and Cornish-Bowden A. (1974) The direct linear plot: new graphical procedure for estimating enzyme kinetic parameters. Biochem. J. 139, 715-720. Glatz J. F. C. and Veerkamp J. H. (1985) Intracellular fatty acid-binding proteins. Int. J. Biochem. 17, 13 22. Haunerland N., Jagschies G., Schulenberg H. and Spener F. (1984) Fatty acid binding proteins: occurrence of two fatty acid binding proteins in bovine liver cytosol and their binding of fatty acids, cholesterol and other lipophilic ligands. Hoppe-Seyler's Z. physiol. Chem. 365, 365 376. Jennesss R. (1974)The composition of milk. In Lactation: A Comprehensive Treatise (Edited by Larson B. L. and Smith V. R.), Vol. 3, pp. 3 I07. Academic Press, New York. Kates M. (1972) Techniques of Lipidology: Isolation, Analysis and Identification of Lipids. American Elsevier Publishing Co., Inc., New York. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680q585. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Mavis R. D., Finkelstein J. N. and Hall B. P. (1978) Pulmonary sufactant synthesis. A highly active micro° somal phosphatidate phosphohydrolase in the lung. J. Lipid Res. 19, 467-477. Merrill C. R., Goldman D., Sedman S. A. and Ebert M. H. (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science, Wash. 211, 1437-1438. Mishkin S., Stein L., Gatmaitan Z. and Arias |. M. (1972)
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H . D . WHETSTONE et al.
The binding of fatty acids to cytoplasmic proteins: binding to Z protein in liver and other tissues of the rat. Biochem. biophys. Res. Commun. 47, 997-1003. Morrow F. D. and Martin R. J. (1983) Quantitation of hepatic fatty acid binding proteins by post-chromatographic ligand binding assay. J. Lipid Res. 24, 324-331. Ockner R. K. and Manning J. A. (1976) Fatty acid binding protein. Role in esterification of absorbed long chain fatty acid in rat intestine. J. clin. Invest. 58, 632 641. Ockner R. K., Manning J. A. and Kane J. P. (1982) Fatty acid binding protein. Isolation from rat liver, characterization, and immunochemical quantification. J. biol. Chem. 257, 7872-7878. Ockner R. K., Manning J. A., Poppenhausen R. B. and Ho H. K. L. (1972) A binding protein for fatty acids in cytosol of intestinal mucosa, liver, myocardium and other tissues. Science 177, 5f~58. O'Doherty P. J. A. and Kuksis A. (1975) Stimulation of triacylglycerol synthesis by Z protein in rat liver and intestinal mucosa. FEBS Lett. 60, 256-258. Parodi P. W. (1982) Positional distribution of fatty acids in the triglyceride classes of milk fat. J. Dairy Res. 49, 73 80.
Pervaiz S. and Brew K. (1985). Homology of /~-lactoglobulin, serum retinol-binding protein, and protein HC. Science 228, 335 337. Pharmacia Fine Chemicals (1982) lsoelectric Focusing: Principles and Methods. Pharmacia Fine Chemicals AB, Uppsala, Sweden. 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. Spector A. A. and Fletcher J. E. (1970) Binding of long chain fatty acids to /~-lactoglobulin. Lipid~ 5, 403411. Takahashi K., Odani S. and Ono T. (1983) Isolation and characterization of the three fractions (DE-I, DE-I1 and DE-Ill) of rat-liver Z-protein and the complete primary structure of DE-II. Eur. J. Biochem. 136, 589 601. Whetstone H. D. (1985) Isolation and characterization of fatty acid binding protein from lactating bovine mammary gland. PhD Thesis, University of Illinois, Urbana-Champaign. Wrigley C. W. (1971) Gel electrofocusing. In Methods in Enzymology (Edited by Jakoby W. B.), Vol. 22, pp. 559-564. Academic Press, New York.
0305-0491/86 $3.00 +0.00 Pergamon Journals Ltd
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I D E N T I F I C A T I O N A N D C H A R A C T E R I Z A T I O N OF A F A T T Y A C I D B I N D I N G P R O T E I N IN B O V I N E MAMMARY GLAND* H. D. WHETSTONE,t W. L. HURLEY3~ and C. L. DAVIS Department of Animal Sciences, University of Illinois, Urbana, IL 61801, U.S.A (Tel: 217-333-1327) (Received 20 February 1986)
A fatty acid binding protein (FABP) was isolated from bovine mammary cytosol by gel filtration and ion exchange chromatography. 2. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a mol. wt. of 12,000. Isoelectric focusing showed two bands at pH 5.6 and 5.8. 3. FABP bound long chain fatty acids and their CoA thioesters, but not medium or short chain fatty acids. Affinity constant (Ka) for 18:1 was about 2 micromolar. 4. Endogenously bound fatty acids included 16:0, 18:0 and 18:1, in both covalent and noncovalent association with FABP. 5. Activities of microsomal phosphatidic acid phosphatase, fatty acid:CoA ligase or diacylglycerol acyltransferase were not affected by purified FABP in vitro. Abstract--1.
INTRODUCTION
Properties of fatty acid binding proteins have recently been reviewed (Glatz and Veerkamp, 1985). Fatty acid binding protein (FABP) is a low mol. wt (about 12,000) cytosolic protein which binds fatty acids and acyl CoA thioesters with high affinity. Its presence has been documented in a number of tissues which metabolize fatty acids including rat liver, myocardium, lung, intestinal mucosa, adipose, kidney, adrenal, and other tissues, bovine liver (Glatz and Veerkamp, 1985), and recently in bovine muscle and adipose (Smith et al., 1985). These FABP are thought to participate in intracellular fatty acid transport and utilization. Inhibitors of fatty acid binding to FABP result in marked decrease in fatty acid esterification into triglycerides in a number of in vitro systems including hepatocytes, prefused liver, adipocytes, and everted jejunal sacs. Activities of aeetyl CoA carboxylase, fatty acid:CoA ligase, acyl CoA:glycerol3-phosphate acyltransferase, and diacylglycerol acyltransferase are enhanced in vitro by FABP. However, the exact role of FABP in intracellular lipid metabolism remains unclear. Large quantities of fatty acids are used in synthesis of milk fat by the mammary gland. Milk fat is 98% triglyceride (Jenness, 1974) and in the case of the dairy cow, as much as 1.5 kg of triglyceride may be synthesized daily. Fatty acids used for triglyceride synthesis in ruminants arise from two sources, the de novo synthesis of short-chain saturated fatty acids
*This research was supported in part by USDA Hatch projects 35-0364 and 35-0363 of the Illinois Agricultural Experiment Station. tCurrent address: Eastman Chemicals Division, Eastman Kodak Company, Rochester, NY 14603, USA. ~;To whom correspondence should be addressed.
(C4-C16) from blood-borne volatile fatty acids derived from rumen fermentation, and from uptake of long chain fatty acids (C~6, C~8, C18:1, C~8:2) derived from in situ lipoprotein lipase activity on blood lipoproteins (Dils, 1983). While FABP might be expected to have a major role in mammary function and milk fat synthesis, its presence has not been noted previously in the literature. Here we report the isolation, identification, and characterization of a fatty acid binding protein from bovine mammary gland. MATERIALS AND METHODS
Materials
Sephadex G-25, G-75, and DEAE Sephadex A-25 were products of Pharmacia, Inc., Piscataway, NJ, USA. Aquasol liquid scintillation cocktail, EN3HANCE, [l-t4C] octanoic and oleic acids were obtained from New England Nuclear, Boston, MA, USA. [1-L4C] butyric, laurie, and palmitic acids were purchased from ICN Radiochemicals, Irvine, CA, USA. [l-14C] Palmitoyl-CoA was from Amersham Corp., Arlington Heights, IL, USA. Dithiothreitol (reduced), gelatin (Type I from swine skin, 300 bloom), dextran (clinicalgrade), bovine serum albumin (Fraction V), ovalbumin, trypsinogen, cytochrome c, pentadecanoic acid, methyl pentadecanoate, phosphatidic acid, sodium adenosine triphosphate, and coenzyme A were obtained from Sigma Chemical Co., St. Louis, MO, USA. Activated charcoal (carbon decolorizing neutral Norit) was purchased from Fisher Scientific,Pittsburg, PA, USA. Acrylamide, bisacrylamide, TEMED, and ammonium persulfate were products of Bio-Rad, Richmond, CA, USA. Ampholine ampholytes (pH3.5 10) were products of LKB Instruments, Inc., Gaithersburg, MD, USA. Fatty acids, mono-, di- and triglycerides and thin layer plates (Adsorbosil Plus 1 preparative soft layer Prekotes) were obtained from Applied Science Laboratories, State College, PA, USA. Fat and Oil Reference Mixture # 6 was a product of Supelco, Inc., Bellefonte, PA, USA. All other chemicals were reagent grade. Chloroform and methanol were redistilled prior to
687
use.
688
H . D . WHETSTONE et al.
Preparation o f cytosol Mammary tissue was obtained at slaughter from lactating Holstein cows from the University dairy herd. Production ranged from 5-25 kg of milk per day prior to slaughter. The tissue was minced and homogenized in ice cold Tris-sucrose buffer (30mM Tris, pH 7.2, 0.3 M sucrose, 1 mM DTT, l mM EDTA) (1:2, w/v) in a Sorvall Omnimixer. Whole cells, nuclei, and mitochondria were removed by centrifugation at 10,000 g for 15 min. The resulting supernatant was centrifuged at 100,000g for 2 hr in a Beckman L5-50B ultracentrifuge at 5°C to obtain cytosol and microsomal (pellet) fractions. For experiments requiring lipid-free cytosol, noncovalently bound and free fatty acids were removed from cytosol by extraction with diisopropyl ether (Ockner et al., 1982). Protein concentrations of the microsomes, cytosol, and FABP fractions were determined by the method of Lowry et al. (1951) using BSA as the standard. Preparation o f F A B P Cytosol (15ml, 250-350mg protein) was applied to a Sephadex G-75 column (90 x 2.6 cm) at 5°C. Protein was eluted at 2(~30 ml/hr in 20 mM Tris, pH 7.9. Fractions of 8-9 ml were collected and absorbance at 280 nm was determined. The assay procedure of Morrow and Martin (1983) for fatty acid binding activity was used to locate FABP. The assay was performed as described, except that [14C] oleic acid was used in place of [14C] palmitic acid. Fractions containing FABP from several chromatographic runs were pooled and concentrated (Amicon ultrafiltration cell with YM5 membrane-mol, wt cutoff 5000). Four to 5 ml of this G-75 fractionated FABP were applied to a DEAE Sephadex A-25 column (20 × 1.6 cm) at 25°C. Unbound protein was eluted with 20mM Tris, pH 7.9. The addition of 0.1 M NaCI to the buffer removed the bulk of bound protein. Residual protein was eluted by 0.5 M NaC1. Fractions containing FABP were combined, concentrated and stored at -20°C. Gel electrophoresis and isoelectric focusing Polyacrylamide gel electrophoresis using sodium dodecyl sulfate (SDS) as a denaturant, was as described by Laemmli (1970). Samples were run on 15% acrylamide slab gels, 10.5 x 0.08 cm, with a 5% acrylamide stacking gel. Ten to 15/al (1 #g protein) were electrophoresed per lane. Gels were fixed and silver-stained (Merrill et al., 1981). Isoelectric focusing was carried out in tube gels by procedures of Pharmacia (1982) and Wrigley (1971). A 5% acrylamide gel was prepared containing ampholytes (1/30 Ampholine) pH 3.5-10. Gels were prefocused at 1 mA/gel for 30 min. The anode solution was 0.2% phosphoric acid and Cathode solution was 0.4% ethanolamine. After the sample was applied, the gels were focused for 7 hr at 500 V. A focused gel without sample was cut into 5 mm pieces, each piece placed in one ml distilled water and the pH determined. Proteins were stained with Coomassie blue. Binding o f f a t t y acids and F A B P f a t t y acid composition Binding affinity (Ka) and capacity were determined for oleic acid using the assay procedure of Morrow and Martin (1983). Substrate concentrations used were 2.9, 2.1, 1.1, 0.21, and 0.11 micromolar. Assays were run in triplicate with 45/~g of FABP per tube. Binding constants were estimated by the direct linear plotting procedure of Eisenthal and Cornish-Bowden (1974). Binding of [t4C] labeled fatty acids to FABP was determined by the method of Oekner et al. (1972). One milligram of FABP (G-75 fraction) was incubated with 25 nmol [ ~4C]fatty acid. Bound fatty acids were separated from unbound by gel filtration on Sephadex G-25. Endogenous fatty acid composition of FABP (G-75 or DEAE fractions) was determined using the lipid extraction method of Kates (1972). Extracted fatty acids were isolated by thin layer chromatography and then converted to their
methyl esters by incubation for 2 hr at 90°C with 4% sulfuric acid in methanol. The methyl esters were analyzed by gas-liquid chromatography on a Vista 44 gas chromatography system (Varian Associates, Walnut Creek, CA) employing a stainless steel column (150 × 0.3 cm) packed with 20% diethylene glycol succinate on Chromosorb W. Separation was performed at 172°C. Pentadecanoic acid was added as an internal standard. For determination of covalently bound fatty acids, FABP was extracted as before. The protein pellet was suspended in 6 N HC1, sealed in an ampule and incubated at 60°C for 24 hr. The hydrolysate was extracted 3 times with 4 ml of petroleum ether. Derivatization and analysis of fatty acids were carried out as described for noncovalent fatty acids. Enzyme assays Phosphatidic acid phosphatase (EC 3.1.3.4) activity was measured in bovine mammary microsomes as the release of inorganic phosphate (Mavis et al., 1978). Fatty acid:CoA ligase (EC 6.2.1.3) activity was measured in bovine mammary microsomes as the formation of the hydroxamates by the method of Ockner and Manning (1976). Diacylglycerol acyltransferase (EC 2.3.1.20) activity was measured in bovine liver microsomes (because of insufficient activity present in mammary microsomes; Whetstone, 1985) using the method of O'Doherty and Kuksis (1975). Statistical analyses of FABP or BSA effects on enzyme assays were done using a paired t-test. Linear regression analyses were performed for the levels of FABP and BSA addition. Analysis o f milk and colostrum Fresh milk and colostral samples were centrifuged for 30 min at 15,000g at 25°C to remove particulate matter and the fat layer. The aqueous fraction was treated by filtration through a sintered glass disc. The filtered solutions were subjected to gel filtration chromatography, FABP assay, and electrophoretic analysis. RESULTS AND DISCUSSION T h e elution profile of fatty acid binding in bovine m a m m a r y cytosol after c h r o m a t o g r a p h y o n Sephadex G-75 is s h o w n in Fig. 1. The smaller binding peak represents F A B P which accounts for 3 % of total cytosolic protein a n d has a s p . act. of 7.7 n m o l oleate b o u n d / u n i t A280. The large binding peak includes serum a l b u m i n a n d / ~ - l a c t o g l o b u l i n as determined by polyacrylamide gel electrophoresis (data not shown). F a t t y acid binding to serum a l b u m i n has been observed previously in efforts to purify F A B P (Ockner a n d M a n n i n g , 1976; Smith et al., 1985). The milk protein fl-lactoglobulin, which a c c o u n t s for 3 - 5 % of bovine milk proteins (Jenness, 1974), binds fatty acids at multiple sites (Spector and Fletcher, 1970), a n d is a fatty acid binding c o m p o n e n t unique to the m a m mary gland. F u r t h e r purification o f F A B P material from gel filtration was achieved by D E A E ion exchange c h r o m a t o g r a p h y (Fig. 2). The m a j o r peak o f protein a n d of oleate binding, which eluted with 0 . 1 M N a C I , h a d a sp. act. of 1 2 . 8 n m o l oleate bound/unit A280, a n d accounted for 1% o f the total cytoplasmic protein. Polyacrylamide gel electrophoresis (with SDS) of protein in the m a j o r peak from the D E A E c o l u m n showed one protein with a mol. wt. of a b o u t 12,000 (data n o t shown). This is similar to the size of F A B P identified in o t h e r tissues including rat liver, rat intestine a n d adipose, h u m a n adipose, bovine liver, a n d others (Glatz a n d Veerkamp, 1985). Isoelectric
Bovine mammary gland FABP
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150 200 250 300 ELUTION VOLUME ( M L I
350
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Fig. 1. Bovine mammary cytosol chromatographed on Sephadex G-75 column. Protein elution is shown as the solid line and expressed as absorbance at 280 nm. Binding of [14C] oleate to fractions is shown as the dashed line.
focusing of bovine mammary FABP material after DEAE chromatography showed two closely associated bands focusing at about pH 5.6 and 5.8 (data not shown). This is similar to the pK found for bovine liver FABP when bound to oleic acid (Haunerland et al., 1984), while delipidated FABP in that study separated into two forms focusing at pH 6 and pH 7. Multiple forms of FABP from rat liver cytosol also have been identified (Glatz and Veerkamp, 1985). Differences between some of these rat liver FABP forms were due to bound fatty acids (Takahashi et aL, 1983).
Long chain fatty acids and their CoA derivatives were bound by FABP (Table 1) but short and medium chain fatty acids were not. A binding capacity of 33.6 nmol/mg FABP was determined representing a ratio of 0.5 fatty acid bound per molecule FABP. The affinity constant for oleic acid was determined to be 2.14 x 10 -6 M, similar to that found for rat liver FABP (Kin = 2.8 x 10 -6 M; Mishkin et al., 1972). Selective binding to long chain fatty acids also had been observed for other isolated FABP (Glatz and Veerkamp, 1985). In this study, palmitate was the most extensively bound (Table 1). This could be
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Fig. 2. Sephadex G-75 purified FABP chromatographed on DEAE Sephadex A-25 column. Protein elution is shown as the solid line and expressed as absorbance at 280 nm. Binding of [ 14C] oleate to fractions is shown as the dashed line. Arrows indicate change in buffer salt concentration. FABP elutes in the presence of 0.1 M NaCI.
690
H, D. WHETSTONEet al. Table 1, Binding of fatty acids to FABW ~om bovine mammarycytosol Percent of added f a t t y acid bounde
Fatty acid b
nmoles fatty acid/mg protein o
Fatty acidb
4:0 (3)
0.35
8:0 (2)
0.92 ±
0.53
16:1
3.02 ±
12:0 (2)
1.82 ±
0.58
18:0
13.77 ±
1.37
56.28 ± 8.46
18:1
1.02 ±
0.89
16:0 (2)
± 0.10
Table 3, Composition of covalently attached fatty acid of F A B W ~ o m b o v i n e m a m m a r y cytosol
18:1 (4)
41.42 ±
1.37
16:0-CoA (2)
29.66 ±
1,64
~One mg FABP from Sephadex G-75 per assay,
hFatty acid carbons:double bonds. Number of determinations in parentheses. "Mean ± SEM of label eluting with protein.
a reflection of the diminished proportion of unsaturated fatty acids available to the ruminant due to the microbial fermentation in the rumen. The affinity constant for oleate determined here for the mammary FABP is about one order of magnitude greater than that for free fatty acid binding to the primary site of fl-lactoglobulin (Spector and Fletcher, 1970). It has not been established whether fl-lactoglobulin has a role in binding or transport of fatty acids intracellularly. Similarity between fl-lactoglobulin structure and the structure of human serum retinol binding protein recently has been demonstrated (Pervaiz and Brew, 1985). Bovine FABP isolated from mammary cytosol had noncovalently associated fatty acids (Table 2), with the predominant fatty acids being oleate and palmirate, while no arachidonate was detected. The ratio of fatty acids nonconvalently bound per FABP molecule was 0.11 for the DEAE preparation. The relative binding affinities shown in Table 1 reflect the relative
Table 2. Composition of endogenous, noncovalently associated fatty acids of FABP a from bovine mammary cytosol Fatty acidb
G-75e
DEAEd
nmo'l'es fatty acid/mg protein 14:0
0.39 ± 0.20
ND
16:0
0,65 ± 0.12
2.08
16:1
0.21 ± 0.03
0.46
18:0
0.27 ± 0.13
1.00
18:1
0.39 ± 0.10
5.29
18:2
0.12 ± 0.04
0.06
18:3
0.05 ± 0.02
0.02
2,07
8.91
Total fatty acids
~FABP from Sephadex G-75 fraction or DEAE
fraction. ~Fatty acid carbons:double bonds. ~Mean _+ SEM of 3 determinations, dOne determination, N D = not detected,
16:0
32.54 ± 15.21
18:2
0.52
15.93 ± 15,94
Total fatty acid
66,28
~FABP from D E A E fraction. hFatty acid carbons:double bonds. CMean + SEM of 3 determinations.
Table 4. Effect of bovine mammary FABP ° and bovine serum albumin on activity of phosphatidic acid phosphatase in mammary microsomes
Incubation
Inorganic phosphate formed nmoles/minb
Microsomes
50 ~g
0.63
+ 0.05
Microsomes
100 ~g
1.30
+ 0.06
Microsomes + FABP
50 vg 50 ~g
0.58
+ 0.02
+ FABP
20 ~g
0.70
+ 0.07
Microsomes + albumin
50 u9 25 ~g
0,68
+ 0.04
aFABP from Sephadex G-75 fraction. hMean + SEM of 3 determinations.
proportions of fatty acids found associated with purified FABP (Table 2). The amount of noncovalently associated fatty acids were quite low, possibly due to losses during the purification procedure or the physiological state of the animal at slaughter, which was unknown. Covalent or tightly associated fatty acids also were found on this mammary FABP (Table 3). The majority of these fatty acids were palmitate, stearate, and linoleate. The ratio of fatty acids bound per FABP molecule was 0.8. Covalently attached fatty acids were found in rat liver FABP preparations (Dempsey et aL, 1981) but not in bovine liver preparations (Haunerland et al., 1984). These fatty acids may increase the hydrophobicity of the molecule and aid in interactions with other lipid components, although the exact role of covalently bound fatty acids is not known. The effects of bovine mammary FABP (DEAE fraction) on activity of microsomal phosphatidic acid phosphatase (Table 4), fatty acid:CoA tigase (Table 5) and diacylglycerol acyltransferase (Table 6) were examined. In contrast to other studies, bovine mammary FABP (or albumin) did not affect these enzymes of lipid metabolism. Ockner and Manning (1976) observed a significant stimulation of fatty acid:CoA ligase activity by FABP using the same
Bovine mammary gland FABP Table 5. Effect of bovine mammary FABPa and bovine serum albumin on the activity of fatty acid:CoA ligase in mammary microsomes Hydroxamate formed nmoles( min/mg microsomal p r o t e i n ~
Incubation
Microsomes
I
m9
21.50 +- 0.75
Microsomes + FABP
I 0.5
mg mg
22.00 ± 0.50
0.75 mg
22.50 + 1.00
+ FABP + FABP
i
mg
20.60 ± 1.12
+ FABP
2
mg
22.40 ± 0.62
Microsomes + albumin
i 1
mg mg
22.10 ± 1.87
+ albumin
2
mg
19.50 ± 0.25
+ albumin
4
mg
17.40 ± 1.60
aFABP from Sepadex G-75 fraction. hMean + SEM of duplicate determinations. Table 6. Effect of bovine mammary FABPa and bovine serum albumin on the activity of diacylglycerol acyltransferase in bovine liver microsomes T r i g l y c e r i d e formed nmoles/15 min~
Incubation
Microsomes
2.0 mg
0.47
± 0.03
Microsomes + FABP
2.0 mg 0.3 mg
0.50
± 0.04
+ FABP
0.6 mg
0.489 ± 0.02
Microsomes 2.0 mg + albumin 1.3 m9
0.32
+ 0.06
aFABP from Sephadex G-75 fraction. hMean _+SEM of 3 determinations. assay (with rat intestinal microsomes) and quantities of F A B P used in this study. O ' D o h e r t y and Kuksis (1975) used the same assay (with rat liver and intestinal microsomes) for triacylglycerol synthesis as described here and obtained large increases in enzyme activity with addition of FABP. The reason for the discrepancy is not known, but difference in tissue is a probable explanation. In the case of diacylglycerol acyltransferase, bovine liver microsomes were used due to insufficient activity with bovine m a m m a r y microsomes (Whetstone, 1985). In incubations of bovine m a m m a r y microsomes and cytosol Bennink (1973) found that of the lipid synthesized, at most 10% was triglyceride, while in other species it accounted for up to 86%. A linear decrease (not statistically significant) in activity of fatty acid : C o A ligase was observed with increasing amounts of albumin, suggesting that fatty acids bound to F A B P may have been more readily available to those enzymes than fatty acids bound to albumin. Examination of skim milk or colostral proteins fractionated by gel filtration chromatography showed considerable fatty acid binding activity (data not shown). Polyacrylamide gel electrophoresis of fractions with binding activity indicated that this primarily was due to the presence of #-lactoglobulin. C.B,P 8 5 , ' 3 ~ M
691
By these methods, F A B P was not detected in either the skim milk or colostral preparations. Bovine m a m m a r y F A B P has many properties in c o m m o n with other m a m m a l i a n FABP; however its role in m a m m a r y gland lipid metabolism and milk fat synthesis remains to be determined. Determination of the fatty acid composition and levels of F A B P in m a m m a r y tissue from cows producing different a m o u n t of milk fat and at different stages of lactation may provide an insight into this role. M a m m a r y F A B P also might be involved in positioning of fatty acids on the milk fat triglyceride molecule, where there is a substantial preference of position by different fatty acids (Parodi, 1982). For example, blood derived palmitate has a different pattern of esterification in milk fat triglycerides than palmitate synthesized in the gland (Dimick et al., 1966), and F A B P may function specifically in the intracellular transport and positioning of fatty acids arising from breakdown of lipoprotein triglycerides.
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H . D . WHETSTONE et al.
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Pervaiz S. and Brew K. (1985). Homology of /~-lactoglobulin, serum retinol-binding protein, and protein HC. Science 228, 335 337. Pharmacia Fine Chemicals (1982) lsoelectric Focusing: Principles and Methods. Pharmacia Fine Chemicals AB, Uppsala, Sweden. 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. Spector A. A. and Fletcher J. E. (1970) Binding of long chain fatty acids to /~-lactoglobulin. Lipid~ 5, 403411. Takahashi K., Odani S. and Ono T. (1983) Isolation and characterization of the three fractions (DE-I, DE-I1 and DE-Ill) of rat-liver Z-protein and the complete primary structure of DE-II. Eur. J. Biochem. 136, 589 601. Whetstone H. D. (1985) Isolation and characterization of fatty acid binding protein from lactating bovine mammary gland. PhD Thesis, University of Illinois, Urbana-Champaign. Wrigley C. W. (1971) Gel electrofocusing. In Methods in Enzymology (Edited by Jakoby W. B.), Vol. 22, pp. 559-564. Academic Press, New York.