Fatty Acid Binding Protein: Stimulation of Microsomal Phosphatidic Acid Formation

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 341, No. 1, May 1, pp. 112–121, 1997 Article No. BB979957

Fatty Acid Binding Protein: Stimulation of Microsomal Phosphatidic Acid Formation1 Christopher A. Jolly,* Timothy Hubbell,* William D. Behnke,† and Friedhelm Schroeder*,2 *Department of Physiology and Pharmacology, Texas A & M University, TVMC, College Station, Texas 77843-4466; and †Department of Molecular Genetics, University of Cincinnati Medical Center, M.L. 0524, Cincinnati, Ohio 45267-0524

Received December 12, 1996, and in revised form February 14, 1997

The effect of fatty acid binding proteins (FABPs) on two key steps of microsomal phosphatidic acid formation was examined. Rat liver microsomes were purified by size-exclusion chromatography to remove endogenous cytosolic fatty acid and fatty acyl-CoA binding proteins while recombinant FABPs were used to avoid cross-contamination with such proteins from native tissue. Neither rat liver (L-FABP) nor rat intestinal fatty acid binding protein (I-FABP) stimulated liver microsomal fatty acyl-CoA synthase. In contrast, L-FABP and I-FABP enhanced microsomal conversion of [14C]oleoyl-CoA and glycerol 3-phosphate to [14C]phosphatidic acid by 18- and 7-fold, respectively. The mechanism for this stimulation, especially by IFABP, is not known. However, several observations presented here suggest that, like L-FABP, I-FABP may interact with fatty acyl-CoA and thereby stimulate enzyme activity. First, I-FABP decreased microsomal membrane-bound oleoyl-CoA. Second, oleoylCoA displaced I-FABP bound fluorescent fatty acid, cis-parinaric acid, with Ki of 5.3 mM and 1.1 sites. Third, oleoyl-CoA decreased I-FABP tryptophan fluorescence with a Kd of 4.2 mM. Fourth, oleoyl-CoA red shifted emission spectra of acrylodated I-FABP, a sensitive marker of I-FABP interactions with ligands. In summary, the results demonstrate for the first time that both L-FABP and I-FABP stimulate liver microsomal phosphatidic acid formation by enhancing synthesis of phosphatidate from fatty acyl-CoA and glycerol 3-phosphate. q 1997 Academic Press Key Words: fatty acid binding protein; phosphatidic acid; microsomes; liver; intestine; fatty acyl-CoA.

1 This work was supported in part by a grant from the United States Public Health Service, National Institutes of Health (DK41402). 2 To whom correspondence should be addressed.

Increasing observations indicate that both L-FABP3 and I-FABP enhance the esterification of fatty acids into triglycerides and phospholipids. Expression of LFABP in transfected fibroblasts stimulated fatty acid uptake and oleic acid esterification into neutral lipids and even more so into phospholipids (1–4). This resulted in a net increase in cellular phospholipid mass (3, 5). In contrast, expression of I-FABP in transfected fibroblasts did not enhance fatty acid uptake but, nevertheless, resulted in selective incorporation of oleic acid into neutral lipids such as triglycerides and cholesteryl esters at the expense of incorporation into phospholipids (1, 2), such that net cellular mass of these neutral lipids increased (2). Furthermore, LFABP and I-FABP enhanced selective incorporation of oleic acid within the phospholipid fraction, primarily into phosphatidylcholine and phosphatidylethanolamine (1–3). Thus, both L-FABP and I-FABP stimulated esterification of fatty acids into phosphatidic acid-derived triglycerides and phospholipid subclasses in intact cells. The two major steps leading to intracellular incorporation of fatty acids into phosphatidic acid are the activation of fatty acid to fatty acyl-CoA (fatty acyl-CoA synthetase) and the incorporation of fatty acyl-CoA into glycerol 3-phosphate to form phosphatidic acid (glycerol-3-phosphate acyltransferase and lysophosphatidate acyltransferase) [reviewed in (6)]. The longchain fatty acyl-CoA synthetase is found mainly in the endoplasmic reticulum and mitochondria of mammalian cells. The rate-limiting step in the sequential acylation of glycerol 3-phosphate is that catalyzed by glycerol-3-phosphate acyltransferase to form monoacylglycerol phosphate. In liver this enzyme is distributed 3 Abbreviations used: FABP, fatty acid binding protein; L-FABP, liver fatty acid binding protein; I-FABP, intestinal fatty acid binding protein; ACBP, acyl-CoA binding protein; CoASH, coenzyme A; DTT, dithiothreitol.

112

AID

0003-9861/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

ABB 9957

/

6b33$$$261

03-27-97 17:43:42

arca

FABPS STIMULATE PHOSPHATIDIC ACID FORMATION

nearly equally between microsomes and mitochondria, while in other tissues it is predominantly microsomal. The microsomal glycerol-3-phosphate acyltransferase utilizes a variety of saturated and unsaturated fatty acyl-CoAs, while the mitochondrial enzyme utilizes saturated acyl-CoAs. A separate enzyme, monoacylglycerol-3-phosphate acyltransferase, catalyzes the second acylation to form phosphatidic acid. The latter enzyme is selective for unsaturated fatty acyl-CoAs in both microsomes and mitochondria. More important, in contrast to microsomes the activity of monoacylglycerol phosphate acyltransferase is very low in mitochondria. Thus, the bulk of triglycerides and phospholipids are formed primarily in the microsomal membrane fraction. The effect of the cytosolic fatty acid and/or fatty acylCoA binding proteins on these microsomal enzymatic activities is unclear. For example, early work utilizing native L-FABP isolated from liver reported both stimulation (7–12) and inhibition (13). Likewise, early reports with native L-FABP and native I-FABP showed stimulation of glycerol-3-phosphate acyltransferase (14, 15), while a subsequent observation with recombinant L-FABP and recombinant I-FABP suggested that only the former protein stimulated this activity (16). Even the view that native L-FABP and/or I-FABP modulated the above activities was challenged by the observation that early preparations of native FABPs may have been contaminated with acyl-CoA binding protein (ACBP) and that the latter may have been responsible for the activities [reviewed in (17, 18)]. Recent data demonstrating that ACBP stimulates the mitochondrial long-chain fatty acyl-CoA synthetase are consistent with this possibility (19), while other data with recombinant L-FABP clearly demonstrate that this protein stimulates microsomal glycerol-3-phosphate acyltransferase (16). To complicate matters, the FABPs, especially L-FABP, can be trapped in unwashed microsomal preparations and may even, in part, associate with liver microsomal membranes (20, 21). This may in part account for the varying degrees of L-FABP stimulation of glycerol-3-phosphate acyltransferase activity observed with unwashed liver microsomes: 1.3-fold (16), 3-fold (22), and 6-fold (20). These results clearly demonstrate the need to further examine the relative roles of I-FABP and L-FABP in the microsomal formation of phosphatidic acid. The purpose of the present investigation was twofold: first, to examine the effect(s) of L-FABP and I-FABP on formation of phosphatidic acid by microsomes isolated from rat liver and further purified by gel-permeation chromatography; second, to determine the potential interaction of fatty acyl-CoA with I-FABP as well as LFABP. The results show that both I-FABP and L-FABP stimulated phosphatidic acid formation 7- and 18-fold, respectively, when the microsomes were further puri-

AID

ABB 9957

/

6b33$$$261

03-27-97 17:43:42

113

fied by gel-permeation chromatography. Furthermore, partitioning and spectroscopic evidence was consistent with both I-FABP and L-FABP interacting with fatty acyl-CoA. MATERIALS AND METHODS Materials. cis-Parinaric acid and acrylodated intestinal fatty acid binding protein were obtained from Molecular Probes (Eugene, OR). cis-Parinaroyl-CoA was synthesized and purified as described earlier (16). [9,10-3H(N)]Oleic acid (10 Ci/mmol) was obtained from Amersham Co. (Arlington Heights, IL). Morpholinopropane sulfonic acid (Mops) was obtained from Serva (New York, NY). Oleoyl-CoA, oleic acid, butylated hydroxytoluene, dithiothreitol (DTT), coenzyme A (CoASH), acyl-coenzyme A synthase, ethylenediaminetetraacetic acid (EDTA), adenosine 5*-triphosphate (ATP), NaF, tricine, and (hydroxymethyl)-aminomethane (Tris) were obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals were reagent grade or better. Silica gel 60 thin-layer chromatography plates were from Merck Inc. Sephacryl S-300 beads were from Pharmacia (Uppsala, Sweden). Low molecular weight standards (6.2, 14.3, 18.4, 29, 43 kDa) were from Sigma Chemical Co. (St. Louis, MO). Protein purification. Purification of recombinant L-FABP and IFABP from the Escherichia coli strain carrying plasmids pJBL2 and pIFABPexp6, respectively, was done as described (23, 24). The purified FABPs were delipidated also as described earlier (25) unless otherwise noted. The protein concentration was determined (26) and corrected according to amino acid analysis (23, 24). The Bradford protein assay overestimates the concentration of L-FABP 1.69-fold and I-FABP 1.07-fold. Isolation of rat liver microsomes. Microsomes, prepared from male Sprague–Dawley rat livers (16, 27), were subsequently washed by gel filtration using a 100-ml column volume of Sephacryl S-300 beads as described earlier (28). SDS–polyacrylamide gel electrophoresis and Western immunoblotting. All procedures for quantitative electrophoresis and determination of L-FABP and ACBP in liver microsomes were performed as described earlier (29) except that photographs of the immunoblots were obtained with an IS-500 gel-documentation system (Alpha-Innotech, Leandro, CA). In each image, protein bands from sample lanes were quantitated by comparison with protein standards loaded on the same blot in adjacent lanes by using NIH Image 1.60 on a Power Macintosh 8100/80av computer. The program was written by Mr. Wayne Rasband at the U.S. National Institutes of Health and is available by Internet by anonymous FTP from zippy.nimh.gov or on floppy disk from NYIS (Springfield, VA). L-FABP and ACBP were expressed as ng/mg protein in the sample. Microsomal enzyme activity. The effect of L-FABP and I-FABP on rat liver microsomal fatty acyl-CoA synthetase was examined by an in vitro assay described in Burrier et al. (12). Each reaction contained 2 mM ATP, 0.75 mM CoASH, 0.5 mM MgCl2 , 100 mM NaCl, 10 mM Tris–HCl (pH 7.4), 50 mg microsome protein, 5 mg I-FABP or L-FABP protein, and 5 mCi/mmol (50 mM) oleic acid in a total volume of 200 ml. This assay was conducted using free [3H]oleic acid as a substrate instead of [3H]oleic acid bound to unilammelar liposomes. Phosphatidic acid biosynthesis from glycerol 3-phosphate was assayed as described earlier (16) except as follows: Sephacryl S-300washed microsomes and 40 mM [14C]oleoyl-CoA (sp act 27,500 dpm/ nmol) were used. Also, phosphatidic acid was separated by one-dimensional thin-layer chromatography using silica gel 60 (Merck) plates in a solvent system containing chloroform/methanol/acetic acid/double-distilled water (50:37.5:3.5:2, v/v). Oleoyl-CoA partitioning. The ability of L-FABP and I-FABP to influence oleoyl-CoA and oleic acid partitioning between aqueous buffer and microsomes was performed essentially as previously de-

arca

114

JOLLY ET AL.

scribed (12). Experiments were conducted at 257C in a 100-ml reaction volume containing 70 mM Tris–HCl (pH 7.4), 10 mM MgCl2 , 4 mM ATP, 0.8 mM CoA, 4 mM DTT, 40 mM [14C]oleoyl-CoA, 10 mg microsomal protein, and 20 mM L-FABP or I-FABP. Under these conditions microsomal acyl-CoA hydrolase activity is partially inhibited (12). After 5 min, samples were cooled to 07C and microsomes were pelleted by centrifugation at 50,000g for 15 min. The supernatant was removed and lipids were extracted from both the supernatant (soluble) and the pellet (membrane bound) by the method of Folch et al. (30). The aqueous phase of the lipid extraction represented the fatty acyl-CoA, while the organic phase contained free fatty acid. Fluorescence spectroscopy. Fluorescence emission spectra for cisparinaric acid, cis-parinaroyl-CoA, I-FABP tryptophan, L-FABP tyrosine, and acrylodated I-FABP were obtained at 257C in 1-cm cuvettes with a PC1 photon-counting spectrofluorometer (ISS Instruments, Champaign, IL). The excitation wavelength for cis-parinaric acid and cis-parinaroyl-CoA was 324 nm. The excitation wavelength for I-FABP and L-FABP was 280 nm. Acrylodated I-FABP was excited at 390 nm. Excitation bandwidth was 4 nm; emission bandwidth was 10 nm. Emission spectra were obtained from 350 to 550 nm for cis-parinaric acid and cis-parinaroyl-CoA; 300 to 500 nm for I-FABP and L-FABP; and 400 to 600 nm for acrylodated I-FABP. No corrections of emission spectra were made for monochromator wavelength dependencies. However, the inner filter effect was avoided through use of dilute solutions with absorbance õ0.12 at the wavelength of excitation. Light scatter was avoided by use of dilute solutions, cutoff filters, and/or narrow slit widths. Ligand binding to I-FABP and L-FABP. Binding parameters of both cis-parinaric acid and cis-parinaroyl-CoA to respective proteins were determined at 247C as described earlier (16). The protein concentration was maintained constant 0.1 mM unless otherwise stated. The ligand concentration was varied as described in the figure legends. Light scattering was eliminated with GG-375 sharp cutoff emission filters (Janos Technology Inc., Townsend, VT). The inner filter effect could be neglected because the maximum absorbance of the samples was below 0.12 in all cases. Binding of oleoyl-CoA and CoASH to I-FABP and L-FABP was monitored by determination of tryptophan and tyrosine quenching, respectively. The binding data were obtained at 247C and binding parameters of these ligands to respective proteins were determined by fitting the data to simple rectangular hyperbolas as described earlier (16, 31). Unless otherwise stated the protein concentration was maintained constant at 0.1 mM while the ligand concentration was varied. Fluorescence emission was recorded as described above except that emission was monitored through a 290- to 305-nm interference filter. Binding of oleic acid, oleoyl-CoA, and CoASH to acrylodated IFABP was performed as described earlier for oleic acid by the manufacturer (Molecular Probes).

RESULTS

Liver microsomal content of endogenous L-FABP and ACBP. Although microsomes isolated from rat liver by standard techniques undergo several wash steps, significant trapped or membrane-associated cytosolic proteins (e.g., L-FABP and ACBP) may remain to influence basal phosphatidic acid synthesis activity. SDS–PAGE gels of microsomes isolated by standard techniques revealed the presence of proteins in this 10–14 kDa molecular weight range (Fig. 1A). Western blots of these gels with antisera to ACBP did not detect any ACBP in the microsomes isolated by standard techniques (Fig. 1B). Western blots of these gels incubated

AID

ABB 9957

/

6b33$$$261

03-27-97 17:43:42

FIG. 1. Western blots of L-FABP and ACBP in rat liver microsomes. All procedures were as described under Materials and Methods. (A) Coomassie-stained SDS–PAGE of microsomes as described under Materials and Methods. Lanes 1–7 indicate 1, low-molecularweight markers; 2–4, 25, 125, and 5 mg microsomes isolated by standard techniques; and 5–7, 25, 125, and 5 mg microsomes isolated by standard techniques followed by Sephacryl S-300 gel permeation chromatography. (B) Western blot of microsomes incubated with rabbit anti-ACBP antisera. Lanes 1–9 indicate 1–3 100, 50, and 10 ng ACBP standard, respectively; 4–6 25, 125, and 5 mg microsomes isolated by standard techniques; 7–9 25, 125, and 5 mg microsomes isolated by standard techniques followed by Sephacryl S-300 gel permeation chromatography. (C) Western blot of microsomes incubated with rabbit anti-L-FABP. Lanes are as indicated for (B).

with antisera to L-FABP detected significant amounts of L-FABP (3.8 ng/mg microsomal protein) in microsomes isolated from rat liver by standard techniques (Fig. 1C). Therefore, gel-permeation chromatography of microsomes was used to improve the removal of trapped proteins (28). Western blots of microsomes further purified by Sephacryl S-300 gel-permeation chromatography revealed the absence of ACBP (Fig. 1B) and reduced the level of L-FABP by nearly 60% to 1.6 ng/mg microsomal protein. Since Sephacryl S-300 gelpermeation chromatography reduced the content of endogenous L-FABP in microsomes isolated from rat livers, microsomes washed by Sephacryl S-300 chromatography were used in all subsequent experiments. Effect of L-FABP and I-FABP on microsomal acylCoA synthetase. The effects of I-FABP and L-FABP on microsomal fatty acyl-CoA synthetase were examined using [14C]oleic acid as a substrate. In the absence of FABPs the initial rate of fatty acyl-CoA formation was 11% conversion/min with maximal conversion of 40% by

arca

FABPS STIMULATE PHOSPHATIDIC ACID FORMATION

FIG. 2. Effect of I-FABP and L-FABP on liver microsomal fatty acyl-CoA synthase. Microsomal fatty acyl-CoA synthase assays were performed as described under Materials and Methods. The assay measures conversion of [3H]oleic acid to [3H]oleoyl-CoA. Symbols: control, open circles; I-FABP, solid triangles; L-FABP, solid circles. Values represent means { SE, n Å 3. An asterisk refers to P õ 0.05 compared to control at the same time point.

30 min (Fig. 2, top curve). L-FABP and I-FABP significantly inhibited fatty acyl-CoA synthetase by 5–10 min incubation such that by 10 min L-FABP and I-FABP inhibited the enzyme by 28% (P õ 0.05) and 38% (P õ 0.05), respectively (Fig. 2, center and bottom). Since fatty acyl-CoA synthetase is already an end-product (acyl-CoA) inhibited enzyme (32, 33), this would suggest that L-FABP and/or I-FABP may inhibit the enzyme by binding substrate fatty acid rather than stimulating the enzyme through removal of inhibitory end-product acylCoA. Therefore, these FABPs do not release microsomal fatty acyl-CoA synthetase from oleoyl-CoA end-product inhibition by sequestering acyl-CoA. Effect of L-FABP and I-FABP on microsomal formation of phosphatidic acid. The effects of I-FABP and L-FABP on microsomal phosphatidic acid formation were examined using [14C]oleoyl-CoA and glycerol 3phosphate as substrates. The oleoyl-CoA concentration of 40 mM was not inhibitory (PA synthesis at 40 and 20 mM was 85 pmol/min/mg protein versus 50 pmol/min/ mg protein, respectively; data not shown). Incorporation of [14C]oleoyl-CoA into phosphatidic acid reflects glycerol-3-phosphate acyltransferase activity. In the absence of added L-FABP or I-FABP, basal activity of microsomal glycerol-3-phosphate acyltransferase, the rate-limiting step in microsomal phosphatidic acid formation (6, 20), was only 85 pmol/min/mg protein (Figs. 3A and 3B). Addition of 20 mM L-FABP maximally in-

AID

ABB 9957

/

6b33$$$261

03-27-97 17:43:42

115

creased the glycerol-3-phosphate acyltransferase enzyme activity 18-fold from 85 to 1332 pmol/min/mg microsomal protein (Fig. 3A). At this concentration the molar ratio of oleoyl-CoA:L-FABP was 2:1, confirming earlier observations that L-FABP maximally binds 2 mol of fatty acyl-CoA per mole of L-FABP (16). Interestingly, 10 mM I-FABP resulted in maximal phosphatidic acid synthesis (Fig. 3B). I-FABP stimulated microsomal glycerol-3-phosphate acyltransferase 7-fold from 80 to 521 pmol/min/mg microsomal protein (Fig. 3B). At this concentration the molar ratio of oleoyl-CoA:IFABP was 1:1. In an earlier report from this laboratory, the use of unwashed microsomes obscured these effects such that L-FABP only stimulated glycerol-3-phosphate acyltransferase in unwashed microsomes by 1.3-fold and the increase by I-FABP was not detectable (16). The present data clearly indicate benefits of utilizing Sephacryl S-300-washed liver microsomes and suggest that I-FABP stimulates the microsomal incorporation of [14C]oleic acid from [14C]oleoyl-CoA into glycerol 3phosphate to form phosphatidic acid. This further suggests that I-FABP may interact with fatty acyl-CoA. The following experiments were performed to examine this possibility. Influence of L-FABP and I-FABP on oleic acid and oleoyl-CoA. FABP activities can be measured by their ability to extract fatty acids from microsomes. In the absence of added FABPs, ú95% of oleic acid was bound to microsomes. Both L-FABP and I-FABP shifted the partitioning of oleic acid from microsomes toward the soluble fraction. Both L-FABP and I-FABP increased soluble oleic acid levels from õ5% of total to 38 and 26% of total, respectively (Fig. 4A). In the absence of added FABPs, ú96% of oleoyl-CoA is bound to the microsomal membrane fraction (Fig. 4B). This would suggest that L-FABP and/or I-FABP may stimulate microsomal glycerol-3-phosphate acyltransferase by binding/extracting oleoyl-CoA from microsomes and increasing oleoyl-CoA availability to the enzyme. Previous observations have shown that LFABP extracts fatty acyl-CoA from microsomal membranes (12), consistent with the ability of L-FABP to bind fatty acyl-CoA (16). This observation was confirmed where addition of L-FABP decreased the membrane-bound acyl-CoA by 38% and increased the soluble acyl-CoA levels fivefold (Fig. 4B). I-FABP acted in a similar fashion but was half (18% versus 38%) as effective in extracting microsomal membrane-bound oleoyl-CoA. While I-FABP increased the soluble levels of oleoyl-CoA, this difference was not statistically significant, suggesting that the I-FABP solubilized oleoylCoA was rapidly utilized by microsomal enzymes. Competition between fatty acyl-CoA and fatty acid for L-FABP and I-FABP ligand binding site(s). Prelimi-

arca

116

JOLLY ET AL.

FIG. 3. Dose-dependent up-regulation of phosphatidic acid biosynthesis by L-FABP and I-FABP. Liver microsomes were prepared as described under Materials and Methods. Each assay was performed in a total volume of 0.1 ml containing 735 mM glycerol 3-phosphate, 80 mM NaF, 15 mM dithiothreitol, 40 mM [14C]oleoyl-CoA, and 10 mg of microsomal protein in 70 mM Tris, pH 7.5, in the presence of 0, 5, 10, 20, 30, or 34 mM L-FABP (A) or 0, 2.5, 5, 10, 20, 30, 35, or 40 mM I-FABP (B). Values represent means of three to six determinations.

nary results using a direct fluorescent binding assay suggested that L-FABP, but not I-FABP, bound cisparinaroyl-CoA (16). The basis for this assay was that fatty acyl-CoA binding could be detected as an increase in cis-parinaroyl-CoA fluorescence intensity. This was observed for L-FABP but not for I-FABP. However, the lack of increased cis-parinaroyl-CoA fluorescence intensity with I-FABP could simply have been due to the fact that the fatty acyl-CoA binding site in I-FABP differed from that in L-FABP (where fatty acid and fatty acyl-CoA competed for the same binding site; see below). Therefore, interaction of L-FABP and I-FABP with fatty acyl-CoA was reexamined by testing the ability of these proteins to displace bound fatty acid. Both L-FABP and I-FABP bind cis-parinaric acid with high fluorescence emission intensities (23, 24). Therefore, competition studies were performed to displace cis-parinaric acid bound to L-FABP or I-FABP (Fig. 5). Oleoyl-

CoA displaced L-FABP bound cis-parinaric acid (Fig. 5A). Increasing concentrations of oleoyl -CoA displaced cis-parinaric acid from L-FABP (Fig. 5A) with a Ki of 12.7 mM and 2.1 sites. The displacement effectiveness of the two major constituents of the oleoyl-CoA molecule, namely oleic acid and CoASH, was compared to that of oleoyl-CoA. Oleic acid was twice as effective (P õ 0.01) as oleoyl-CoA in displacing L-FABP bound cis-parinaric acid, while CoASH was ineffective (Table I). When this experiment was repeated with cis-parinaroyl-CoA bound to L-FABP (instead of cis-parinaric acid), both oleic acid and oleoyl-CoA displaced L-FABP bound cis-parinaroyl-CoA to a similar extent, while CoASH was again ineffective (Table I). Since CoASH by itself did not displace fluorescent ligand bound to L-FABP (Table I), this would suggest that the more hydrophobic acyl part of the oleoyl-CoA appeared necessary for the displacement activity of oleoyl-CoA.

FIG. 4. Alteration of oleoyl-CoA partitioning by L-FABP and I-FABP. The partitioning of oleic acid (A) and oleoyl-CoA (B) between microsomal membranes and soluble fractions was performed as described under Materials and Methods. Briefly, [14C]oleoyl-CoA (4 nmol) was incubated with microsomal membranes for 5 min at 257C. The membrane and aqueous phases were then separated by centrifugation and a lipid extraction was performed on each phase. Each value represents the mean of three determinations.

AID

ABB 9957

/

6b33$$$261

03-27-97 17:43:42

arca

117

FABPS STIMULATE PHOSPHATIDIC ACID FORMATION

Oleoyl-CoA also displaced I-FABP bound cis-parinaric acid (Fig. 5B). Increasing concentrations of oleoylCoA displaced bound cis-parinaric acid from I-FABP with a Ki of 5.3 mM and 1.1 sites (Fig. 5B). Interaction of oleoyl-CoA with I-FABP: Intrinsic tryptophan fluorescence. A direct fatty acyl-CoA binding assay, taking advantage of the intrinsic fluorescence of I-FABP tryptophan, was utilized as shown previously for other fatty acyl-CoA binding proteins (34, 35). The

TABLE I

Displacement of L-FABP Bound cis-Parinaric Acid and cis-Parinaroyl-CoA by Nonfluorescent Ligands Competing ligand

Displacement of cis-parinaric acid

Displacement of cis-parinaroyl-CoA

Oleic acid (18:1) Oleoyl-CoA CoASH

81.6 { 7.8% SE 44.8 { 5.2% SE 5.9 { 7.9% SE

84.7 { 2.4% SE 81.2 { 2.3% SE 5.5 { 1.8% SE

Note. Fluorescence displacement binding assays were performed at 377C in 2 ml of 25 mM potassium phosphate buffer, pH 7.4 cisParinaric acid or cis-parinaroyl-CoA at 0.3 mM binding to 0.1 mM L-FABP was displaced by 3.0 mM of each ligand. Exciting wavelength was 324 nm with emission measured with a GG 375-nm cutoff filter in place. Values represent means { SE.

FIG. 5. Displacement of cis-parinaric acid from I-FABP and LFABP. Oleoyl-CoA displacement of bound cis-parinaric acid was performed at 247C as described under Materials and Methods. (A) 0.24 mM L-FABP / 0.50 mM cis-parinaric acid; (B) 0.4 mM I-FABP / 1.0 mM cis-parinaric acid. Each point represents the mean net fluorescence intensity of two assay tubes plotted versus the oleoyl-CoA concentration. Fluorescence signal of controls (no protein) was identical to each assay tube of a given oleoyl-CoA concentration and was subtracted from the signal of each assay tube to give net fluorescence. Displacement curves, Ki , and the number of binding sites on the L-FABP and I-FABP were determined using the GraphPad (ISI Software, Philadelphia, PA) competition curve-fitting program.

AID

ABB 9957

/

6b33$$$261

03-27-97 17:43:42

effect of oleoyl-CoA on the intrinsic fluorescence emission of I-FABP tryptophan was determined. I-FABP tryptophan, excited at 280 nm, exhibited a broad fluorescence emission from 300 to 450 nm (Fig. 5). The maximal emission intensity near 327 nm (Fig. 5) was consistent with the I-FABP tryptophan being localized in a hydrophobic environment (36, 37). Addition of oleoyl-CoA to I-FABP decreased the I-FABP tryptophan emission by up to 25% (Fig. 6A). Equivalent concentrations of the two molecular components of oleoylCoA, namely oleic acid and CoASH, decreased I-FABP tryptophan emission only 8 and 13%, respectively (Figs. 6B and 6C, respectively). Thus, both the fatty acyl chain and the CoASH moieties contributed to decreased fluorescence intensity of I-FABP tryptophan. It should be noted that this decrease was not due to a direct interaction of oleoyl-CoA, oleic acid, or CoASH with the I-FABP tryptophan residue. When added in the range of 0–20 mM, none of these ligands quenched the fluorescence intensity of the water-soluble tryptophan analog N-acetyltryptophanamide or of tyrosine in buffer (data not shown). Oleoyl-CoA (Fig. 7A) and CoA (Fig. 7B) binding curves, based on I-FABP tryptophan intensity changes, were saturable. On transformation of the binding data to rectangular hyperbola binding isotherms, the resultant Kd s were very similar: 4.2 { 1.0 mM (n Å 3) and 2.0 { 1.0 mM (n Å 3) for oleoyl-CoA and CoASH, respectively (Fig. 7). Fatty acid, acyl-CoA, and CoA interaction with acrylodated I-FABP. The interaction of fatty acyl-CoA with I-FABP was examined through use of acrylodated I-FABP. The fluorescence emission spectrum of acrylodan alone in aqueous buffer is insensitive to addition of fatty acid, fatty acyl-CoA, or CoASH (data not shown). However, in acrylodated I-FABP acrylodan is covalently linked to Lys27 of I-FABP. This acrylodated-Lys27 residue is localized on the I-FABP surface near the opening of the I-FABP fatty acid binding pocket such that the acrylodan is inserted into the I-FABP fatty

arca

118

JOLLY ET AL.

acid binding pocket, resulting in a blue-shifted emission spectrum, as expected (Fig. 8). Ligands capable of displacing the acrylodan from the I-FABP binding pocket shift the acrylodan residue from the I-FABP binding pocket toward the aqueous as indicated by a red-shifted acrylodated I-FABP fluorescence emission spectrum (Fig. 8). Addition of oleoyl-CoA decreased the emission of acrylodated I-FABP near 432 nm up to 63% (Fig. 8). Concomitantly, oleoyl-CoA induced an increase in acrylodated I-FABP emission near 505 nm, up to 325% (Fig. 8). Because an isosbestic point was not obtained during the binding reaction, this would suggest the presence of more than two acrylodated I-FABP states in solution. Either the acrylodated

FIG. 7. Concentration dependence of I-FABP tryptophan emission quenching by oleoyl-CoA and CoASH. The I-FABP tryptophan (1.2 mM) maximum emission intensity was plotted as a function of (A) oleoyl-CoA or (B) CoASH concentration. Values represent means { SE (n Å 3).

I-FABP fatty acid binding site and fatty acyl-CoA binding site were not identical or some intermediate state was obtained. Therefore, it was not possible to construct oleoyl-CoA binding curves to acrylodated I-FABP to obtain correct binding parameters. Nevertheless, the method clearly shows that oleoyl-CoA does interact with acrylodated I-FABP. DISCUSSION

FIG. 6. Oleoyl-CoA quenching of I-FABP tryptophan emission. IFABP (1.2 mM) was excited at 280 nm and fluorescence emission spectra were obtained at 247C in the presence of increasing (0.15–6 mM) oleoyl-CoA (A), (0.15–6 mM) oleic acid (B), or (10 mM) CoASH (C). The bottom curve in (A) shows the effect of adding 6 mM oleic acid to a sample of 1.2 mM I-FABP already containing 6 mM oleoyl-CoA.

AID

ABB 9957

/

6b33$$$261

03-27-97 17:43:42

The data show that L-FABP and I-FABP interact with fatty acyl-CoA and stimulate phosphatidic acid synthesis in vitro. This was not due to cross-contaminating ACBP, another fatty acyl-CoA binding protein first isolated as a contaminant of FABPs prepared by early chromatographic procedures [reviewed in (17, 18)]. Earlier data obtained in vitro on the effects of liver and intestinal fatty acid binding proteins on microsomal fatty acyl-CoA synthetase and phosphatidic acid

arca

FABPS STIMULATE PHOSPHATIDIC ACID FORMATION

FIG. 8. Oleoyl-CoA alters acrylodated I-FABP fluorescence emission. Emission spectra were obtained as described under Materials and Methods. Top curve, 0.1 mM acrylodated I-FABP. Additional curves from top to bottom were 0.1 mM acrylodated I-FABP / 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, or 6.4 mM oleoyl-CoA, respectively.

biosynthesis were equivocal, especially with regard to I-FABP due to (a) potential cross-contamination of native FABPs with ACBP or (b) the presence of trapped ACBP and/or FABPs in microsomes isolated by standard methods yielding high basal GPAT activity (20, 21). Previously published basal activities of 10 (16), 0.6 (22), and 0.5 (20) nmol/min/mg microsomal protein are 66- to 3-fold higher than the basal activity observed in the present work, 0.15 nmol/min/mg microsomal protein using microsomes purified by Sephacryl S-300 gelpermeation chromatography. The high basal activity in the previous report from our laboratory may have obscured the effects of recombinant I-FABP on GPAT activity (16). The data presented herein also differ from our previous data in that the maximal GPAT activity in the presence of recombinant L-FABP is 10-fold lower. The specific reason for this discrepancy is not clear, but several possibilities may be considered. First, the additional gel filtration of the microsomes may remove other proteins (enzymes) which play a role in GPAT activity or contribute to the phosphatidic acid pool. Second, other phospholipids which could incorporate oleate via phospholipid fatty acyl chain remodeling enzymes may be present in the phosphatidic acid band on the thin-layer chromatography system used earlier (16), thereby yielding a higher activity. The data presented herein are consistent with IFABP as well as L-FABP binding fatty acyl-CoA. This conclusion is based on partitioning of oleoyl-CoA between microsomal membranes and the aqueous buffer, displacement of bound fatty acid, spectral changes in I-FABP tryptophan, and spectral shifts in acrylodated I-FABP. The data with I-FABP tryptophan fluorescence were especially useful since essentially the same technique was used to demonstrate fatty acyl-CoA

AID

ABB 9957

/

6b33$$$261

03-27-97 17:43:42

119

binding to ACBP, a small 10-kDa cytosolic protein completely unrelated to the FABPs (34, 35). The Kd s obtained by these methods were consistent with I-FABP binding long-chain fatty acyl-CoA with Kd s in the 4.2– 5.3 mM range. Thus, I-FABP bound long-chain acylCoAs with affinities about 2- to 10-fold lower compared to its binding of fatty acids (23, 38, 39). As shown herein, this similarity in affinities is reflected in acylCoAs competing effectively with the I-FABP fatty acid binding site. In an earlier report from this laboratory it was shown that fatty acyl-CoA increased the I-FABP tryptophan limiting anisotropy and ‘‘wobbling in a cone’’ angle, also indicative of fatty acyl-CoA interacting with I-FABP (40). It should be noted that these observations are in contrast to a preliminary fatty acylCoA binding study from our laboratory suggesting that L-FABP, but not I-FABP, binds fatty acyl-CoA (16). However, that study utilized the Lipidex-1000 fatty acid binding assay adapted for measurement of [3H]oleoyl-CoA binding to I-FABP and L-FABP. Unfortunately, the Lipidex-1000 assay has a number of limitations (41) that can preclude detection of acyl-CoA binding to I-FABP compared to L-FABP. For example, under one set of conditions using the Lipidex-1000 assay it was demonstrated that L-FABP did not bind acyl-CoA (17), while under another set of conditions with the Lipidex-1000 binding assay L-FABP bound acyl-CoA with a 2:1 stoichiometry (16). Since I-FABP and L-FABP differ considerably in their properties, pI, etc. (42), under the specific assay conditions optimized for L-FABP the binding of [3H]oleoyl-CoA to I-FABP may not necessarily be observed. With regard to the relative physiological significance of L-FABP and I-FABP binding fatty acyl-CoA and stimulating microsomal phosphatidic acid formation in vitro, several factors must be considered [reviewed in (18)]. First is the relative affinities of the fatty acylCoA binding proteins. There has been some confusion about the binding affinity for acyl-CoA binding to ACBP [reviewed in (18, 43)]. However, more recent papers (44)4 report extensive data on acyl-CoA binding to ACBP. These and previous data (45) show that ACBP binding affinity increases linearly with chain length. Calculation of the Kd for palmitoyl-CoA binding to ACBP by linear extrapolation of the microcalorimetry data yields a Kd of approximately 0.5 nM. The previously reported Kd of 10013 M (46) is incorrect due to an error in the published method for calculating Kd s from microcalorimetry; the correct value is 2 nM (J. Knudsen, personal communication).4 Therefore, the Kd for binding oleoyl-CoA to ACBP appears to be at least three orders of magnitude lower than that of I-FABP. This would suggest that essentially all the cellular

4

J. Knudsen, Biochem. J. 323, 1–12, 1997.

arca

120

JOLLY ET AL.

fatty acyl-CoA may be bound to ACBP and no effect of I-FABP (or even L-FABP) expression should be noted. Second, fatty acyl-CoA binding proteins may interact with microsomal membranes. As shown herein, LFABP, but not ACBP, was associated with microsomes. Third is the relative amounts of fatty acyl-CoA. Typical tissue contents of fatty acyl-CoA are near 108–248 nmol/g tissue (47, 48). However, this concentration may vary over an 18-fold range depending on tissue status. For example, in starvation liver fatty acyl-CoA may be nearly 4-fold higher than normal, while in hepatotoxicity the fatty acyl-CoA content may be 5-fold lower [reviewed in (18)]. Fourth is the relative amounts of the acyl-CoA binding proteins. Intestinal L-FABP and IFABP are expressed at 400–600 nmol/g tissue, while ACBP is present at about an 8-fold lower concentration than I- or L-FABP (18). Since I-FABP has one acylCoA binding site, the total acyl-CoA binding capacity of intestinal I-FABP content exceeds the fatty acyl-CoA content by 2-6 fold under normal conditions and 10- to 30-fold in hepatotoxicity. Similar considerations hold for L-FABP, which has two fatty acyl-CoA binding sites, in intestine and in liver. Therefore, the fatty acylCoA binding capacity of FABPs in liver and intestine normally exceeds the fatty acyl-CoA contents of these tissues by 4- to 18-fold, respectively. If the affinity of ACBP is three or more orders of magnitude higher than L-FABP or I-FABP, then nearly all the fatty acyl-CoA should be complexed by ACBP. In contrast, if the affinities of FABPs and ACBP are in a similar range, as suggested by most reports (18), then FABPs may play a role in fatty acyl-CoA utilization in vivo. Fifth is the relative competition for fatty acyl-CoA by binding proteins and membranes. The amount of cellular fatty acyl-CoA bound to the respective fatty acyl-CoA binding proteins in situ is not known, but will certainly be influenced by the relative Kd s of the three fatty acylCoA binding proteins for fatty acyl-CoAs as shown herein and earlier (16, 17) and by the concentration of other competing ligands, e.g., fatty acids. For example, fatty acids can displace bound fatty acyl-CoA from LFABP (16) but not from I-FABP (Fig. 5) or ACBP (17). Both L-FABP and I-FABP, as well as ACBP, could be important contributors in the determination of intracellular pools of unbound fatty acyl-CoAs, ligands that affect the activities of many physiological processes [reviewed in (18)]. Sixth, the relative effects observed in vitro may not reflect the situation in intact cells. However, under conditions where ACBP expression was unaltered in transfected L-cells (18), the expression of either L-FABP (1–4) or I-FABP (1, 2) (at levels in the range of those for ACBP) stimulated fatty acid esterification into neutral and/or phospholipids. Thus, these findings in vitro are consistent with effects of I-FABP and L-FABP on fatty acyl-CoA metabolism in intact cells.

AID

ABB 9957

/

6b33$$$261

03-27-97 17:43:42

In summary, the data establish that L-FABP and IFABP stimulate microsomal glycerol-3-phosphate acyltransferase, inhibit microsomal fatty acyl-CoA synthetase, and interact with fatty acyl-CoAs. I-FABP binds fatty acyl-CoAs and fatty acids with similar affinity and binds fatty acyl-CoAs with higher affinity than does LFABP. I-FABP is unlikely to be involved in fatty acid metabolism by releasing microsomal fatty acyl-CoA synthase from end-product inhibition by fatty acylCoA. Finally, the findings presented herein suggest a basis whereby both L-FABP and I-FABP may stimulate esterification of fatty acids into glycerides in intact transfected cells expressing these proteins. REFERENCES 1. Prows, D. R., Murphy, E. J., and Schroeder, F. (1995) Lipids 30, 907–910. 2. Prows, D. R., Murphy, E. J., Moncecchi, D., and Schroeder, F. (1996) Chem. Phys. Lipids 84, 47–56. 3. Murphy, E. J., Prows, D. R., Jefferson, J. R., and Schroeder, F. (1996) Biochim. Biophys. Acta, 191–196. 4. Schroeder, F., Jefferson, J. R., Powell, D., Incerpi, S., Woodford, J. K., Colles, S. M., Myers-Payne, S., Emge, T., Hubbell, T., Moncecchi, D., Prows, D. R., and Heyliger, C. E. (1993) Mol. Cell. Biochem. 123, 73–83. 5. Jefferson, J. R., Powell, D. M., Rymaszewski, Z., Kukowska-Latallo, J., Lowe, J. B., and Schroeder, F. (1990) J. Biol. Chem. 265, 11062–11068. 6. Vance, J. E., and Vance, D. E. (1990) Experientia 46, 560–569. 7. McCormack, M., and Brecher, P. (1987) Biochem. J. 244, 717– 723. 8. Peeters, R. A., In’t Groen, M. A., de Moel, M. P., van Moerkerk, H. T., and Veerkamp, J. H. (1989) Int. J. Biochem. 21, 407–418. 9. Ockner, R. K., and Manning, J. A. (1976) J. Clin. Invest. 58, 632– 641. 10. Wu-Rideout, M. Y., Elson, C., and Shrago, E. (1976) Biochem. Biophys. Res. Commun. 71, 809–816. 11. Haq, R. U., Shrago, E., Christodoulides, L., and Ketterer, B. (1985) Exp. Lung. Res. 9, 43–55. 12. Burrier, R. E., Manson, C. R., and Brecher, P. (1987) Biochim. Biophys. Acta 919, 221–230. 13. Noy, N., and Zakim, D. (1985) Biochemistry 24, 3521–3525. 14. Mishkin, S., and Turcotte, R. (1974) Biochem. Biophys. Res. Commun. 57, 918–926. 15. Burnett, D. A., Lysenko, N., Manning, J. A., and Ockner, R. K. (1979) Gastroenterology 77, 241–249. 16. Hubbell, T., Behnke, W. D., Woodford, J. K., and Schroeder, F. (1994) Biochemistry 33, 3327–3334. 17. Knudsen, J. (1990) Mol. Cell. Biochem. 98, 217–223. 18. Gossett, R. E., Frolov A. A., Behnke, W. D., Kier, A. B., and Schroeder, F. (1996) Lipids 31, 895–918. 19. Rasmussen, J. T., Rosendal, J., and Knudsen, J. (1993) Biochem. J. 292, 907–913. 20. Bordewick, U., Heese, M., Borchers, T., Robenek, H., and Spener, F. (1989) Biol. Chem. Hoppe Seyler 370, 229–238. 21. Woodford, J. K., Behnke, W. D., and Schroeder, F. (1995) Mol. Cell. Biochem. 152, 51–62. 22. Vancura, A., and Haldar, D. (1992) J. Biol. Chem. 267, 14353– 14359.

arca

FABPS STIMULATE PHOSPHATIDIC ACID FORMATION 23. Nemecz, G., Hubbell, T., Jefferson, J. R., Lowe, J. B., and Schroeder, F. (1991) Arch. Biochem. Biophys. 286, 300–309. 24. Nemecz, G., Jefferson, J. R., and Schroeder, F. (1991) J. Biol. Chem. 266, 17112–17123. 25. Glatz, J., and Veerkamp, J. (1983) Anal. Biochem. 132, 89–95. 26. Bradford, M. (1976) Anal. Biochem. 72, 248–254. 27. Lichtenstein, A. H., and Brecher, P. (1980) J. Biol. Chem. 255, 9098–9104. 28. Tangen, O., Jonsson, J., and Orrenius, S. (1973) Anal. Biochem. 54, 597–603. 29. Jefferson, J. R., Slotte, J. P., Nemecz, G., Pastuszyn, A., Scallen, T. J., and Schroeder, F. (1991) J. Biol. Chem. 266, 5486–5496. 30. Folch, J., Lees, M., and Sloane-Stanley, G. H. (1957) J. Biol. Chem. 226, 497–509. 31. Schroeder, F., Myers-Payne, S. C., Billheimer, J. T., and Wood, W. G. (1995) Biochemistry 34, 11919–11927. 32. Suzue, G., and Marcel, Y. L. (1972) J. Biol. Chem. 247, 6781– 6783. 33. Oram, J. F., Wenger, J. I., and Neely, J. R. (1975) J. Biol. Chem. 250, 73–78. 34. Knudsen, J., Hojrup, P., Hansen, H. O., Hansen, H. F., and Roepstorff, P. (1989) Biochem. J. 262, 513–519. 35. Mikkelsen, J., and Knudsen, J. (1987) Biochem. J. 248, 709– 714.

AID

ABB 9957

/

6b33$$$261

03-27-97 17:43:42

121

36. Lakowicz, J. R. (1983) in (Lakowicz, J. F., Ed.), Plenum Press, New York. 37. Eftink, M. (1994) Biophys. J. 64, 482–501. 38. Lowe, J. B., Sacchettini, J. C., Laposata, M., McQuillan, J. J., and Gordon, J. I. (1987) J. Biol. Chem. 262, 5931–5937. 39. Richieri, G. V., Ogata, R. T., and Kleinfeld, A. M. (1992) J. Biol. Chem. 267, 23495–23501. 40. Frolov, A., and Schroeder, F. (1997) Biochemistry 36, 505–517. 41. Vork, M. M., Glatz, J. F., Surtel, D. A., and Van der Vusse, G. J. (1990) Mol. Cell. Biochem. 98, 111–117. 42. Paulussen, R. J. A., and Veerkamp, J. H. (1990) Subcell. Biochem. 16, 175–226. 43. Frolov, A., Cho, T. H., Billheimer, J. T., and Schroeder, F. (1996) J. Biol. Chem. 271, 31878–31884. 44. Faergeman, N. J., Sigurskjold, B. W., Kragelund, B. B., Andersen, K. V., and Knudsen, J. (1996) J. Biochem. 35, 14118–14126. 45. Rosendal, J., Ertbjerg, P., and Knudsen, J. (1993) Biochem. J. 290, 321–326. 46. Rasmussen, J. T., Faergeman, N. J., Kristiansen, K., and Knudsen, J. (1994) Biochem. J. 299, 165–170. 47. Woldegiorgis, G., Spennetta, T., Corkey, B. E., Williamson, J. R., and Shrago, E. (1985) Anal. Biochem. 150, 8–12. 48. Brunengraber, H., Boutry, M., and Lowenstein, J. M. (1978) Eur. J. Biochem. 82, 373–384.

arca