©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Triacylglycerol Synthesis by Purified Triacylglycerol Synthetase of Rat Intestinal Mucosa
ROLE OF ACYL-CoA ACYLTRANSFERASE (*)

Richard Lehner (§) , Arnis Kuksis (¶)

From the (1) Banting and Best Department of Medical Research, C. H. Best Institute, Toronto, Ontario, Canada M5G 1L6

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The activities of the proposed triacylglycerol synthetase complex, acyl-CoA ligase, acyl-CoA acyltransferase (AAT), monoacylglycerol acyltransferase (MGAT), and diacylglycerol acyltransferase (DGAT), coeluted upon Cibacron blue 3GA-agarose affinity chromatography of detergent-solubilized rat intestinal microsomes. The AAT activity is associated with a 54-kDa protein, that binds covalently an acyl group from acyl-CoA via a thiol ester linkage (Lehner, R. and Kuksis, A.(1993) J. Biol. Chem. 268, 24726-24733). Reagents that prevent the acyl-AAT formation inhibit triacylglycerol synthesis as does the removal of AAT from the complex by immunoprecipitation. In the absence of mono- and diacylglycerol acceptors, the acyl group is transferred to water. It is proposed that triacylglycerol synthesis proceeds via a sequential transfer of acyl groups from acyl-CoA ligase to the AAT, from which they are passed to the mono- and diacylglycerol acyltransferases for incorporation into the di- and triacylglycerols depending on the availability of the acyl acceptors.


INTRODUCTION

Dietary fat (triacylglycerol) is converted by pancreatic lipase to 2-monoacylglycerols and free fatty acids prior to its absorption by the intestinal villus cells. In the enterocyte, the lipolysis products enter the predominant route for acylglycerol biosynthesis in the absortive cells, the monoacylglycerol pathway (1) . The sequential intracellular reesterification of 2-monoacylglycerol to triacylglycerol has been proposed to be catalyzed by a triacyl-glycerol synthetase complex (2) consisting of MGAT,() DGAT, and of a fatty acid activating enzyme, acyl-CoA ligase. Because of the important role of these ezymes in fat absorption, there is considerable interest in molecular characterization of the individual components of the enzyme complex as well as in elucidation of their regulation and mechanism of action. A 16-fold purification of the membrane-bound complex from hamster intestinal microsomes has been reported (2) . A 10-fold purification of the complex was also obtained by hydrophobic chromatography from hamster (3) and rat (4) intestinal membranes.

We have recently purified to homogeneity a microsomal long chain AAT and obtained polyclonal antibodies against the 54-kDa enzyme (5) . Here we report on the possible role of AAT in the triacylglycerol synthetase complex isolated by affinity chromatography. The present study shows that a covalent binding of the acyl group by AAT is required for acylglycerol biosynthesis, since inhibition of acylation of AAT prevents triacylglycerol biosynthesis. Furthermore, triacylglycerol synthesis by the synthetase complex is decreased by the removal of AAT from the complex by chromatography on a protein A-agarose containing covalently bound polyclonal antibodies raised against purified AAT.


EXPERIMENTAL PROCEDURES

Materials

Oleoyl-CoA, ATP, CoASH, trioleoylglycerol, phosphatidylcholine (egg yolk), phospholipase C (Bacillus cereus type V), CHAPS, bovine serum albumin (fatty acid free), Trizma base, Cibacron blue 3GA-agarose, NEM, iodoacetamide, and DIDS were purchased from Sigma. Econo-Pac 10 DG desalting columns, DTT, reagents and molecular weight standards for SDS-PAGE were from Bio-Rad. Silica gels G and H (Merck 60 G and H) and Aquacide III were obtained from Terochem Laboratories Ltd. (Mississauga, Ontario). [2-H]Glycerol trioleate (2 Ci/mmol), [1-C]oleic acid (52 mCi/mmol) and CytoScint were from ICN Biochemicals Canada Ltd. (Montreal, Quebec). [9,10-H]Oleic acid (8.9 Ci/mmol), [1-C]oleoyl-CoA (52 mCi/mmol), L-[C]glycerol 3-phosphate (120 mCi/mmol), and EnHance were purchased from DuPont NEN. All other chemicals and solvents were of reagent grade or better quality and were obtained from local suppliers.

2-Oleoyl-[2-H]glycerol (3.3 mCi/mmol) was prepared by digestion of radiolabeled [2-H]glycerol trioleate with porcine pancreatic lipase (6) . 1,2-Dioleoyl-rac-[2-H]glycerol (0.25 mCi/mmol) and sn-1,2-diacylglycerols were prepared by Grignard degradation of [2-H]glycerol trioleate (6) and by phospholipase C digestion of egg yolk phosphatidylcholine, respectively.

Preparation of Microsomal Membranes

Male rats (Wistar, Charles River Canada Inc., La Salle, Quebec) weighing 250-300 g were fed adlibitum with standard diet. They were anesthetized with diethyl ether and exsanguinated via their abdominal aortae. The upper two-thirds of the small intestine were removed and rinsed with 0.9% NaCl, 2 mM Hepes pH 7.1, and the mucosal scrapings were obtained as described by Hoffman and Kuksis (7) . Homogenization and low speed centrifugation procedures were adapted from a method by Pind and Kuksis (8) . The scrapings from three to four rats were suspended in approximately 200 ml of 300 mM mannitol, 5 mM EDTA, 5 mM Hepes pH 7.1 and homogenized in a Waring blender, set at low speed (power setting of 50 on a Powerstat) for 30 s. The homogenate was then gently filtered through a single layer of gauze (Nu Gauze, Johnson and Johnson Inc., Toronto, Ontario), followed by a double layer of 111-µm pore size polyethylene mesh (Spectromesh PE, Spectrum Medical Industries Ltd., Los Angeles, CA) to remove mucus and fat particles. The homogenate was centrifuged at 1,500 g for 5 min. Supernatant was centrifuged at 25,000 g for 10 min, and the postmitochondrial supernatant was further centrifuged at 106,000 g for 1 h to pellet microsomal membranes. Lumenal contents were released from the membranes by treatment with 1 mM Tris-HCl, pH 8.8. Microsomes were washed by homogenization in 5 ml of 50 mM potassium phosphate (pH 7.4), 0.5 M KCl, 1 mM EDTA, and 2 mM DTT with seven strokes of a motor-driven Potter-Elvehjem homogenizer followed by centrifugation at 106,000 g for 1 h. All steps were carried out at 4 °C. Washed microsomes were suspended in 50 mM potassium phosphate (pH 7.4), 1 mM EDTA, and 2 mM DTT to give a final concentration of protein between 6 and 7 mg/ml.

Solubilization Procedure

KCl was added to microsomes to achieve a final salt concentration of 0.2 M. The membranes were incubated with detergents for 30 min on ice. The solubilized crude enzyme extract was recovered with a Pasteur pipette after centrifugation at 106,000 g for 1 h.

Purification of a Triacylglycerol Synthetase Complex

Microsomes (30 mg of protein) were solubilized with 0.6% CHAPS as described above and desalted by passage through a Econo-Pac 10DG column. 15-20 mg of the solubilized extract were loaded on 1 5-cm Cibacron blue 3GA-agarose column equilibrated with 20 mM potassium phosphate (pH 7.4), 2 mM DTT, 0.5% CHAPS (Buffer A). The column was washed with 14 ml of Buffer A and was subsequently eluted with 10 ml of Buffer A containing 10 mM ATP (Buffer B) followed by 10 ml of Buffer B containing 0.8 M NaCl.

Enzyme Assays

The activities of the triacylglycerol synthetase complex were assayed using radioactive mono- and diacylglycerols as substrates or radiolabeled oleoyl-CoA. The enzyme fractions (1-10 µg of protein) were incubated at 37 °C for 10 min with 60 µM 2-oleoyl-[2-H]glycerol (3.3 mCi/mmol) or 150 µM 1,2-dioleoyl-rac-[2-H]glycerol (0.25 mCi/mmol) suspended in CHAPS and 30 µM oleoyl-CoA or [1-C]oleoyl-CoA (2.5 mCi/mmol) and nonradioactive mono- and diradylglycerols in a final volume of 0.5 ml of 25 mM Tris-HCl (pH 7.4), 1 mM DTT, and 2 mg/ml bovine serum albumin. The concentration of CHAPS in the assay mixture was 0.04%. The reaction was terminated by the addition of 4 ml of chloroform/methanol, 2:1 (v/v) (9) . After extraction and separation of lipids by TLC using a solvent system of heptane/isopropyl ether/acetic acid, 60:40:4 (v/v/v), free fatty acids, mono-, di-, and triacylglycerols were detected by brief exposure of the plate to iodine vapor. The lipid areas were scraped into scintillation vials containing CytoScint, and the amount of radioactivity was determined by scintillation counting (10) .

The presence of the acyl-CoA ligase activity in the purified complex was assayed by the ability of the preparations to synthetize di- and triacylglycerols from 2-monoacylglycerols using in situ acyl-CoA-generating system where the oleoyl-CoA in the incubation mixture was replaced with 0.2 mM [C]oleic acid (specific activity, 0.5 mCi/mmol), 20 mM ATP, 2.5 mM CoASH, and 4 mM MgCl. The reaction was initiated by the addition of the enzyme fraction and terminated after a 10-min incubation at 37 °C by chloroform/methanol, 2:1 (v/v), and the amount of radioactivity in di- and triacylglycerols was determined as described above. Alternatively, synthesis of radiolabeled oleoyl-CoA from [C]oleic acid in the presence of ATP and CoASH was examined according to the method of Banis and Tove (11) .

Glycerol-3-phosphate acyltransferase activity was determined using [C]glycerol 3-phosphate and oleoyl-CoA as described previously with slight modification (12) . The assay mixture contained (final volume, 0.5 ml) 70 mM Tris-HCl (pH 7.4), 1 mM DTT, 1 mg of bovine serum albumin, 50 µM oleoyl-CoA, and 250 µM [C]glycerol 3-phosphate (specific activity, 0.4 mCi/mmol). After a 10-min incubation at 37 °C, lipids were extracted as described above. The amount of radioactivity in phosphatidic acid and neutral lipids was determined after separation of the lipid components by TLC. The TLC plate was first developed halfway in chloroform/methanol/ammonium hydroxide/water, 65:35:1:3 (v/v/v/v) followed by neutral lipid system as described above for the triacylglycerol synthetase assay. The respective lipid areas were scraped into vials, and radioactivity was determined by scintillation counting.

Monoacylglycerol lipase and AAT were assayed by monitoring 2-oleoyl-[2-H]glycerol hydrolysis in the absence of acyl-CoA and [C]oleic acid release from [C]oleoyl-CoA in the absence of acyl acceptors, respectively. The reaction mixture and assay conditions as well as the TLC solvent system were identical to those used for measuring monoacylglycerol acyltransferase activity.

Inhibition Assays

Purified triacylglycerol synthetase fractions were preincubated for 30 min on ice or at 37 °C with indicated inhibitors. After the preincubation, oleoyl-CoA, radiolabeled 2-oleoylglycerol/1,2-dioleoyl-rac-glycerol or radiolabeled oleoyl-CoA with nonlabeled acyl acceptors were added, and the assay was continued at 37 °C. Incubations were terminated after 10 min, and radioactivity in di- and triacylglycerols was determined as described above.

Fatty Acid Labeling of Proteins

1-5 µg of Cibacron blue 3GA-agarose purified, dialyzed, and concentrated triacylglycerol synthetase were incubated at 37 °C with 50 µM [C]oleic acid (5 mCi/mmol), 20 mM ATP, 2.5 mM CoA, or with 50 µM [C]oleoyl-CoA (5 mCi/mmol) in Tris-HCl buffer (pH 7.4), 4 mM MgCl, 20 mM KF, 1 mM DTT in a final volume of 20 µl for 20 min. Incubation was stopped by the addition of electrophoresis sample buffer, samples were boiled, and electrophoresis under nonreducing conditions, using 10% polyacrylamide gels was performed according to the procedure of Laemmli (13) . Gel was stained with Coomassie Blue and destained overnight in 5% methanol and 7.5% acetic acid. Gel was then washed for 10 min in glacial acetic acid, soaked in 20% (w/v) 2,5-diphenyloxazole in acetic acid for 1.5 h, followed by a 30-min water wash, dried under vacuum, and exposed to Kodak X-Omat x-ray film at -80 °C for 4-6 days (14) .

Preparation of Immunoaffinity Columns

Covalently linked antibody-protein A agarose columns were prepared essentially as described by Schneider et al.(15) . Polyclonal antibodies against purified microsomal AAT from rat intestinal mucosa or IgGs from preimmune serum were bound to protein A agarose beads, the beads were washed twice with 10 column volumes of 0.2 M sodium borate (pH 9.0), and the IgGs were cross-linked to the protein A using 20 mM dimethylpimelimidate in 10 volumes of 0.2 M sodium borate (pH 9.0). The cross-linking reaction was stopped after 30 min by incubating the beads with 10 column volumes of 0.2 M ethanolamine (pH 8.0) followed by extensive washing with Tris-buffered saline.

Immunoprecipitation

10 µg of purified triacylglycerol synthetase was mixed with 0-50 µl of protein A-bound IgG (preimmune and immune) for 1 h. The gel was let to settle, and the supernatant was assayed for MGAT, DGAT, and AAT activities.

Other Methods

Protein bands after SDS-PAGE were stained using Coomassie Blue R-250 or silver stained by method of Rabilloud et al. (16) .

Protein concentrations were determined after precipitation with deoxycholate and trichloroacetic acid (17) by bicinchonic acid assay (Pierce Chemical Co.).

Unilamellar phosphatidylcholine or total rat liver microsomal phospholipid vesicles were prepared by sonication of 10 mg of phospholipid/ml of 25 mM Tris (pH 7.8) for 30 min on ice followed by ultracentrifugation at 42,000 rpm (TI 70 rotor) for 3 h. The translucent supernatant was recovered with Pasteur pipette, and the phospholipid concentration determined by gas-liquid chromatography after phospholipase C digestion using tridecanoylglycerol as internal standard (18) .


RESULTS

Isolation of Enzyme Complex

The zwitterionic detergent CHAPS was most effective in solubilizing the enzyme activities comprising the triacylglycerol synthetase complex. DGAT has been previously shown to be more labile and more susceptible to inactivation by different reagents than MGAT and acyl-CoA ligase (4, 19) . CHAPS at detergent/protein (w/w) ratio of 1:1 and final concentration of 0.6% solubilized on average 52% of DGAT and 70% of MGAT and AAT activities. Higher concentrations of the detergent were inhibitory to all complex activities. Incubation with 1% CHAPS at detergent/protein ratio of 3:1 yielded only 15% of soluble DGAT activity and resulted in only 60% recovery of the original microsomal activity. Zwittergent 3-14 treatment of the microsomal membranes or extraction of microsomes with n-heptane resulted in a complete inactivation of the acyltransferase activities. Presence of CHAPS in the enzyme assays was not inhibitory, provided the detergent concentration was kept below its Cys(Cm) and the detergent/protein weight ratio was 1:1 or lower.

Fig. 1 shows a typical elution profile obtained by chromatography of the 0.6% CHAPS-solubilized microsomal extract on a Cibacron blue 3GA-agarose column. Approximately 90% of the protein was found in the flow-through. Some protein devoid of acyltransferase activity was eluted with 10 mM ATP, while the triacylglycerol synthetase activity was eluted with 0.8 M NaCl. 10% polyacrylamide gels, silver-stained after SDS-PAGE, showed several protein bands with the predominant polypeptides migrating at apparent molecular mass between 52-72 kDa (Fig. 2). 80% of the synthetase activity chromatographed with approximately 5% of the applied protein. Although the chromatography on the dye-agarose yielded an estimated 20-fold purification of the complex from solubilized microsomes based on the protein and specific activities of the enzyme components, it also led to substantial inactivation of the complex since only a 30% recovery of the original solubilized activity was obtained. We did not succeed in reconstitution of the solubilized activity by combining various column fractions or by adding increasing amounts of sonicated phospholipids isolated from rat liver microsomes. No significant activation of the solubilized and affinity purified complex by added phospholipid was observed (Fig. 3A). At concentration of 200 µg/ml, MGAT activity was slightly stimulated while DGAT activity was almost 50% inhibited. Inactivation of both enzymes occurred at higher phosphatidylcholine concentrations. Recovery of enzyme activities from the protein bands after SDS-PAGE (20) or native gel (SDS substituted with Triton X-100, Lubrol-PX, or CHAPS) electrophoresis was not successful. The Cibacron blue A-purified fraction contained the MGAT, DGAT, acyl-CoA ligase and AAT activities. The complex was found to be devoid of any monoacylglycerol lipase activity, which was detected in the flow-through fractions or of phosphatidic acid phosphohydrolase activity, which was removed during washing of microsomes with salt. However, it is possible that our preparations contain inactivated glycerol-3-phosphate acyltransferase. The enzyme activities in the isolated complex were stable for at least 1 week at -20 °C or 3 days at +4 °C, during which time dialysis and additional handling was performed.


Figure 1: Elution profile of 0.6% CHAPS-solubilized rat villus cell microsomes chromatographed on Cibacron blue 3GA-agarose. 1.5-ml fractions were collected. Activity was measured for triacylglycerol formation from 2-monoacylglycerol and oleoyl-CoA () as described under ``Experimental Procedures.'' Arrows indicate changes of elution buffers (i.e. ATP and NaCl). Protein () was determined using BCA assay.




Figure 2: SDS-PAGE profile of proteins at different stages of purification. MIC, washed microsomal membranes; SOLMIC, solubilized microsomes; BLUEA, triacylglycerol synthetase active fraction purified by the dye-ligand chromatography. 2 µg of protein was electrophoresed in a 10% SDS-polyacrylamide gel and visualized by silver staining. Molecular mass standards are indicated in the left margin.




Figure 3: Inactivation of triacylglycerol synthetase. Affinity-purified triacylglycerol synthetase was incubated with various concentrations of unilammelar phosphatidylcholine vesicles (A), NEM (B), and DIDS (C) for 30 min on ice. Enzyme activities were then assessed using 1-10 µg of purified triacylglycerol synthetase, 60 µM 2-oleoyl-[2-H]glycerol, 30 µM oleoyl-CoA, 1 mM DTT, 2 mg/ml bovine serum albumin, and 25 mM Tris-HCl in a final volume of 0.5 ml. After a 10-min incubation at 37 °C, the radioactivity in the lipid was quantitated as described under ``Experimental Procedures.'' Values plotted are the percentage of noninhibited values for the MGAT () and DGAT () from two independent experiments performed in duplicate.



Essential Nature of Sulfhydryl Groups

Presence of DTT in our assay buffer protected the enzymes and stimulated both the acyltransferase (MGAT and DGAT) and AAT activities, indicating that free sulfhydryl groups of cysteine residues were involved in the acyl transfer reactions. Preincubation of the purified fractions with NEM, which specifically binds sulfhydryl groups, had an inhibitory effect on both MGAT and DGAT activities, with the latter being more sensitive to the reagent (Fig. 3B). Approximately 30 and 60% inhibition of MGAT and DGAT activities, respectively, was evident at 0.5 mM NEM even in the presence of 1 mM dithiothreitol. Similar inhibition values were obtained for AAT activity. At 5 mM NEM, 40% of the MGAT and about 90% of DGAT activities were inhibited. Iodoacetamide, which like NEM is a specific sulfhydryl-directed agent, also displayed inhibitory effect on both MGAT and DGAT as well as on AAT activities. Preincubation with DIDS, which interacts with free amino groups of basic amino acid residues, resulted in 80% inhibition of MGAT and 95% inhibition of DGAT activities at 50 µM concentration (Fig. 3C), demonstrating the potential involvement of a free amino group in the acyl transfer reactions.

Evidence for Acyl-CoA Hydrolysis Prior to Acyl Transfer in Acylglycerol Synthesis

We have investigated the mechanism of the acyl transfer reaction by incubations of the purified complex with radiolabeled oleoyl-CoA and nonradioactive 2-oleoylglycerol (Fig. 4A) or diacylglycerol (Fig. 4B). In the absence of a proper acyl acceptor, only hydrolysis (transfer to water) of acyl-CoA occurred. However, in the presence of 2-monoacylglycerol or diacylglycerol, the amount of radioactivity in the free fatty acid was greatly diminished in favor of di- and triacylglycerol synthesis. The decrease of label in the free fatty acid fraction in the presence of 2-monoacylglycerol or 1,2-diacylglycerol did not result from an inhibition of AAT by the acylglycerols since monoacylglycerols and diacylglycerols at the used concentrations do not alter the AAT activity (5) . The acceptor-independent acyl-CoA hydrolysis is suggestive of ping-pong kinetics of acyl transfer. This sequential ordered mechanism is also characterized by the occurrence of a covalent enzyme-substrate intermediate (21) .


Figure 4: Assay of triacylglycerol synthetase with [1-C]oleoyl-CoA and nonlabeled acyl acceptors. Incubations and lipid extractions were carried out as described under ``Experimental Procedures.'' The extracted lipids were spotted on Polygram Sil N-HR thin-layer plates (Machery-Nagel, Germany), developed first in chloroform/methanol 2:1 (v/v) to a height of one-third of the plate, and then taken to the top with heptane/isopropyl ether/acetic acid, 60:40:4 (v/v/v). The TLC plate was sprayed with EnHance and exposed to Kodak X-Omat x-ray film at -80 °C for 2 days. DG, diacylglycerol; FFA, free fatty acid; TG, triacylglycerol.



Formation of Acylproteins by Purified Triacylglycerol Synthetase from Intestinal Mucosa

Incubation of Cibacron blue 3GA-agarose-purified and Aquacide III-concentrated complex with [C]oleic acid in the presence of ATP and CoA or with [C]oleoyl-CoA resulted in rapid and selective labeling of the 54-kDa AAT (Fig. 5). Because the protein acylation requires the formation of acyl-CoA (5) , the incorporation of the label from oleic acid in the presence of ATP and CoA also directly demonstrates the presence of acyl-CoA ligase in the complex.


Figure 5: Fatty acid labeling of purified triacylglycerol synthetase. 10 µg of triacylglycerol synthetase was incubated for 20 min at 37 °C with [1-C]oleic acid, 20 mM ATP, and 2.5 mM CoASH (A) or [1-C]oleoyl-CoA (B), and the acylproteins were analyzed by SDS-PAGE and fluorography as described under ``Experimental Procedures.''



Inhibition of Acyl-Enzyme Formation

Preincubation of the enzyme complex with NEM, iodoacetamide, and DIDS at their maximal inhibitory concentrations resulted in complete abolishment of incorporation of the labeled fatty acid into the polypeptide (Fig. 6).


Figure 6: Inhibition of acylation of the 54-kDa AAT. Purified triacylglycerol synthetase (40 µl of 0.5 mg/ml preparation) was preincubated for 30 min at 37 °C with 10 µl of phosphate buffer (A), 50 mM NEM (B), 2 mM DIDS (C), 250 mM iodoacetamide (D) (all inhibitors were dissolved in 20 mM phosphate buffer, pH 7.4). After the preincubation, [1-C]oleoyl-CoA was added, and the incubation was continued for additional 20 min. Reactions were stopped by addition of 50 µl of 2 concentrated electrophoresis sample buffer, and the incorporation of radioactivity into proteins was analyzed by SDS-PAGE and fluorography as described under ``Experimental Procedures.''



Immunoprecipitation of AAT from the Purified Complex

Incubation of purified triacylglycerol synthetase with protein A-bound anti-AAT antibodies resulted in substantial depletion of the enzyme from the complex as assessed by both enzyme assays and SDS-PAGE. In addition to 60 ± 5% decrease of the AAT activity, corresponding decreases of MGAT (67 ± 3%) and DGAT (68 ± 5%) activities were observed (Fig. 7).


Figure 7: Immunoprecipitation of AAT activity from the triacylglycerol synthetase complex by anti-AAT polyclonal antibodies. Purified triacylglycerol synthetase (10 µg) was mixed with 0-50 µl of protein A-bound preimmune or anti-AAT IgG for 1 h. Immune complexes bound to protein A-agarose were pelleted, and the activities of AAT, MGAT, and DGAT in the supernatant were determined. The results are expressed as the percent of the activities recovered in control incubations. The values represent averages of three separate experiments performed in duplicates.




DISCUSSION

A thorough investigation of the biosynthetic pathways of glycerolipids in intestinal mucosa has been hindered by the absence of the purified enzymes. Because tight lipid-protein interactions are necessary for optimal activity of many of these enzymes, attempts to solubilize and purify them have met with only partial success. Detergent extraction of microsomal membranes mostly led to unstable enzyme preparations with substantial loss of activities (2, 3, 4) . The ionic detergents (sodium cholate, SDS) present at their solubilizing concentrations have severe inhibitory effect on DGAT and nonionic detergents (Triton and Zwittergent series, octyl glucoside) although effective in extracting the membrane proteins, yield largely inactive enzymes. Some of the nonionic detergents can be thought of as fatty acid analogues, as they are composed of long aliphatic tail and a polar head group and may compete for the active site required for fatty acid binding, thus inhibiting the enzymatic activity.

We have solubilized these enzymes from their membranous environment by the zwitterionic detergent CHAPS, which combines the properties of ionic and nonionic detergents. In addition to preserving substantial enzymic activity, CHAPS is stable over a wide range of pH and can be easily removed by dialysis. CHAPS could also be used at low concentration (0.05%) as a dispersing agent for the hydrophobic and amphiphilic lipid acceptors in the triacylglycerol synthetase activity assays.

Gel filtration of CHAPS-solubilized microsomes suggested that the enzyme components of the triacylglycerol synthetase complex were dissociated from each other because the ratio of MGAT and DGAT activity was not the same before and after chromatography (results not shown). In addition, the observed molecular mass of 100 kDa for the eluted activities does not seem to be sufficient to account for undissociated complex. Gel filtration of hamster (2) and rat (4) triacylglycerol synthetase showed elution of the synthetase activity at higher molecular mass. However, these procedures were carried out in the absence of detergents and may have represented recoveries of aggregated proteins.

The in vitro products of triacylglycerol synthesis from monoacylglycerol and acyl-CoA differ from those obtained in vivo by much higher diacylglycerol/triacylglycerol ratios. While in vivo little diacylglycerol intermediate is discernible, accumulation of diacylglycerols is readily observed in vitro when either microsomal or purified enzyme fractions are employed. It has been reported (22, 23, 24) that rat liver and intestinal diacylglycerol acyltransferase require a cytoplasmic protein of low molecular mass for activation, while Cianflone et al.(25) have purified such a protein from plasma. In our assays, the addition of total or fractionated cytosolic fraction (106,000 g supernatant) to rat intestinal microsomal or purified preparations also enhances total triacylglycerol synthesis from monoacylglycerol and acyl-CoA; however, the ratios of diacylglycerol intermediates and triacylglycerol products remain largely unchanged.() With the purified synthetase, the label in free fatty acid (acyl-CoA hydrolysis) accounts for up to 50% of the reaction product, while with microsomes, the label in fatty acid corresponds to less than 20% of total. Also, DG/TG ratio is higher in the solubilized preparations, which suggests impaired channeling of intermediates.

We have exploited the benefits of solubilization by CHAPS in the purification of triacylglycerol synthetase by dye-ligand affinity chromatography, a procedure used successfully in the purification of rat liver microsomal acyl-CoA ligase (26) and recently of rat intestinal AAT (5) . The dye-ligand is thought to interact directly with specific substrate and cofactor binding sites, although ionic and hydrophobic forces also contribute to the protein adsorption. The Cibacron blue 3GA (Blue A) ligand was the most effective in retaining the complex, but other ligands (Red A, Green A, Blue B) also were able to bind the complex to some extent. Orange A and control agarose (no dye) did not retain the complex, thus indicating the specificity of interaction between the gel matrix and the proteins. Until now, no direct interactions have been detected between the enzymes proposed to form the triacylglycerol synthetase complex, and the complex may only be formed in situ. The existence of such complex is supported by the low amount of the diacylglycerol intermediate observed in vivo. This is consistent with channeling of intermediates along the metabolic pathway.

The triacylglycerol synthetase complex purified by dye chromatography was composed of four major polypeptides with apparent molecular masses between 52 and 72 kDa, which were also observed in the preparations by Manganaro and Kuksis (4) . A single-sized unit of 72 ± 4 kDa was required for rat liver microsomal DGAT activity as analyzed by radiation inactivation (27) , and a monoclonal antibody reacting with a 60-kDa protein on Western blots immunoprecipitated rat liver DGAT activity (28) . On the other hand, a molecular mass of 1,539 kDa was reported for DGAT purified from germinating soybeans (29) . A dissociation of a 37-kDa polypeptide apparently possessing MGAT activity from the 50-56-kDa region by gel filtration in denaturing 6 M guanidine was reported (30) , suggesting that some acyltransferases may function as multimers. Recently, a 40-fold purification of an apparently tissue-specific MGAT isoenzyme from solubilized microsomes of neonatal rat liver was obtained (31) . Contrary to the MGAT of intestinal mucosa (30) , the liver enzyme does not appear to interact with an anion-exchange medium, suggesting that it is a basic protein (31) . Gel filtration of the partially purified liver MGAT led to loss of the enzyme activity (31) . The authors attributed this to tight binding of the enzyme to the Sephacryl medium even in the presence of detergent. The triacylglycerol synthetase complex purified by the dye-ligand chromatography does not contain monoacylglycerol lipase or phosphatidic acid phosphohydrolase activities. It may contain glycerol-3-phosphate acyltransferase as we were not able to confirm the absence or presence of this enzyme because of its inactivation by the detergent. A 54-kDa protein was identified as a potential candidate for GPAT in adipose tissue by selective modification with radiolabeled iodoacetate (32) . These authors, however, did not take into consideration that other enzymes may be susceptible to the same covalent modification as they may also possess reactive cysteine residues. It is possible, that the adipose tissue 54-kDa protein is an isoenzyme of AAT that we purified from rat intestinal mucosa. The intestinal AAT is acylated by incubation with acyl-CoA, and this modification is blocked by preincubation of the enzyme with sulfhydryl binders, including iodoacetamide. Although AAT activities in various subcellular fractions of mammalian tissues (33-38) have been described and purified, the biological function(s) of these enzymes have remained unknown. It was suggested that the enzyme may play an important role in lipid metabolism by controlling the chain length of synthesized fatty acid (39) , modifying the product specificity of fatty acid synthetase (40) , or controlling the intracellular concentrations of acyl-CoA esters (35) . Our results suggest that AAT may be actively involved in regulation of acylglycerol biosynthesis. Immunoprecipitation of the enzyme activity from the purified triacylglycerol synthetase complex decreased the levels of total triacylglycerol synthesis. Electrophoretic analysis of the immunoprecipitate showed that only the 54-kDa AAT was bound to the beads. This indicates that the components of the complex were resolved from each other in the presence of detergent. It also explains the much higher rate of acyl-CoA hydrolysis in the purified synthetase compared with the membrane-bound enzymes where acyl-CoA is much more efficiently utilized for the synthesis of di- and triacylglycerols. It thus appears that the enzyme participates in the acyl group transfer to suitable acyl acceptor molecules. Acyl-CoA hydrolysis is a necessary event in the esterification reaction, and the enzyme may represent an acyl-CoA binding subunit of a hetero-oligomeric complex catalyzing acylglycerol biosynthesis. In the absence of acyl acceptors acyl-CoA is transferred to water. The involvement of AAT in the synthesis of cholesterol esters by rat liver microsomal acyl-CoA:cholesterol acyltransferase has also been recently suggested (41) . The formation of an acylthioenzyme and acceptor independent acyl-CoA hydrolysis is consistent with a sequential ordered ping-pong acyl transfer mechanism (Fig. 8). In such model, the first step would constitute transfer of acyl-CoA from acyl-CoA ligase to AAT, resulting in the formation of a covalent acyl-enzyme intermediate. The second step would involve an acyl transfer from AAT to monoacylglycerol in a reaction catalyzed by MGAT. The intermediate diacylglycerol would then be channeled to DGAT, and an acyl transfer from the acyl-AAT would complete the TG synthesis.


Figure 8: Possible ping-pong mechanism of triacylglycerol synthesis by the triacylglycerol synthetase complex of rat intestinal mucosa. E, acyl-CoA ligase; E, acyl-CoA acyltransferase; E, monoacylglycerol acyltransferase; E, diacylglycerol acyltransferase.



In addition to the potential involvement of cysteine residue(s) in the acylation process, basic guanidinium groups of arginyl residues reported to form salt bridges with the phosphoanions of CoA (42, 43) may also play an important role since DIDS (a basic amino acid side chain modifier) inhibits both diacylglycerol and triacylglycerol synthesis.


FOOTNOTES

*
This work was supported by the Medical Research Council of Canada, Ottawa, Ontario and the Heart and Stroke Foundation of Ontario, Toronto, Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Laboratoire de Lipolyse Enzymatique, UPR 9025, CNRS, 31, Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.

To whom correspondence should be addressed: Banting and Best Dept. of Medical Research, University of Toronto, 112 College St., Toronto, Ontario, Canada M5G 1L6. Tel.: 416-978-2590; Fax: 416-978-8528.

The abbreviations used are: MGAT, monoacylglycerol acyltransferase; DGAT, diacylglycerol acyltransferase; AAT, acyl-CoA acyltransferase; NEM, N-ethylmaleimide; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DIDS, 4,4`-diisothiocyanostilbene-2,2`-disulfonate; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; TLC, thin-layer chromatography.

R. Lehner and A. Kuksis, unpublished data.


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