From the
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.
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,
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.
2-Oleoyl-[2-
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 [
Glycerol-3-phosphate acyltransferase activity was
determined using [
Monoacylglycerol lipase and
AAT were assayed by monitoring
2-oleoyl-[2-
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) .
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.
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 (
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
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) ,
(
)
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.
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 En
Hance were purchased from DuPont NEN. All
other chemicals and solvents were of reagent grade or better quality
and were obtained from local suppliers.
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) .
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) .
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.
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) .
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.
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 En
Hance 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.
0.05%) as a dispersing
agent for the hydrophobic and amphiphilic lipid acceptors in the
triacylglycerol synthetase activity assays.
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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.