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INTRODUCTION |
Biosynthesis of diacylglycerol is shown to occur by the sequential
acylation of glycerol-3-phosphate (1-3). The first enzyme in this
pathway, G3P1
acyltransferase, catalyzes the formation of lysophosphatidic acid
(LPA). LPA can also be synthesized by acylation followed by the
reduction of dihydroxyacetone phosphate that is catalyzed by
dihydroxyacetone phosphate acyltransferase (4) and the
NADPH-dependent acyl-dihydroxyacetone phosphate reductase
(5), respectively. LPA is shown to induce a wide range of activities in
animal systems (6-8). LPA can be metabolized through dephosphorylation
by a soluble LPA phosphatase to form monoacylglycerol (MAG) or acylated to phosphatidic acid (PA) by LPA acyltransferase. LPA phosphatase has
been identified (9), purified (10), and cloned (11) from animal
systems. PA is the precursor for diacylglycerol (DAG) and anionic
phospholipids. PA phosphatase catalyzes the dephosphorylation of PA to
form DAG, which is the immediate precursor for triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine. DAG is also an
important signal molecule that activates protein kinase C (12). DAG can
also be derived directly from phospholipids by the action of
phospholipase C (13).
Alternatively, DAG can be synthesized by the esterification of MAG by
acyl-CoA:MAG acyltransferase (EC 2.3.1.22). This enzyme has been
proposed to be important for fat absorption in human small intestine
(14-16). Another acyltransferase, acyl-CoA-independent MAG
acyltransferase, has been purified to homogeneity from rat intestinal
mucosa (17), whereas acyl-CoA-dependent MAG acyltransferase has not been purified from any source. All the acyltransferases in
these pathways are membrane-bound, and most of them use acyl-CoA as a
primary acyl donor (1-3).
We identified DAG biosynthetic activity from the soluble fraction of
developing peanut (Arachis hypogaea) cotyledons, and the
enzyme involved was purified to apparent homogeneity by successive column chromatographic procedures and characterized. This is the first
report of the purification of acyl-CoA-dependent MAG acyltransferase.
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EXPERIMENTAL PROCEDURES |
Materials--
[1-14C]Palmitoyl-CoA (54 mCi
mmol
1), [9,10-3H(N)]trioleoylglycerol (10 Ci mmol
1), [glycerol-U-14C]PA (100 mCi
mmol
1),
[[2-palmitoyl-9,10-3H]phosphatidylcholine (92.3 Ci
mmol
1) and [2- 3H]G3P (12 Ci
mmol
1), were obtained from Perkin Elmer Biosystems. Prep
grade Superdex 75 (26/60) and Superdex 200 (10/30) FPLC columns,
octyl-Sepharose 4 Fast Flow matrix, and gel filtration molecular mass
standards were purchased from Amersham Pharmacia Biotech. Protein assay reagents were obtained from Pierce. Thin layer chromatography plates
were from Merck. All other reagents were obtained from Sigma. MAG was
purified by preparative silica-TLC and quantified colorimetrically (18,
19). Field grown-developing peanut (Arachis hypogaea L.)
cotyledons were harvested at 20-25 days after flowering and used
either fresh or stored at
80 °C until further use.
Lipid Extraction--
Lipids were extracted from 10 g of
frozen immature seeds by grinding the tissue in liquid nitrogen to a
fine powder in mortar and pestle (20). The powder was extracted with 20 ml of boiling isopropyl alcohol. The mixture was then centrifuged
briefly, supernatant was removed, and the extraction repeated twice.
The pooled isopropyl alcohol extracts were brought to dryness on a
rotary evaporator. The tissue residue was then reextracted twice with
38 ml of chloroform/methanol/10% acetic acid (1:2:0.8, v/v). After
centrifugation, the supernatant was added to the isopropyl alcohol
extract, and 20 ml each of chloroform and water were added to the
mixture. The biphasic system was mixed and centrifuged. The lower
chloroform phase was removed and dried in a rotary evaporator. The
lipid residue was dissolved in chloroform/methanol (1:1, v/v) and
stored at
20 °C.
Preparation of Subcellular Fractions--
Differential
centrifugation was used to fractionate intracellular components (21).
Either fresh or frozen immature seeds (100 g) were ground in a
prechilled mortar and pestle with 10 g of acid-washed sand and 250 ml of buffer consisting of 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10 mM KCl, 1 mM
MgCl2, 1 mM
-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg ml
1
leupeptin, and 0.25 M sucrose. The extract was passed
through two layers of cheesecloth and centrifuged at 3,000 × g for 10 min. The supernatant was centrifuged at 18,000 × g for 15 min. The 18,000 × g supernatant
was further centrifuged at 150,000 × g for 2.5 h.
The pellets were resuspended in small volume of buffer containing 20 mM Tris-HCl, pH 7.0, and 1 mM
-mercaptoethanol. All these operations were performed at 4 °C.
All the fractions were assayed for acyltransferase activities. Protein
concentrations were determined by the bicinchoninic acid method (22)
using bovine serum albumin as the standard. For the purification of MAG
acyltransferase, the homogenate was centrifuged directly at 18,000 × g for 30 min, and the supernatant was then centrifuged at
150,000 × g for 2.5 h. The 150,000 × g supernatant (soluble fraction) was used as the source for
MAG acyltransferase.
Enzyme Assays--
The assay mixtures consisted of 50 mM Tris-HCl, pH 7.0, 20 µM
[1-14C]palmitoyl-CoA (100,000 dpm), 15-45 µg enzyme,
and 50 µM MAG (1-oleoyl) in a total volume of 100 µl.
The incubation was carried out at 30 °C for 10 min and stopped by
the addition of 400 µl of CHCl3/CH3OH (1:2,
v/v). Following lipid extraction by the modified method of Bligh and
Dyer (23), the lower chloroform-soluble materials were separated by TLC
on 250 µm silica gel G plates either using petroleum ether/diethyl
ether/acetic acid (70:30:1, v/v) or chloroform/methanol/water
(98:2:0.5, v/v) as the solvent system (24). The lipids were visualized
with iodine vapor, and the spots of DAG scraped off for determination
of radioactivity by liquid scintillation counting.
Purification of MAG Acyltransferase--
All operations were
conducted at 4 °C except the FPLC purification step, which was
conducted at ambient temperature. Buffer A contained 50 mM
Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM KCl, 1 mM MgCl2, 1 mM
-mercaptoethanol,
and 0.1 mM phenylmethylsulfonyl fluoride.
Octyl-Sepharose Chromatography--
Solid ammonium sulfate was
added to bring the soluble fraction to 1 M followed by
centrifugation to make the solution clear and then loaded onto an
octyl-Sepharose column (4.4 × 15 cm) that had been
pre-equilibrated with 1 M ammonium sulfate in Buffer A with
a flow rate of 1 ml min
1. The column was washed with the
same buffer to remove unbound proteins until the effluent had very low
absorbency at 280 nm. The enzyme was eluted with 200 ml of a
linear-reversed gradient from 1 to 0 M ammonium sulfate in
Buffer A, and fractions of 5 ml were collected. The active fractions
were pooled and dialyzed against Buffer A.
Blue-Sepharose Chromatography--
Active fractions from the
octyl-Sepharose were combined, dialyzed against Buffer A, and applied
onto a blue-Sepharose (cibacron blue A) column. The column was eluted
with a 0-1 M NaCl gradient.
Size Exclusion Chromatography--
The fractions eluted at
0.35-0.4 M NaCl from the blue-Sepharose were concentrated
using a Centricon (30-kDa cut-off) concentrator and filtered. The
filtrate was applied onto a preparative Superdex 75 FPLC column fitted
with Bio-Rad BioLogic low-pressure chromatography system. The column
was eluted with the same buffer at a flow rate of 5 ml
min
1.
Palmitoyl-CoA-agarose Chromatography--
A 2.5-ml sample of
palmitoyl-CoA-agarose was pre-equilibrated with Buffer A at room
temperature, and the active fractions from the previous column was
mixed with matrix for 90 min at 4 °C. The mixture was then poured
into a column, washed with Buffer A, and eluted with Buffer A
containing 0.25, 0.5, and 1 M NaCl, respectively. The MAG
acyltransferase activity was eluted at 0.5 M NaCl.
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RESULTS |
Subcellular Distribution of DAG Biosynthesis in Immature
Peanuts--
The subcellular distribution of DAG biosynthesis in fresh
peanut cotyledons was studied by isolating the intracellular components by differential centrifugation. DAG biosynthetic capacity was found low
in the membrane fraction because of the presence of active
triacylglycerol biosynthesis (data not shown). However, DAG formation
was higher in 150,000 × g supernatant as compared with
the corresponding microsomal pellet, which is in agreement with the
earlier report on developing rapeseed (25). The pattern of distribution
remained the same even in the frozen tissue, but total DAG biosynthetic
activity decreased 21% after freezing and thawing. These results
suggested that the additional DAG biosynthetic machinery could exist in
the soluble fraction.
Marker Enzyme Activities from Peanut Soluble Fraction--
There
was a significant amount of DAG formation in the soluble fraction, and
this could either be caused by the presence of soluble enzymes or the
presence of nonsedimentable cellular membrane fragments generated
during isolation procedures. Marker enzyme activities were measured to
assess the extent of contamination in the soluble fraction with the
membranes. The activities of succinate dehydrogenase (26), NADH
cytochrome C reductase (27), and vanadium sensitive ATPase (28) were
measured as the marker enzymes for mitochondrial, microsomal, and
plasma membranes, respectively. About 6% (1.2 µmol
min
1 mg
1), 18% (1.3 µmol
min
1 mg
1) and 11% (0.55 µmol
min
1 mg
1) activities of these enzymes were
detected in the soluble fraction, respectively. These results indicated
that the membrane contamination in the soluble fraction was not significant.
Incorporation of [14C]Palmitoyl-CoA into
Diacylglycerol--
Effect of [14C]palmitoyl-CoA
incorporation into DAG was measured both in the soluble and the
membrane fractions of immature peanuts (Fig.
1). Maximum activity was observed at 10 µM in the soluble fraction and at 20 µM
palmitoyl-CoA in the membrane fraction. Exogenous MAG did not alter the
rate and the pattern of incorporation into DAG. To determine the
amounts of MAG in the immature peanut seeds, the total lipid was
extracted as described under "Experimental Procedures." The amounts
of MAG in the immature seeds and the soluble fraction were calculated
to be about 134 nmol/g fresh weight and 2.6-3.1 nmol/mg protein,
respectively.

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Fig. 1.
Effect of palmitoyl-CoA on diacylglycerol
biosynthesis. Incorporation of [14C]palmitoyl-CoA
into DAG was carried out in the presence of 50 µM MAG in
soluble ( ) and membrane ( ) fractions. Each point is the average
of two independent experiments.
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Formation of DAG by the Acylation of MAG--
DAG can be
synthesized either by the dephosphorylation of PA or by the direct
acylation of MAG. To find out the contribution of each step to the
total DAG pool, peanut soluble fraction was treated with various
concentrations of NaF to inhibit phosphatase activity, and these were
used for the incorporation of [14C]palmitoyl-CoA into
DAG. Initially, we studied the effect of various concentrations of NaF
on the dephosphorylation of PA in the membrane fraction and found that
there was no formation of DAG at 20 mM NaF (Fig.
2A). As shown in Fig.
2B, there was only a 25-38% decrease in the incorporation
of [14C]palmitoyl-CoA into DAG in the soluble fraction in
the presence of 20 mM NaF suggesting that the DAG formation
was independent of PA dephosphorylation activity. NaF-treated membrane
fraction showed a profound inhibition of DAG formation indicating the
presence of NaF sensitive PA dephosphorylation activity (Fig.
2C). These results suggest that the soluble fraction has
NaF-insensitive and PA dephosphorylation-independent DAG
biosynthesizing activity in the soluble fraction of immature
peanuts.

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Fig. 2.
Generation of diacylglycerol.
A, effect of NaF on the dephosphorylation of
[14C]PA to DAG was determined in the soluble ( ) and
membrane ( ) fractions. The incorporation of
[14C]palmitoyl-CoA in the presence ( ) or absence ( )
of 20 mM NaF was performed with soluble (B) and
membrane (C) fractions into DAG in the absence of exogenous
acyl acceptor. Each point is an average of three determinations.
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Evaluation of Different Plant Tissues for MAG Acyltransferase
Activity--
Incorporation of [14C]palmitoyl-CoA into
DAG was studied in the soluble and the particulate fractions from the
tissues of leaf and hypocotyl of peanut and immature seed and leaf
tissue of castor (Ricinus communis L.). The MAG
acyltransferase activity was not detected in the soluble fractions of
leaf and hypocotyl, but the enzyme activity was in the membrane
fractions (data not shown). In peanut leaf, the activity was ~48-fold
lower than that of immature seed. These data suggest that MAG
acyltransferase was also present in other plant tissues but at low levels.
Purification of MAG Acyltransferase from Peanut
Cotyledons--
MAG acyltransferase activity was found to be high in
the soluble fraction, and the rate of synthesis of diacylglycerol in the soluble fraction was 24 pmol min
1 mg
1.
Solid ammonium sulfate was added to bring the soluble fraction to 1 M and then loaded onto an octyl-Sepharose column. The
column was eluted with a 1-0 M linear-reversed gradient of
ammonium sulfate (Fig. 3A).
This step was the most effective resulting in a 221-fold purification
of acyltransferase and yielding a 2.6-fold increase in the total
activity. The active fractions from the octyl-Sepharose were loaded
onto a blue-Sepharose column and eluted with a linear NaCl gradient.
The activity was eluted between 0.35 and 0.4 M NaCl (Fig.
3B). The recovery of MAG acyltransferase activity from the
blue-Sepharose column was nearly 77% of that applied. The pooled
active fractions were applied to a preparative Superdex 75 column. The
MAG acyltransferase activity was eluted as a single peak from fraction
27 to 31 (Fig. 3C). The active fractions were pooled and
applied to a palmitoyl-CoA agarose column as the final step. An overall
purification of ~6,608-fold was obtained, and the specific activity
of acyltransferase was 15.86 nmol min
1 mg
1
(Table I). The purified enzyme was
resolved on a 12% SDS-polyacrylamide gel, which showed a single band
with a molecular mass of 43 kDa (Fig. 4).
The native molecular mass of the purified enzyme was found to be 43 kDa
by Superdex 200 column chromatography (data not shown).

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Fig. 3.
Elution profile of MAG acyltransferase with
column chromatography. A, the soluble fraction from
immature peanuts was loaded onto an octyl-Sepharose column that was
pre-equilibrated with 1 M ammonium sulfate. The MAG
acyltransferase was eluted from the octyl-Sepharose column in a
reversed linear gradient of ammonium sulfate (---), and 5-ml fractions
were collected. B, the active fractions from the
octyl-Sepharose were pooled and loaded onto a blue-Sepharose column.
The MAG acyltransferase was eluted in a linear gradient of NaCl (---),
and 1.5-ml fractions were collected. C, active fractions
from the blue-Sepharose column were pooled and loaded onto a
preparative gel filtration column (Superdex 75), and 5-ml fractions
were collected. All the fractions from various column effluents were
assayed for MAG acyltransferase activity ( ) and protein
concentration ( ).
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Table I
Purification of MAG acyltransferase from developing peanut cotyledons
The results are from the summary of purification of the MAG
acyltransferase. Frozen immature seed (100 g) was used for preparing
the soluble fraction. The enzyme activity measurement and the
purification steps are described in "Experimental Procedures."
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Fig. 4.
SDS-polyacrylamide gel electrophoresis
profile of MAG acyltransferase purification. Samples from each
purification step were separated by 12% SDS-polyacrylamide gel
electrophoresis. Lanes 1-5 correspond to the pooled
fractions from steps 1-5 (Table I). Lane Mw represents the
standard molecular mass marker.
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To confirm that the 43-kDa polypeptide corresponds to the MAG
acyltransferase, the purified enzyme was loaded onto a 12%
SDS-polyacrylamide gel in the presence of 0.1% SDS without boiling and
electrophoresed at 4 °C. The gel was cut into 0.5-cm sections, the
protein eluted from the gel with Buffer A and assayed for the enzyme
activity (29). The yield of MAG acyltransferase activity from the gel was low (1.5%), and the activity was associated with the area of the
gel corresponding to the 43-kDa protein (data not shown). These results
indicate that the 43-kDa protein detected on the silver-stained gel
(Fig. 4) was indeed the MAG acyltransferase.
The reaction products formed at each step of purification were analyzed
on a silica-TLC and autoradiographed (Fig.
5). When the soluble fraction was
incubated with [14C]palmitoyl-CoA in the presence of
1-MAG (16:0), formation of 1-acyl-, 1,2-diacyl-, and 1,3-diacyl- and
triacylglycerols was observed suggesting the presence of many different
acylation activities. The active fractions eluted from the
octyl-Sepharose column showed minor amounts of other acylation
activities, which diminished in further purification steps as shown in
Fig. 5.

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Fig. 5.
Autoradiography of the TLC profile of the
reaction products formed at each step of purification. The enzyme
was assayed using [14C]palmitoyl-CoA and 1-MAG (16:0),
and the products formed were chromatographed using petroleum
ether/diethyl ether/acetic acid (70:30:1, v/v; A) and
chloroform/methanol/acetic acid (98:2:0.5, v/v; B) as the
solvent systems. Lane 1 represents heat-inactivated soluble
fraction; lanes 2-6 correspond to the active fractions from
steps 1-5 (Table I).
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Characteristics of Peanut MAG Acyltransferase--
The enzyme
activity was linear with respect to time and protein concentrations,
and the pH optimum of the MAG acyltransferase was found to be 7.0. The
enzyme was specific for MAG and did not utilize any other acyl acceptor
such as glycerol, G3P, LPA, or lysophosphatidylcholine. The effect of
various detergents on the MAG acyltransferase activity was studied
(data not shown). In the presence of 0.3% Triton X-100, the enzyme
activity was reduced to 58%. At 20 mM concentration of the
zwitterionic detergent (CHAPS), the enzyme lost its activity
completely. The activity of MAG acyltransferase was reduced to 50% in
the presence of 40 mM octylglucoside (data not shown).
Substrate Dependence of MAG Acyltransferase--
The substrate
specificity of MAG acyltransferase was studied by providing
monoacylglycerol of varying chain lengths and position of fatty acid as
the substrate. The MAG acyltransferase activity was highest for 1-MAG
(16:0) and lower for 2-MAG (16:0). The initial rate of reaction was
high for 1-MAG (18:1) but the Vmax was small when compared with MAG containing saturated acyl chains. The rate of
reaction declined sharply after 10 µM of 1-MAG (18:1).
The 1-MAG (16:0) and 1-MAG (18:1) had higher
Vmax values and the Km values were 16.39 and 5.65 µM, respectively. The other
monoacylglycerols had lower Vmax and apparent
Km values (Fig.
6A). These results suggest
that the MAG acyltransferase preferentially use
sn-1-monoacylglycerols.

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Fig. 6.
Lineweaver-Burk plot of MAG acyltransferase
activity toward monoacylglycerols and acyl-CoAs. A, the
enzyme activity was measured as a function of MAG concentration, and
the concentration of palmitoyl-CoA (20 µM) was kept
constant. B, the enzyme activity was measured as the
function of acyl-CoA concentrations, and
1-palmitoyl-sn-glycerol (20 µM) was kept
constant. Each point is the average of two determinations.
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MAG acyltransferase activity was the highest for palmitoyl-CoA when
compared with stearoyl-, and oleoyl-CoAs, but the initial rate of
reaction was higher for oleoyl-CoA. The apparent
Km values for palmitoyl-CoA, oleoyl-CoA, and
stearoyl-CoA were 17.54, 9.35, and 25.64 µM, respectively
(Fig. 6B). Competition studies with myristoyl-CoA and
lauroyl-CoA showed that the medium-chain acyl-CoAs and acetyl-CoA were
not good substrates for the MAG acyltransferase (data not shown). Based
on the Lineweaver-Burk plots, apparent Vmax and
Km values for acyl acceptors and acyl donors at
the standard assay conditions were obtained as summarized in Table
II.
Effect of Fatty Acids, Phospholipids, and Sphingoid Bases on MAG
Acyltransferase Activity--
Effect of fatty acids on the MAG
acyltransferase activity was studied using C8 to
C18 and C18:1 (Fig. 7A). Oleic acid
stimulated the MAG acyltransferase activity at concentrations below 15 µM, but at the higher concentrations, the activity was
reduced. Palmitic acid at concentrations between 20 and 50 µM had a stimulatory effect on the enzyme activity, and
other fatty acids had no significant effect on the MAG acyltransferase
activity. Phosphatidylcholine and phosphatidic acid had stimulatory
effects on the purified MAG acyltransferase activity at lower
concentrations from 2.5 to 15 µM, whereas at higher
concentrations no stimulatory effect was observed (Fig.
7B). Phosphatidylethanolamine
and phosphatidylinositol had no effect on the enzyme activity. At lower
concentrations of 2.5-7.5 µM, sphingosine and
sphingomylein activated the acyltransferase activity (Fig.
7C). The derivative of sphingosine, dehydrosphingosine showed lower stimulatory effects when compared with
sphingosine. Dehydrosphingosine had no effect on the MAG
acyltransferase activity.

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Fig. 7.
Effect of fatty acids, phospholipids, and
sphingoid bases on MAG acyltransferase activity. The MAG
acyltransferase activity was measured under the standard assay
conditions using [14C]palmitoyl-CoA and
1-palmitoyl-sn-glycerol in the presence of various
concentrations of fatty acids (A), phospholipids
(B), and sphingoid bases (C). Each point is the
average of two determinations.
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DISCUSSION |
The biosynthesis of diacylglycerol is known to occur via the
Kennedy pathway in the microsomal membranes (1-3, 30). The present
study deals with the first identification, purification, and
characterization of a soluble MAG acyltransferase from oilseeds. The
soluble DAG biosynthesizing activity was observed in immature peanut
cotyledons. The presence of a few soluble enzymes that provide
precursors for lipid biosynthesis have been reported. A soluble G3P
acyltransferase has been isolated from cocoa seed (31). PA phosphatase
is found to be localized both in the soluble and the membrane fractions
of Saccharomyces cerevisiae (32, 33) and higher plants (34).
A soluble DAG biosynthetic activity has been demonstrated in developing
rapeseed (25). LPA phosphatase (9, 10), DAG kinase (35, 36), inactive
choline cytidyltransferase (37), and active ethanolaminephosphate
cytidyltransferase (38) have also been found in the cytosol of animal systems.
The incubation of NaF-treated soluble fraction with
[14C]PA did not generate DAG; however, about 62-75% of
[14C]palmitoyl-CoA incorporation into diacylglycerol was
observed in the immature peanuts. The synthesized DAG did not originate from the hydrolysis of either triacylglycerol or phosphatidylcholine. These results provide evidences for an alternate enzymatic step for the
synthesis of DAG. In this study, we show that DAG is synthesized by the
acylation of MAG by acyl-CoA-dependent acyltransferase and
this enzyme has not been purified from any source. The following observations revealed that the MAG acyltransferase is present in the
soluble fraction. (i) The activity is associated with 150,000 × g supernatant. (ii) The enzyme is permeable in the gel
filtration column, and (iii) the MAG acyltransferase is purified to
homogeneity by successive column chromatographic separations without
detergent. The role of this enzyme in intracellular processes and its
regulation has yet to be elucidated. It appears that different tissues
express isozymes with respect to subcellular location. For example,
leaf enzyme is found in the particulate fraction, but the soluble
form is found in immature seeds.
We have purified to apparent homogeneity a MAG acyltransferase activity
from developing peanut cotyledons by successive chromatographic procedures. The key to the successful purification was the initial step
of octyl-Sepharose column chromatography, and this step gave a 221-fold
purification with a 2.6-fold increase in the total activity. The
increase in total enzyme activity could be attributed to the
elimination of an inhibitor or the enzymes competing for palmitoyl-CoA.
The remaining purification steps showed a successive increase in the
specific activity and the fold purification.
The purified peanut MAG acyltransferase showed the highest activity
with palmitoyl-CoA, but oleoyl-CoA had a lower
Km when compared with palmitoyl- or
stearoyl-CoAs. Characterization of the partially purified MAG
acyltransferase from rat liver (14), intestinal mucosa (39), and
adipocytes (40) has also showed higher activity with palmitoyl-CoA.
Unlike rat MAG acyltransferase (14), peanut enzyme showed a preference
to sn-1-monoacylglycerol over the sn-2 isomer.
This observation could also be attributed to the possible acyl
migration from sn-2 to sn-1 of MAG during conditions of storage, assay, or extraction (41). However, we are not
certain about either of the two possibilities. Kinetic experiments
showed that the overall catalytic efficiencies
(Vmax/Km) for 1-acyl
acceptor was higher than that of the 2-acyl acceptor suggesting the
1-acyl acceptor was a good substrate. The analysis of acyl donors
showed that the catalytic efficiency for oleoyl-CoA and palmitoyl-CoA
were comparable.
The activity of most of the lipid biosynthetic enzymes is dependent on
or modulated by the various lipid cofactors. In developing oilseeds,
lipid biosynthesis is highly active, and various metabolic intermediates are accumulated during seed development. All these intermediates either stimulate or inhibit the enzymes involved in lipid
metabolism. The characterization of the purified peanut MAG
acyltransferase indicated that the lower concentrations of phospholipids and oleic acid stimulated the activity. Apart from oleic
acid, palmitic acid also showed an activation effect on peanut MAG
acyltransferase. In contrast to our results, it was shown in partially
purified hepatic MAG acyltransferase that the higher concentrations of
fatty acid inhibited the enzyme activity (15). Sphingosine was shown to
inhibit rat hepatic MAG acyltransferase (42), but the peanut enzyme was
activated in the presence of lower concentrations of sphingosine, and
no inhibition was observed at higher concentrations.
Identification of MAG acyltransferase in peanut indicates the presence
of the MAG pathway for DAG biosynthesis. It has been proposed in animal
systems that the MAG pathway may play an important role in the
regulation of lipid metabolism by controlling the chain length of fatty
acids (43) or controlling the intracellular concentrations of acyl-CoA
esters (44) or facilitating selective retention of essential fatty
acids during hepatic oxidation (13). In plants, the MAG pathway may be
involved in the synthesis of triacylglycerol and may also provide a
regulatory link between signal transduction and synthesis of complex
lipids. Another possibility is that the MAG pathway contributes to a
separate intracellular pool of DAG for different sets of metabolic
reaction (45). The identification of the MAG acyltransferase has
significant implications in understanding the regulation of di- and
triacylglycerol biosynthesis in plants.