 |
INTRODUCTION |
Cardiolipin (CL)1 is a
major membrane glycerophospholipid of mammalian mitochondria and is
localized exclusively to mitochondria (1-5). The synthesis of CL
occurs via the CDP-DG pathway in mammalian tissues (6, 7). Phosphatidic
acid is converted to CDP-DG, which is then condensed with
sn-glycerol-3-phosphate to form PG phosphate, which is
rapidly dephosphorylated to PG. Finally, PG condenses with another
CDP-DG molecule to form CL. CL is localized to mitochondria and is
required for the reconstituted activity of a number of key mammalian
mitochondrial enzymes involved in cellular energy metabolism, including
cytochrome c oxidase (8), carnitine palmitoyltransferase
(9), creatine phosphokinase (10), pyruvate translocator (11),
tricarboxylate carrier (12), mitochondrial glycerol-3-phosphate
dehydrogenase (13), phosphate transporter (14), ADP/ATP carrier (15),
and the ATP synthase (16). In addition, several recent studies have
implicated CL loss in the regulation of mitochondrial-mediated
apoptosis (reviewed in Ref. 17). Thus, the appropriate content of CL is
an important requirement for activation of enzymes involved in
mitochondrial respiration and in the control of programmed cell death.
CL is unique among glycerophospholipids in that it contains four fatty
acyl side chains. The proportion of CL symmetrical molecular species is
50-65%, and the four acyl positions are occupied by monounsaturated
and diunsaturated chains of 16-18 carbons in length (18). The
hydrophobic double unsaturated linoleic diacylglycerol species appears
to be the important structural requirement for the high protein binding
affinity of CL (19). Alteration in molecular species composition of CL
may alter activities of the electron transport chain enzymes. For
example, the activity of delipidated rat liver cytochrome c
oxidase was reconstituted by the addition of CL (20). The specific
activity of the reconstituted cytochrome c oxidase varied
markedly and significantly with different molecular species of CL. In
addition, peroxidation of CL in rat basophile leukemia cells lead to an
increase in cytochrome c release, a primary event in
mitochondrial-mediated apoptosis (21). Thus, in addition to CL content,
the CL molecular species composition may regulate mitochondrial
respiratory performance and programmed cell death. The CL de
novo biosynthetic enzymes show little acyl species selectivity
(22, 23). Hence, CL must be rapidly remodeled to achieve the molecular
composition of CL observed in the mitochondrial membrane. The
deacylation-reacylation cycle was first described by William Lands
several decades ago (24). However, it was not until 1990 that a
deacylation-reacylation cycle for the molecular remodeling of
endogenous CL in rat liver mitochondria was proposed (25). Recently, we
characterized the MLCL AT activity responsible for the acylation of
MLCL to CL in the rat heart and mammalian tissues and proposed a model
of cardiolipin molecular remodeling (26). In the current study we
describe a procedure for the purification of MLCL AT activity from pig
liver mitochondria. In addition, we have characterized the purified
enzyme activity, raised a polyclonal antibody to the protein, and shown
that the protein is expressed in response to thyroid hormone. This
study represents the first purification and characterization of a MLCL
AT from any organism.
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EXPERIMENTAL PROCEDURES |
Materials--
Pig livers were obtained from freshly slaughtered
pigs from the local abattoir. Sprague-Dawley rats (125 g) were obtained from Central Animal Care Services, University of Manitoba (Winnipeg, Manitoba, Canada). Treatment of rats conformed to the guidelines of the
Canadian Council on Animal Care. [1-14C]Linoleoyl-CoA and
[1-14C]palmitoyl-CoA were obtained from American
Radiolabeled Chemicals Inc. (St. Louis, MO).
[1-14C]Oleoyl-CoA and [Na125I] were
obtained from Dupont (Mississauga, Ontario, Canada). Acyl-CoAs were
obtained from Serdary Research Laboratories (Englewood Cliffs, NJ).
MLCL (a mixture of
1'(1-acyl-sn-glycerol-3-phosphoryl)-3'-(1",2"-diacyl-sn-glycerol-3-phosphoryl)glycerol and
1'-(1,2-diacyl-sn-glycerol-3-phosphoryl)-3'-(1"-acyl-sn-glycerol-3-phosphoryl)glycerol), produced by phospholipase A2 hydrolysis of bovine heart CL,
was obtained from Avanti Polar Lipids (Alabaster, AL). The purity of
the MLCL substrate was checked by two-dimensional thin layer chromatography as described previously (27). The fatty acyl molecular
species composition of the MLCL substrate was examined as described
previously (28) and was comprised mainly of linoleic (90.3%) and oleic
(8.6%) acids. Ecolite scintillation mixture was obtained from ICN
Biochemicals (Costa Mesa, CA), and thin layer plates (Silica Gel 60, 0.25-mm thickness) were obtained from Fisher Scientific (Winnipeg,
Manitoba, Canada). Kodak X-OMATTM SA film was used for
autoradiography and Western blot analysis. Precision Plus Protein
Standards were obtained from Bio-Rad. All other biochemicals were of
analytical grade and obtained from either Fisher Scientific (Edmonton,
Alberta, Canada) or Sigma Chemical Co. (St. Louis, MO) or the source
indicated in the purification protocol.
Assay of MLCL AT Activity--
For assay of MLCL AT activities,
fractions (1-50 µg of protein) were incubated for 30 min at 25 °C
in 50 mM Tris-HCl, pH 7.0, 30-170 µM
[1-14C]linoleoyl-CoA (specific radioactivity, 14,200 dpm/nmol) at pH 7.0, and 30-480 µM MLCL in a final
volume of 0.35 ml. The MLCL substrate in chloroform was dried under
nitrogen and resuspended in double-distilled water via sonication in a
bath sonicator for 45 min prior to addition to the assay mixture. The
temperature of the bath sonicator was maintained at 4 °C by ice. The
reaction was initiated by the addition of the radioactive acyl-CoA
substrate and terminated by the addition of 3 ml of chloroform:methanol (2:1, v/v). 0.8 ml of 0.9% KCl was added to facilitate phase
separation. The aqueous phase was removed, and the organic phase was
washed with 2 ml of chloroform:methanol:0.9% NaCl (3:48:47, v/v). The resulting organic fraction was dried under nitrogen and resuspended in
25 µl of chloroform:methanol (2:1, v/v). A 20-µl aliquot of the
resuspended organic phase was placed on a thin layer plate, and CL was
separated from other phospholipids using a two-dimensional separation
described previously (27). The silica gel corresponding to CL was
removed and placed in a plastic scintillation vial, and 5 ml of
scintillant was added. Radioactivity incorporated into CL was examined
~24 h later using a liquid scintillation counter. MLCL AT activity
was taken as radioactivity incorporated into CL in the presence of the
MLCL substrate minus radioactivity incorporated into CL in the absence
of the MLCL substrate. In some experiments, 0.3 mM
lysophospholipid (lysophosphatidylglycerol, lysophosphatidylcholine,
lysophosphatidylinositol, lysophosphatidylserine, lysophosphatidic
acid, or lysophosphatidy-lethanolamine) was included in the
assay mixture in place of MLCL. In other experiments,
[1-14C]oleoyl-CoA or [1-14C]palmitoyl-CoA
(similar specific radioactivity as [1-14C]linoleoyl-CoA)
was substituted for [1-14C]linoleoyl-CoA. In other
experiments, MLCL AT activity was determined in the presence of a fixed
concentration of MLCL and variable concentrations of linoleoyl-CoA or
in the presence of a fixed concentration of linoleoyl-CoA and variable
concentrations of MLCL. In other experiments, the purified enzyme was
incubated for up to 1 h with 40 µM linoleoyl-CoA and
0.13 mM 5,5'-dithiobis(2-nitrobenzoic acid) in the absence
of MLCL in the standard assay, and the absorbance at 412 nM
was determined. The formation of CoA was calculated from a standard
curve (29). In other experiments, MLCL AT was preincubated at 50 °C
for up to 10 min, and enzyme activity was assayed as described above.
For the pH titration curve, MLCL AT was assayed at pH range between 5.0 and 9.0. Protein was determined as described previously (30).
Purification of MLCL AT--
Pig liver (3.2 kg) was ground in a
Hobart meat grinder. 3.8 liters of buffer A (10 mM
Tris-HCL, pH 7.4, 0.25 M sucrose, 10 mM
2-mercaptoethanol, 2 mM EDTA) was added, and the mixture
was homogenized in a Polytron® (Kinematica, Switzerland) for 3 min (medium speed). The crude homogenate was centrifuged at 700 × g for 50 min. The pellet was discarded, and the supernatant
was filtered through glass wool. The supernatant was then centrifuged at 16,000 × g for 30 min. The resulting crude
mitochondrial pellet was washed with 1.5 liters of buffer A and
recentrifuged. The washed pellet was resuspended in 400 ml of buffer A
and allowed to sit overnight at 4 °C. The crude mitochondrial
fraction was then homogenized in a Polytron for 3 min (medium speed)
and then freeze-dried.
Butanol Extraction--
Butanol (250 ml) was added to 35 g
of freeze-dried crude mitochondria in a beaker and stirred for 25 min
using a magnetic stirrer. The mixture was centrifuged at full speed for
10 min in a tabletop centrifuge. The supernatant was removed, and the pellet was re-extracted with 200 ml of butanol. The pellet was washed twice with 150 ml of acetone and dried with a stream of nitrogen. Double-distilled water (200 ml) and 100 ml of homogenizing buffer were added to the butanol-extracted pellet and mixed for 3 min
using a Polytron at low speed. The mixture was centrifuged at
40,000 × g for 40 min, and the resulting supernatant
(250 ml) used for hydroxyapatite chromatography.
Hydroxyapatite Chromatography--
The supernatant above was
loaded onto a column containing 80 ml of hydroxyapatite gel (Bio-Gel,
HT Gel from Bio-Rad) that had been pre-equilibrated with buffer A. The
gel was washed stepwise with 1.5 liter of buffer A containing 0.2 M sodium phosphate (monobasic, hydrated). The enzyme was
eluted with buffer A containing 0.42 M sodium phosphate.
The eluted enzyme (250 ml) was dialyzed against 4 liters of
double-distilled water overnight. 450 ml of the dialyzed protein was
freeze-dried to a final weight of 4.5 g.
Preparative SDS-PAGE--
The freeze-dried protein from above (1 g) was dissolved in 1.5 ml of double-distilled water and 1 ml of
Laemmli sample buffer (Sigma, 2× concentrate) and centrifuged at
maximum speed in a tabletop centrifuge through a 0.22 µM
Centricon filter. The filtered protein was subjected to
Preparatory Gel Electrophoresis (Model 491 Prep Cell) using 40 ml of
7.5% acrylamide (separating gel) and 10 ml of 4% acrylamide (stacking
gel). The gel was run at a constant current of 20 mA overnight. The
elution buffer contained 25 mM Tris-HCl, pH 7.4, and 0.19 M glycine. Fractions (6 ml) were collected at a rate of 20 min per fraction. The eluted fractions with the highest MLCL AT
activity were pooled and freeze-dried. An aliquot of the freeze-dried
eluate from the hydroxyapatite purification step (55 µg) and aliquots
of the eluted protein fraction 30, pooled fractions 32-33, and pooled
fractions 34-36 from the preparative SDS-PAGE step (0.2-1.0 µg)
along with protein standards (6.25 µg) were subjected to SDS-PAGE on
mini-gels (Bio-Rad) using 7.5% acrylamide (separating gel) and 4%
acrylamide (stacking gel). Gels were stained with Coomassie Blue. In
some experiments, the pooled fractions 34-36 from the preparative
SDS-PAGE step were subjected to non-denaturing-PAGE using 7.5%
acrylamide (separating gel) and 4% acrylamide (stacking gel) to remove
SDS and MLCL AT activity determined in the gel slices.
Two-dimensional Electrophoresis--
Purified MLCL AT (pooled
fractions 34-36 from the preparative SDS-PAGE step) (0.2 µg) was
subjected to two-dimensional electrophoresis. The first dimension
isoelectric focusing step was performed using ready-made IPG 13 Cm
strips (Amersham Biosciences) with a pH 3-10 gradient. The protein was
focused overnight on an IPGhor (Amersham Biosciences) isoelectric
focusing unit. The protein was then separated in the second dimension
by SDS-PAGE on a 15- × 15-cm gel with 12% acrylamide using a standard
electrophoresis unit (Bio-Rad) with 6.25 µg of molecular mass
standards. The gel was silver-stained, and the molecular mass and
isoelectric point of the purified MLCL AT were calculated.
Polyclonal Antibody Preparation--
A polyclonal antibody to
the purified 74-kDa protein was raised in New Zealand White rabbits at
National Biologicals Inc., Winnipeg, Canada. Excised gel slices from
the preparative SDS-PAGE step (pooled fractions 34-36) containing the
purified acyltransferase (~15 µg each) were utilized for biweekly
intramuscular injections. Antiserum was collected biweekly. The
antibody was purified on a protein A-Sepharose affinity column
(Amersham Biosciences) and used for the immunological studies.
Western Blot Analysis--
The crude mitochondrial fraction (56 µg) and the purified MLCL AT fraction obtained from the preparative
SDS-PAGE step (10 µg) were subjected to SDS-PAGE according to the
Laemmli procedure using 7.5% separating gels (31). Protein from the
separating gel was blotted onto polyvinylidene difluoride membranes and
incubated with either preimmune serum or with the anti-MLCL AT
polyclonal antibody (dilution, 1:500 dilution). Identification was
according to the ECL Western blotting Analysis System (Amersham
Biosciences) using goat anti-rabbit IgG labeled with horseradish
peroxidase as secondary antibody (dilution, 1:1000). In other
experiments, crude liver mitochondrial fractions were prepared from
euthyroid and thyroxine-treated rats as previously described (32), and 100 µg of the fractions were subjected to SDS-PAGE according to the
Laemmli procedure using 10% separating gels (31), and Western blot
analysis using the polyclonal antibody was performed as described above.
12-[(4-Azidosalicyl)amino]dodecanoyl-CoA Photoaffinity Labeling
of MLCL AT--
12-[(4-Azidosalicyl)amino]dodecanoic acid (ASD) and
12-[(4-azidosalicyl)amino]dodecanoyl-CoA (ASD-CoA) were synthesized
as described (33). ASD-CoA was iodinated with Na125I as
described (34). Photoaffinity labeling was performed by incubation of
the purified 74-kDa protein with [125I]ASD-CoA at room
temperature in a dark room. The reaction mixture contained 1.9 µCi of
[125I]ASD-CoA (9.15 × 106 cpm/µmol)
in 20 mM Tris-succinate (pH 6.0), 40 µM EDTA,
and 4.2% glycerol (v/v) and 0.2 µg of purified MLCL AT in a final
volume of 215 µl. Cross-linkage of the photoaffinity probe to the
enzyme was induced by exposing the mixture to ultraviolet light. A
hand-held ultraviolet lamp (Model UVS-54, Ultraviolet Productions Inc., San Gabriel, CA) was held a distance of 5 cm from the sample for 15 min. 10% Trichloroacetic acid was added to stop the reaction, and the mixture was incubated at
20 °C for 15 min. The
precipitated protein was sedimented at 10,000 × g for
5 min, and the pellet was resuspended in 15 µl of 0.1 M
NaOH. SDS-PAGE sample buffer (15 µl) was added to the sample.
The sample was subjected to SDS-PAGE using 10% acrylamide as described
(35). The gel was dried, and the labeled protein band identified by
autoradiography. In some experiments, the purified protein was
photoaffinity-labeled as described above except
[125I]ASD-CoA was replaced with various concentrations of
ASD-CoA and MLCL AT activity determined in this fraction.
 |
RESULTS |
Purification of Pig Liver MLCL AT--
The purification protocol
for MLCL AT activity is outlined in Table
I. Pig liver was homogenized, and the
mitochondrial fraction was prepared and subjected to butanol
extraction. The freeze-dried butanol extract was subjected to
hydroxyapatite chromatography followed by preparative SDS-PAGE. The
elution profiles of MLCL AT activity from hydroxyapatite chromatography
and preparative SDS-PAGE steps are shown in Fig.
1. The result was the isolation of a
single protein band of 74-kDa molecular mass (Fig.
2).
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Table I
Purification of monolysocardiolipin acyltransferase
MLCL AT was purified as described under "Materials and Methods."
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Fig. 1.
Elution profiles of MLCL AT activity from
hydroxyapatite chromatography and preparative SDS-PAGE.
A, crude mitochondria prepared from pig liver was butanol
extracted and subjected to hydroxyapatite chromatography and protein
and MLCL AT activity determined in the eluted fractions.
Arrow indicates 0.42 M sodium phosphate elution.
B, fractions containing the highest MLCL AT activity were
pooled and subjected to preparative SDS-PAGE and MLCL AT activity
determined in the eluted fractions. One unit = 1 pmol of CL formed
per minute.
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Fig. 2.
Purification of MLCL AT. Crude
mitochondria prepared from pig liver was butanol-extracted, subjected
to hydroxyapatite chromatography followed by preparative SDS- PAGE.
Fractions were analyzed by SDS-PAGE and stained with Coomassie Blue as
described under "Materials and Methods." Lane 1,
molecular mass markers (myosin, 198 kDa; -galactosidase, 115 kDa;
bovine serum albumin, 93 kDa; ovalbumin, 49.8 kDa) indicated on the
left; lane 2, hydroxyapatite chromatography;
lane 3, fraction 30 from preparative SDS-PAGE; lane
4, pooled fractions 32 and 33 from preparative SDS- PAGE;
lane 5, pooled fractions 34-36 from preparative SDS-PAGE.
Molecular mass of MLCL AT is indicated on the right. A
representative gel is depicted.
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Properties of MLCL AT--
The purified protein was subjected to
two-dimensional isoelectric focusing followed by SDS-PAGE. The
isoelectric point was determined to be 5.4. We investigated whether the
purified enzyme bound acyl-CoA. The purified enzyme was incubated with
[125I]ASD-CoA and photoaffinity cross-linked to the
enzyme. As seen in Fig. 3
(inset), [125I]ASD-CoA was bound to the 74-kDa
purified protein. The purified enzyme was then incubated with ASD-CoA
and exposed to ultraviolet light, and then the MLCL AT activity was
determined. MLCL AT activity of the purified protein was inhibited in a
concentration-dependent manner by ASD-CoA (Fig. 3). Thus,
the purified protein bound acyl-CoA and ASD-CoA inhibited enzyme
activity.

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Fig. 3.
Photoaffinity labeling of MLCL AT with
[125I]ASD-CoA and inhibition of MLCL AT activity by
ASD-CoA. Purified MLCL AT was subjected to SDS-PAGE and
photoaffinity-labeled with [125I]ASD-CoA as described
under "Materials and Methods." Cross-linkage of the photoaffinity
probe to the enzyme was induced by exposing the mixture to ultraviolet
light, and the labeled protein band at 74 kDa was identified by
autoradiography (arrow). A representative gel is depicted in
the inset. Purified MLCL AT was incubated with various
concentrations of ASD-CoA as described under "Materials and
Methods." Cross-linkage of the photoaffinity probe to the enzyme was
induced by exposing the mixture to ultraviolet light, and MLCL AT
activity was subsequently determined. Results represent the mean of two
determinations. MLCL AT activity was 9987 pmol/min/mg of protein and
taken as 100%. The results between samples differed by less than
15%.
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We examined the pH profile, substrate specificity, and heat
inactivation profile of purified MLCL AT. The purified protein exhibited a pH optimum at 7.0 (Fig. 4).
The purified protein utilized [1-14C]linoleoyl-CoA,
[1-14C]oleoyl-CoA, and [1-14C]palmitoyl-CoA
as substrates, and enzyme activity was 10-fold greater with the
unsaturated fatty-acyl CoA substrates compared with palmitoyl-CoA (data
not shown). The purified protein did not utilize other
lysophospholipids (lysophosphatidylglycerol, lysophosphatidylcholine,
lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, or lysophosphatidic acid) as substrate and
was heat-labile, because preincubation at 50 °C for up to 10 min
reduced enzyme activity by 70% (data not shown).

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Fig. 4.
pH Profile of MLCL AT. MLCL AT was
purified and enzyme activity determined at pH 5.0-9.0. Results
represent the mean of two determinations. The results between samples
differed by less than 15%.
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MLCL AT catalyzes a Bi Bi reaction. To determine the kinetic
mechanism of the enzyme, the effects of varying concentrations of
linoleoyl-CoA and monolysocardiolipin on enzyme velocity were determined. The results were depicted in a double-reciprocal plot (Fig.
5). The parallel lines of the
initial-rate kinetic behavior of MLCL AT was indicative of a ping pong
reaction mechanism (Fig. 5). The calculated Km for
linoleoyl-CoA from the inset of Fig. 5A was 100 µM, and the calculated Km of MLCL from
the inset of Fig. 5B was 44 µM. The
Vmax calculated from the inset of
Fig. 5B was 6802 pmol/min·mg of protein. The formation of
CoA from linoleoyl-CoA was determined in the absence of MLCL. In the
absence of MLCL, CoA was formed from linoleoyl-CoA at a rate consistent
with the formation of an enzyme-linoleate intermediate (Table
II). These results provide further
support for a ping pong reaction mechanism.

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Fig. 5.
The effect of varying concentrations of
linoleoyl-CoA and monolysocardiolipin on initial velocity. Initial
velocity of MLCL AT activity was determined using varying
concentrations of linoleoyl-CoA and monolysocardiolipin. A,
reciprocal velocity of MLCL AT verses reciprocal concentration of MLCL
at varying concentrations of linoleoyl-CoA. Circles, 50 µM; squares, 80 µM;
triangles, 110 µM; inverted
triangles, 170 µM. Inset, 1/initial
velocity at the point where the lines intersect the y-axis
was plotted against 1/linoleoyl-CoA concentration. B,
reciprocal velocity of MLCL AT verses reciprocal concentration of
linoleoyl-CoA at varying concentrations of MLCL. Circles, 30 µM; squares, 60 µM;
triangles, 120 µM; diamonds, 480 µM. Inset, 1/initial velocity at the point
where the lines intersect the y-axis was plotted
against 1/MLCL concentration. Results represent the mean of two
determinations. The results between samples differed by less than
15%.
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Table II
Formation of CoA from linoleoyl-CoA in the absence of MLCL
Purified MLCL AT was incubated in the presence of linoleoyl-CoA in the
absence of MLCL, and the formation of CoA was determined. Results
represent the mean of two determinations performed in duplicate. The
results between samples differed by less than 15%.
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A polyclonal antibody was raised in New Zealand White rabbits from the
purified protein. The polyclonal antibody reacted with the purified
74-kDa protein obtained from the preparative SDS-PAGE step (pooled
fractions 34-36) (lane 3) and from crude pig liver mitochondrial fraction (lane 2) (Fig.
6A). Pre-immune serum did not
react with the 74-kDa protein (data not shown). The polyclonal antibody
was not immunoprecipitating, because incubation of crude mitochondrial
fractions with antibody did not reduce enzyme activity.

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Fig. 6.
Western blot analysis of MLCL AT.
A, crude pig liver mitochondrial fraction and purified MLCL
AT were separated by SDS-PAGE using 7.5% separating gels then probed
for antibody binding with polyclonal antibody to MLCL AT as described
under "Materials and Methods." Lanes: blank lane
1, no protein; lane 2, crude mitochondrial fraction;
lane 3, purified MLCL AT from preparative SDS-PAGE.
B, crude rat liver mitochondrial fractions were prepared
from euthyroid and hyperthyroid rats then separated by SDS-PAGE using
10% separating gels then probed for antibody binding with polyclonal
antibody to MLCL AT. Lane 1, euthyroid; lane 2,
hyperthyroid. Molecular mass is indicated on the right.
Representative gels are depicted.
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Previously we demonstrated that cardiac MLCL AT activity was elevated
in crude mitochondrial fractions prepared from thyroxine-treated rats.
Rats were injected with thyroxine (250 µg/kg/day) or saline (euthyroid control) for 5 consecutive days. Crude rat liver
mitochondrial fractions were prepared, and then equal amounts of
mitochondrial protein from thyroxine-treated or euthyroid rats were
subjected to SDS-PAGE. Western blot analysis was then performed using
the polyclonal antibody. As seen in Fig. 6B, expression of
MLCL AT protein was elevated in thyroxine-treated rats compared with
euthyroid control. The bands were analyzed by scanning the film with a
densitometer, and relative intensities of the bands were determined by
Scion Image software. The relative intensity of the band in lane
2 of Fig. 6B was 1.8-fold higher than lane
1. Thus, MLCL AT protein expression is regulated by thyroid hormone.
 |
DISCUSSION |
In this study, we have purified a unique and previously
unidentified MLCL AT activity from pig liver mitochondria to apparent homogeneity. The protein exhibits a molecular mass of 74 kDa and acylates MLCL to CL with linoleoyl-CoA. Purified MLCL AT is
heat-labile, has Km values of 44 µM
for MLCL and 100 µM for linoleoyl-CoA, and a
Vmax of 6802 pmol/min·mg. Parallel lines
obtained from primary plots indicate a ping pong reaction mechanism. In
support of this, CoA is formed from linoleoyl-CoA at a rate consistent
with the formation of an enzyme-linoleate intermediate. The initial
purification step utilized butanol extraction to remove lipids from the
protein. Although no degree of purification was obtained, this step
allowed reconstitution of enzymatic activity subsequent to
freeze-drying. Previously, we demonstrated that different detergents
inhibited in vitro MLCL AT activity to varying degrees in
crude rat heart mitochondrial fractions (26). Because SDS was required
for purification of MLCL AT in the terminal preparative SDS-PAGE step,
it is possible that the observed degree of purification is an
underestimate of the actual value.
The pH optimum of purified MLCL AT was pH 7.0. This value falls within
the range of the normal physiological pH observed within respiring
mitochondria (36). In addition, two-dimensional isoelectric focusing/SDS-PAGE indicated an isoelectric point of pH 5.4. We had
previously demonstrated that various acyl-CoAs could compete with
[14C]CL formation from [14C]oleoyl-CoA and
[14C]linoleoyl-CoA in crude rat heart mitochondria (26).
This was confirmed in the ASD-CoA experiments. The enzyme-bound
acyl-CoA as indicated in photoaffinity cross-linking studies with
[125I]ASD-CoA. In addition, cross-linking of the enzyme
with ASD-CoA inhibited in vitro enzyme activity in a
concentration-dependent manner. Thus, purified MLCL AT
binds acyl-CoA substrates. Substrate specificity studies of the
purified enzyme indicated that purified MLCL AT utilized exclusively
MLCL and not other lysophospholipids as substrate. In addition,
purified MLCL AT utilized unsaturated acyl-CoAs (oleoyl-CoA,
linoleoyl-CoA) to a much greater degree (10-fold preference) than a
saturated acyl-CoA (palmitoyl-CoA). This may explain the enrichment of
the unsaturated fatty acyl molecular composition of CL observed
in vivo (18). Finally, a polyclonal antibody raised in
rabbits to MLCL AT reacted with the protein in crude mitochondrial
fractions prepared from pig liver.
Subsequent to de novo biosynthesis, CL is rapidly remodeled
by deacylation followed by reacylation (25, 26). The molecular species
composition of CL is responsive to changes in diet (37). In addition,
dietary modification of the molecular species composition of CL has
been shown to alter the oxygen consumption in cardiac mitochondria (20,
37). Hence, regulation of the activities of the enzymes involved in CL
remodeling may play a key role in regulation of CL function and
mitochondrial respiration. It is well documented that thyroid hormone
regulates mitochondrial respiration (38). Previously we demonstrated
that treatment of rats with thyroxine for 5 days elevated cardiac CL
levels, CL synthase, and MLCL AT activities (32, 39). Mitochondrial
phospholipase A2 and lysophosphatidylglycerol
acyltransferase activities were unaltered under these conditions (32).
In the current study, the polyclonal antibody prepared against purified
pig liver MLCL AT cross-reacted with the protein in crude rat liver
mitochondrial fractions. In addition, expression of MLCL AT protein was
elevated in rat liver mitochondrial fractions prepared from
thyroxine-treated rats indicating that MLCL AT expression is regulated
by thyroid hormone. Because CL synthase exhibits little fatty acyl
species specificity (23) and thyroid hormone did not affect
mitochondrial phospholipase A2 activity but elevated CL
synthase and MLCL AT activities (32), it is possible that MLCL AT could
serve as a control point for the regulation of the remodeling of newly synthesized CL in mammalian tissues.