From the
We have previously reported that long chain acyl-CoA
thioesterase activity was induced about 10-fold in rat liver
mitochondria, when treating rats with the peroxisome proliferator
di(2-ehtylhexyl)phtalate (Wilcke M., and Alexson S. E. H(1994) Eur.
J. Biochem. 222, 803-811). Here we have characterized two
enzymes which are responsible for the majority of long chain acyl-CoA
thioesterase activity in mitochondria from animals treated with
peroxisome proliferators. A 40-kDa enzyme was purified and
characterized as a very long chain acyl-CoA thioesterase (MTE-I). The
second enzyme was partially purified and characterized as a long chain
acyl-CoA thioesterase (MTE-II). MTE-I was inhibited by
p-chloromercuribenzoic acid, which implicates the importance
of a cysteine residue in, or close, to the active site. Antibodies
against MTE-I demonstrated the presence of immunologically related
acyl-CoA thioesterases in peroxisomes and cytosol. High expression of
MTE-I was found in liver from peroxisome proliferator treated rats and
in heart and brown fat from control and induced rats. Comparison of
physical and catalytical characteristics of the enzymes studied here
and previously purified acyl-CoA thioesterases suggest that MTE-I and
MTE-II are novel enzymes.
Palmitoyl-CoA hydrolases (EC 3.1.2.2) cleave activated fatty
acids (acyl-CoAs) to the corresponding free fatty acid and CoASH. The
enzyme activity is widely distributed among organisms and cell types
and is found in several cellular compartments. Some of these activities
have been characterized
(1, 2, 3, 4) .
The most carefully studied thioesterases are those acting on the
multienzyme complex of fatty acid synthesis (reviewed in Ref. 5).
Although it is tempting to speculate that acyl-CoA thioesterases may be
potential regulators of fatty acid energy metabolism, a thioesterase
capable of hydrolyzing palmitoyl-protein thioesters of H-Ras and
Several reports in the literature have
indicated that rat liver mitochondria contain long chain acyl-CoA
thioesterase activity that is regulated by peroxisome
proliferators
(8, 9, 10, 11) . One rat
liver mitochondrial palmitoyl-CoA thioesterase has been purified from
untreated rats
(12) . Peroxisome proliferating drugs have been
shown to induce long chain acyl-CoA thioesterase activity also in rat
liver cytosol
(8, 13, 14) , and to a lesser
extent in peroxisomes
(9) , but not in the endoplasmic
reticulum
(15) . Therefore, in line with our studies on the
structure and function of acyl-CoA
thioesterases
(11, 16, 17, 18, 19) ,
the aim of the present study was to investigate whether the increased
mitochondrial activity emanated from novel enzymes. Our results show
that rat liver mitochondria contain several isoforms of long chain
acyl-CoA thioesterase and in the present report two novel enzymes were
characterized.
Estimation of kinetic parameters
were performed by fitting the experimental data to the Michaelis-Menten
equation:
On-line formulae not verified for accuracy
The unknown parameters were numerically solved by the computer
program Kaleida Graph 3.0 for Apple Macintosh. When high substrate
concentrations inhibited enzyme activity, the corresponding data points
were ommited from the calculations.
The activity in control
mitochondria was increased from 12 to 18 nmol
MTE-I was purified to apparent homogeneity according to the
procedure described under ``Experimental Procedures.'' The
effective purification step was the separation by Mono Q anion-exchange
chromatography, where most of the total protein did not bind to the
column (Fig. 2A). The activity eluted close after the
onset of the salt gradient with only minor impurities present. The
remaining contaminating proteins were removed from the enzyme
preparation by the subsequent size exclusion chromatography
(Fig. 2B). The fractions containing activity could be
stored frozen in small aliquots containing 20% glycerol at -70
°C until used for characterization. SDS-PAGE analysis showed that
the final preparation contained a single band after staining with
Coomassie Brilliant Blue (or silver), corresponding to a molecular mass
of 45 kDa (Fig. 2C). Chromatography of this preparation
on a µRPC C2/C18 PC 3.2/3 reversed phase column (Pharmacia)
revealed only one peak detected at 280 nm (Fig. 2D).
This multistep purification resulted in a 23-fold enrichment over the
mitochondrial matrix fraction, as summarized in . Assuming
that the novel thioesterase does not contribute to more than about
30-40% (as calculated from the integrated area of the
corresponding peak in Fig. 1) of the total mitochondrial matrix
myristoyl-CoA thioesterase activity, the purification of this
particular enzyme was 50-75-fold over the starting matrix
fraction. The estimated size of the purified protein by size exclusion
chromatography corresponded to a molecular mass of 40 kDa,
demonstrating that the enzyme was monomeric.
The fate of fatty acids in cellular metabolism depends on
several physiological or pathological factors. Under normal conditions
fatty acids are mainly
In
this report we show that the increased mitochondrial acyl-CoA
thioesterase activity during treatment of rats with peroxisome
proliferators can mainly be correlated to two isoactivities. As
determined by size exclusion chromatography, these two activities are
due to proteins with molecular masses of about 40 and 110 kDa,
respectively. The acyl-CoA chain length specificity of the purified
40-kDa enzyme showed that it represents a novel very long chain
acyl-CoA thioesterase. A similar acyl-CoA thioesterase (ACH2) was
recently purified from the liver cytosol of DEHP-treated rats. We
purified this enzyme and compared its properties to MTE-I. Based on the
molecular mass difference of 2 kDa, different substrate specificities,
and organ distributions, we conclude that they are different enzymes.
Our results, and the results of Yamada and co-workers
(14) ,
indicate that rat liver contains two structurally related acyl-CoA
thioesterase protein families of 40- and 110-kDa enzymes, which are
induced by peroxisome proliferators in cytosol and mitochondria. In
peroxisomes though, we can detect an induced 40-kDa, but not a 110-kDa,
acyl-CoA thioesterse. Cloning of the respective genes will provide
information on structural similarities and mechanisms for specific
targeting for their proper localization in the different compartments
of the cell.
The 110-kDa enzyme was partially purified and
characterized as a long chain acyl-CoA thioesterase. Comparison of the
substrate specificities of the long chain and very long chain
thioesterases with the substrate specificities of mitochondrial long
chain and very long chain acyl-CoA
dehydrogenase
(30, 31) , showed striking similarities
(Fig. 6). In addtion, the calculated K
It is now well
established that there are several, also extraperoxisomal, enzymes
involved in metabolism of fatty acids that are strongly induced by
peroxisome proliferators. Apparently, a member of the steroid receptor
superfamily which is activated by peroxisome proliferators (peroxisome
proliferator-activated receptor) (42) mediates the induction of several
lipid metabolizing enzymes and is implicated to play an important role
in the regulation of lipid metabolism, as recently reviewed by Keller
et al.(43) . It is likely that a relatively large
number of lipid-metabolizing enzymes will be found to be regulated by
peroxisome proliferators or carboxylic acids in a similar manner.
Taken together, the results presented here makes the very long and
long chain acyl-CoA thioesterases of interest for future studies on
regulation of mitochondrial fatty acid metabolism and functional
aspects of induction by peroxisome proliferators. Thus, molecular
cloning of the corresponding cDNAs and genes will provide useful tools
to elucidate the roles of different acyl-CoA thioesterases in cellular
metabolism and regulation of their gene expression.
A matrix fraction of rat liver
mitochondria was used as starting material for the purification. Long
chain acyl-CoA thioesterase activity was measured as described under
``Experimental Procedures'' with 25 µM myristoyl-CoA as substrate.
Thioesterase activity with acyl-CoAs of different chain lengths were
measured at 10 different concentrations between 1 and 100
µM. The activity was measured as described under
``Experimental Procedures'' using approximately 0.6 µg of
purified MTE-I and partially purified MTE-II from the phenyl-Sepharose
pool, corresponding to an amount that hydrolyzed 50 µM myristoyl-CoA at a rate of 3 nmol
We thank Susanna Engberg for skillful technical
assistance and Shahparak Zaltash for the characterization of the
peroxisomal isoenzyme.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-subunits of heterotrimeric G proteins as well as acyl-CoA was
recently discovered
(6) . However, the physiological functions of
this enzyme are still uncertain since molecular cloning of the cDNA
revealed that the enzyme was destined for the secretory
pathway
(7) , but the results imply that the physiological
function of apparent acyl-CoA thioesterases can be diverse. Generally,
the physiological function of other studied acyl-CoA thioesterases have
not been well established.
Animals
Male Sprague-Dawley rats (Eklunds, Stockholm, or ALAB,
Sollentuna, Sweden) were used. The rats were fed a standard pellet diet
(R3, Ewos, Södertälje, Sweden) with or without
di(2-ethylhexyl)phthatlate (DEHP)(
)
(2% w/w) or
clofibrate (0.5% w/w) for 10-14 days. Food and tap water were
supplied ad libitum. Animals were fasted overnight and killed
by CO
-anesthesia followed by decapitation.
Materials
SDS and 30% acrylamide/bis solution (37.5:1) were obtained
from Bio-Rad. Clofibrate (Klofiran) was purchased from Orion (Espoo,
Finland). DEHP was obtained from Fluka (Buchs, Switzerland).
CM-Sepharose Fast Flow, DEAE-Sepharose Fast Flow, phenyl-Sepharose Fast
Flow, and Percoll were purchased from Pharmacia (Uppsala, Sweden).
Econo-Pac columns were from Bio-Rad. Matrex gel red A was obtained from
Amicon (Lexington, MA). All acyl-CoA esters, phenylmethylsulfonyl
fluoride, p-chloromercuribenzoic acid (pCMB), and diisopropyl
fluorophosphate (DFP) were purchased from Sigma. AlumImject was from
Pierce. All other reagents were of analytical grade and purchased from
Merck (Darmstadt, Germany) or Sigma.
Methods
Preparation of Mitochondria
Livers from four rats (about 60 g) were cut into pieces and
homogenized in a Potter-Elvehjem glass Teflon homogenizer by 10 strokes
with a loose-fitting pistil in a standard homogenization buffer,
consisting of ice-cold 0.25 M sucrose, 10 mM
Tris-HCl, pH 7.4, 0.1% ethanol, and 1 mM EDTA. Homogenates
(20% w/v) were prepared and centrifuged for 10 min at 600
g. The pellets were resuspended in homogenization buffer and
recentrifuged once. The combined supernatants were centrifuged for 10
min at 3,600
g, and the resulting pellet was washed
once (10 min at 3,600
g). The mitochondrial pellet was
suspended in 20 mM potassium phosphate, pH 7.0, containing 0.1
mM dithiothreitol, 0.1 mM benzamidine hydrochloride,
30 mM NaCl and 10% glycerol (v/v) (buffer A), and kept frozen
at -70 °C until use.
Preparation of Mitochondria by Percoll Gradient
Centrifugation
Mitochondria from control and DEHP-treated rats were further
purified on Percoll gradients in principle as described
previously
(20, 21) , except that the mitochondrial
fractions were prepared as described above. A gradient was developed
during centrifugation at 63,000 g for 30 min in a
Sorvall TV-850 rotor. Fractions (2 ml) were collected from the bottom
of the centrifuge tube using a gradient unloader (Nycomed, Oslo,
Norway) connected to a peristaltic pump. These mitochondria were used
in Western blot experiments.
Purification of Mitochondrial Long Chain Acyl-CoA
Thioesterases
Frozen mitochondria (kept in buffer A) were thawed and
sonicated (Heat Systems Sonicator, model XL2020) on ice 6 times for 10
s with 20 s intervals. The sonicated mitochondria were centrifuged for
1 h at 100,000 g, and the resulting supernatant was
used as starting material for chromatography.
Purification of Mitochondrial Very Long Chain Acyl-CoA
Thioesterase (MTE-I)
Column Triplet
A CM-Sepharose column (50
26 mm), a DEAE-Sepharose column (100
26 mm), and a Matrex gel
red A column (50
26 mm) were serially connected to an Econo
System (Bio-Rad) operated at +4 °C. The column triplet was
equilibrated with buffer A, and the mitochondrial matrix fraction was
applied at a flow rate of 120 ml/hour. The triplet was washed with 250
ml of buffer A and subsequently disconnected. The Matrex gel red A
column was washed with an additional column volume of buffer A,
followed by elution of bound proteins with 400 ml of a linear gradient
of 0-1.5 M NaCl in buffer A. Fractions were collected
and assayed for thioesterase activity.
Hydrophobic Interaction Chromatography
A
phenyl-Sepharose column (70 16 mm) was equilibrated with buffer
A containing 0.7 M (NH
)
SO
.
The pooled activity from the Matrex gel red A column was applied to the
column, after addition of solid (NH
)
SO
to a concentration of 0.7 M. The column was washed with
buffer A containing 0.7 M (NH
)
SO
until A
returned to base line. Bound
protein, not containing thioesterase activity, was eluted with buffer
A, and the acyl-CoA thioesterase activity was subsequently eluted by
isocratic elution with 60% ethylene glycol in buffer A, at a flow rate
of 60 ml/h. The fractions containing activity were collected and
pooled. The ethylene glycol fraction was used as material for the
subsequent purification steps which were performed on a SMART
micropurification system (Pharmacia).
Mono Q Chromatography
Buffer exchange of the
sample was performed by applying aliquots of 0.5 ml of the ethylene
glycol fraction to an Econo-Pac P6 cartridge (Bio-Rad) equilibrated in
20 mM Tris-HCl, pH 8.2, containing 10% glycerol (v/v) (buffer
B). A sample corresponding to 250 µl of the phenyl-Sepharose pool
was applied to a Mono Q PC 1.6/5 column (Pharmacia), equilibrated with
buffer B. After washing, bound protein was eluted with a 2-ml linear
gradient of 0-0.3 M NaCl in buffer B. The peak
fractionation mode was used, and the maximal peak fraction volume was
set to 100 µl. Most of the activity eluted in one fraction, which
was subjected to size exclusion chromatography.
Size Exclusion Chromatography
A Superdex 200 HR
10/30 column was equilibrated with 4.3 mM sodium phosphate,
1.4 mM potassium phosphate containing 2.7 mM KCl, and
0.137 M NaCl, pH 7.4 (phosphate-buffered saline), at a flow
rate of 30 ml/h. The fraction from the Mono Q step was loaded onto the
column, and 250-µl fractions were collected and assayed for
thioesterase activity.
Partial Purification of Mitochondrial Long Chain
Acyl-CoA Thioesterase (MTE-II)
Anion-exchange Chromatography
The DEAE column
was disconnected from the column triplet described above and washed
with one column volume of buffer A. The bound protein was eluted with a
400 ml gradient of 0-1.5 M NaCl in buffer A. The pooled
fractions containing acyl-CoA thioesterase activity were subjected to
hydrophobic interaction chromatography.
Hydrophobic Interaction Chromatography
The pooled
activity from the DEAE chromatography was adjusted to 0.7 M
(NH)
SO
and applied to a
phenyl-Sepharose column (70
16 mm), equilibrated with 0.7
M (NH
)
SO
in buffer A, at a
flow rate of 60 ml/h. The column was washed with buffer A containing
0.7 M (NH
)
SO
until
A
returned to base line. After washing with two
column volumes of 0.3 M (NH
)
SO
in buffer A, the thioesterase activity was isocratically eluted
with buffer A containing 40% ethylene glycol. This fraction was used
for characterization of MTE-II.
Purification of Acyl-CoA Thioesterases from Cytosol
and Peroxisomes That Cross-reacted with anti-MTE-I Antibodies
We have partially purified a peroxisomal acyl-CoA
thioesterase activity(
)
which cross-react with
anti-MTE-I. Briefly, the acyl-CoA thioesterase activity from
peroxisomal matrix fraction was unretarded on a column triplet (Econo
Pac, Bio-Rad) consisting of S-Sepharose, Q-Sepharose, and
Blue-Sepharose, equilibrated with 10 mM potassium phosphate,
pH 7.4. The unbound fraction was subjected to adsorbtion chromatography
on hydroxylapatite (Econo Pac HTP), using the same buffer and pH. The
cross-reactive activity was not bound. The buffer in the unbound
fraction was exchanged to 10 mM Tris, pH 8.8, and loaded onto
a Resource Q column, equilibrated with the same buffer. We analyzed
every purification step on SDS-PAGE and on Western blots probed with
anti-MTE-I. The fraction of highest purity was used for comparison with
purified MTE-I. We also purified a DEHP-inducible acyl-CoA thioesterase
from the cytosolic fraction of rat liver (ACH2) according to Yamada and
co-workers
(14) and compared its properties to MTE-I.
Production of Polyclonal Antibodies against MTE-I
About 100 µg of purified MTE-I was mixed with AlumImject
according to the manufacturer's instructions. The emulsion was
injected into 10 sites at the back of a rabbit (Loop strain). After 4
weeks the procedure was repeated with antigen/AlumImject mixture for
booster injections. Ten days after the booster injections the rabbit
was bled from the ear artery and serum was prepared. Partially purified
IgG fraction was prepared by (NH)
SO
precipitation (33% saturation) as described
previously
(22) .
SDS-PAGE and Western Blot Analysis
SDS-PAGE was performed according to Laemmli
(23) . Low
molecular weight standards from Bio-Rad were used for size
determination. Proteins for Western blot analysis were separated by
SDS-PAGE and electrotransferred onto nitrocellulose (NitroPure, Micron
Separation Inc., Westboro, MA). Blots were probed with anti-MTE-I
antibodies and horseradish peroxidase-conjugated secondary antibodies
and visualized by enhanced chemiluminescence (ECL,
Amersham, Buckinghamshire, United Kingdom).
Other Methods
Acyl-CoA thioesterase activity was routinely followed
spectrophotometrically with 5,5`-dithiobis(2-nitrobenzoic acid), as
earlier described
(16) , but omitting Triton X-100.
Alternatively, the decrease in absorbance by cleveage of the thioester
bond was followed at 232 nm using = 4250
M
cm
. The appropriate
acyl-CoA was preincubated, and the reaction was started by addition of
enzyme. Protein was determined according to Bradford, with bovine serum
albumin as standard
(24) .
Induction of Acyl-CoA Thioesterase Activity in
Percoll-purified Mitochondrial Fractions
Liver mitochondria from
peroxisome proliferator-treated rats contained elevated long chain
acyl-CoA thioesterase activity compared to mitochondria from control
rats
(10, 11) , and as confirmed in this study. We
encountered a paradoxical problem when we tried to quantitate the
degree of induction of acyl-CoA thioesterase activity in mitochondrial
fractions isolated from livers of peroxisome proliferator treated rats.
Liver mitochondria and mitochondrial matrix fractions from control and
DEHP-treated rats were prepared by Percoll centrifugation and
sonication as described under ``Experimental Procedures.''
The activity was measured without and with the addition of 0.025%
Triton X-100 to access latency of the activty in intact mitochondria.
At a concentration of 0.05%, the activity of purified MTE-I and
partially purified MTE-II activities were inhibited by 80%, with both
25 µM myristoyl- and palmitoyl-CoA as substrates. However,
0.025% Triton X-100 inhibited the purified enzymes only about 20%. On
the contrary, a Triton X-100 concentration of 0.005% slightly
stimulated the activity (about 10%).
min
mg
by addition of 0.025% Triton X-100.
In DEHP-induced mitochondria the corresponding values were 60 and 73
nmol
min
mg
,
i.e. a 4-fold induction of the activity in the presence of
Triton X-100. The mitochondrial matrix fraction was prepared by
sonication and ultracentrifugation, and the activity was measured. The
activity in the control matrix fraction was 15 nmol
min
mg
, and there was no
effect of addition of 0.025% Triton X-100. In the mitochondrial matrix
fraction DEHP-induced rats, the activity was 126 nmol
min
mg
, which decreased
to 100 nmol
min
mg
by the addition of Triton X-100. The activity was thus induced
about 8.5-fold by DEHP treatment in the mitochondrial matrix fraction.
The recoveries of activity were 67 and 86% in matrix fractions prepared
from mitochondria isolated from control and DEHP-induced rats,
respectively. We conclude that the induction by peroxisome
proliferators is easily underestimated in crude mitochondrial
preparations, due to sensitivity to solubilizing agents, such as Triton
X-100.
Size Exclusion Chromatography of Mitochondrial Matrix
Fractions from Control and DEHP-treated Rats
In order to
investigate the nature of the induced thioesterase activity by DEHP
treatment, size exclusion chromatography was performed on samples of
mitochondrial matrix fractions. Fractions prepared from control rats
showed that most of the myristoyl-CoA thioesterase activity migrated as
a protein with a molecular mass corresponding to about 50 kDa (peak in
fractions 28-29) (Fig. 1). In addition there was a clear
shoulder of activity in fractions 31-32, corresponding to a
molecular mass <40 kDa. Size determination of the components in
mitochondrial matrix fractions from DEHP-treated rats revealed two well
separated acyl-CoA thioesterase activity peaks, corresponding to
molecular masses of about 40 kDa (peak activity in fraction 30) and 110
kDa (peak activity in fraction 22), respectively (Fig. 1).
Earlier reports have not described thioesterase activities of these
sizes, thus the induced activity appears to be due to novel enzymes.
Similar results were obtained with mitochondrial matrix fractions
prepared from clofibrate-treated rats (data not shown). The enzymes
were purified as described under ``Experimental Procedures''
and characterized with respect to some catalytic and physical
properties.
Figure 1:
Size
exclusion chromatography of liver mitochondrial matrix fractions
prepared from control and DEHP-treated rats. Samples (150 µl
containing 1.5 mg of protein) of mitochondrial matrix fractions,
prepared from control and DEHP-treated animals, were subjected to size
exclusion chromatography on Superdex 200 HR 10/30. Fractions of 250
µl were collected and assayed for myristoyl-CoA thioesterase
activity as described under ``Experimental Procedures.'' The
y axis shows the normalized activity that was calculated by
dividing the activity in the fractions with total integrated absorbance
at 280 nm for each experiment. The integrated absorbance was used to
relate the activity to recovered protein. Indicated by arrows are: 1, void volume determined by elution of blue
dextran, and the positions for elution of: 2, m = 200 kDa (-amylase); 3, m =
150 kDa (alcohol dehydrogenase); 4, m = 66 kDa
(albumin); 5, m = 29 kDa (cabonic anhydrase);
6, m = 12.4 kDa (cytochrome
c).
Purification and Characterization of
MTE-I
Analysis of the distribution of myristoyl-CoA thioesterase
activity after chromatography of the mitochondrial matrix fraction on
the CM-DEAE-Matrex gel red A triplet showed that the activity was
roughly evenly bound to the DEAE and the Matrex gel red A columns. The
activity bound to the Matrex gel red A column was eluted and subjected
to hydrophobic interaction chromatography. The activity was eluted with
60% ethylene glycol, and size exclusion chromatography of this fraction
showed that the thioesterase activity (MTE-I) migrated as a single peak
(in fraction 31) corresponding to a molecular mass of about 40 kDa
(data not shown). After elution of the activity that was bound to the
DEAE column, the fractions containing activity were subjected to size
exclusion chromatography. The activity eluted in one peak (in fraction
22) with an estimated molecular mass of about 110 kDa (designated
MTE-II), demonstrating that the DEAE-pool was devoid of MTE-I (data not
shown).
Figure 2:
Purification of MTE-I by chromatography on
a Pharmacia SMART-system. A, an aliquot of the
phenyl-Sepharose pool was chromatographed on Mono Q PC 1.6/5 as
described under ``Experimental Procedures.'' The
myristoyl-CoA thioesterase activity was eluted by a linear NaCl
gradient at a flow rate of 6 ml/h, and fractions were collected using
the ``peak fractionation'' mode. B, the peak
fraction from the Mono Q step was subjected to size exclusion
chromatography on Superdex 200 HR 10/30 as described under
``Experimental Procedures.'' Fractions of 250 µl were
collected and assayed for activity. C, samples from each step
in the purification summarized in Table I were electrophoresed by
SDS-PAGE and stained with Coomassie Brilliant Blue. Lanes 1 and 7 show molecular mass standards corresponding to
97.4, 66.2, 45, and 31 kDa. Lane 2, mitochondrial matrix;
lane 3, Matrex gel red A pool; lane 4,
phenyl-Sepharose pool; lane 5, Mono Q PC 1.6/5 peak fraction;
and lane 6, Superdex 200 HR 10/30 peak fraction. D, a
sample corresponding to the peak fraction from the Superdex column was
applied to a µRPC C2/C18 PC 3.2/3 reversed phase column. The column
was eluted with a 0-30% acetonitrile gradient. The chromatogram
was constructed by subtracting a blank run from the sample
run.
Catalytic Properties of MTE-I
The kinetic
properties of MTE-I obeyed Michaelis-Menten kinetics with the
substrates tested, except for myristoyl-CoA and stearoyl-CoA, as the
enzyme showed substrate inhibition with myristoyl-CoA at concentrations
higher than 25 µM and with stearoyl-CoA at concentrations
higher than 10 µM and weak inhibition at a palmitoyl-CoA
concentration of 100 µM. V and
K
values were calculated for each
substrate by fitting the experimental data to the Michaelis-Menten
equation to numerically solve the unknown parameters ().
Although the values for myristoyl-CoA and especially stearoyl-CoA are
somewhat uncertain, due to substate inhibition, the data suggest that
MTE-I is most active on substrates with chain lengths ranging from
C
-C
. The K
values for the preferred substrates were in the range of 2.9 to
5.8 µM, and V
for the best
substrate, palmitoyl-CoA, was calculated to 4.5 µmol
min
mg
. We therefore
propose the name ``very long chain acyl-CoA thioesterase''
for the new enzyme. The enzyme had a pH optimum between 8 and 9 when
tested with different buffers giving overlapping pH intervals (data not
shown). MTE-I showed no carboxylester hydrolase activity with
nitrophenyl-acetate or nitrophenyl-decanoat (data not shown).
Effect of Chemical Modifiers
MTE-I was not
inhibited by phenylmethylsulfonyl fluoride or DFP which are known to
act as serine esterase inhibitors
(25) (data not shown). The
effect of the sulfhydryl reactive reagent pCMB on the acyl-CoA
hydrolyzing activty was pronounced. After incubation with 0.4, 4, and
40 µM pCMB, the remaning activity was 81, 59, and 5%,
respectively, of the initial activity. Thus, MTE-I is dependent on a
cysteine residue in, or close to, its active site for the thioesterase
activity.
Partial Purification of MTE-II
The acyl-CoA
thioesterase activity (MTE-II) that was retained on the DEAE column was
partially purified by hydrophobic interaction chromatography. Gradient
elution of the phenyl-Sepharose column gave a broad peak of activity
with low recovery (data not shown), and isocratic elution with 40%
ethylene glycol in buffer A was necessary to elute the activity.
Further attempts to purify MTE-II by chromatography on Mono Q,
hydroxylapatite, or size exclusion chromatographies failed due to
substantial loss of activity.
Catalytic Characterization of MTE-II
The partially
purified enzyme showed preference for acyl-CoAs with chain lengths
ranging from C-C
and was less active with
C
-C
and C
-CoA (). No
activity was detected with acetyl-CoA or acetoacetyl-CoA as substrates.
The calculated K
values were in the range
of 3.9-5.5 µM for the good substrates
().
Western Blot Analysis of Subcellular Fractions of Liver
and Organ Distribution Using Anti-MTE-I
Western blot analysis on
the induction of MTE-I was performed with liver mitochondria, purified
by Percoll gradient centrifugation, from control and DEHP-treated rats
(Fig. 3). The antibody reacted with a single band in mitochondria
isolated from DEHP-treated rats, corresponding to purified MTE-I in
size. There was no signal in the samples of control rat liver
mitochondria. However, prolonged exposure of the blot gave a weak
signal in the sample containing a 5-fold amount of control
mitochondrial protein (Fig. 3, inset). The antibody
against MTE-I did not recognize the partially purified MTE-II. To
verify the mitochondrial localization of MTE-I, Western blot analysis
was carried out on Nycodenz gradient-purified peroxisomes,
mitochondria, and microsomes, isolated as described
earlier
(11) , and on liver cytosolic fractions from control and
DEHP-treated rats. Surprisingly, peroxisomal and cytosol fractions from
DEHP-treated rats also contained a band that reacted with anti-MTE-I.
The microsomal fraction did not contain cross-reactive antigens. We
purified the cytosolic thioesterase that was cross-reactive and found
to be identical to ACH2, recently described by Yamada and
co-workers
(14) . We also partially purified and characterized
the peroxisomal cross-reactive isoenzyme (peroxisomal acyl-CoA
thioesterase), which has not been previously described. Western blot
analysis with anti-MTE-I on these enzyme preparations showed that the
cross-reactive proteins in cytosol and peroxisomes were slightly larger
on SDS-PAGE (47 kDa compared to 45 kDa for MTE-I)
(Fig. 4A). The inducible cytosolic ACH2 and the
partially purified peroxisomal acyl-CoA thioesterase from peroxisomes
were analyzed for chain length specificity (Fig. 4B).
The main difference between MTE-I and the cytosolic and the peroxisomal
enzymes was the pronounced preference for longer acyl-CoAs of MTE-I.
Based on the different molecular masses, the different chain length
specificities and the different chromatographic behavior, we conclude
that MTE-I is different from ACH2 and peroxisomal acyl-CoA
thioesterase, although they are structurally related.
Figure 3:
Western
blot analysis of MTE-I in isolated mitochondria. Mitochondria were
isolated from livers of control and DEHP-treated rats, electrophoresed
by SDS-PAGE, and blotted onto a nitrocellulose membrane. The membrane
was incubated with anti-MTE-I IgG, and the probed bands were visualized
after incubation with peroxidase-conjugated secondary antibody using
the Amersham enhanced chemoluminiscence kit. Lanes 1 and
6, 20 ng of MTE-I; lane 2, 10 µg of mitochondrial
protein isolated from control rat liver; lane 3, 50 µg of
mitochondrial protein isolated from control rat liver; lane 4,
10 µg of mitochondrial protein isolated from clofibrate-treated rat
liver; lane 5, the amount of partially purified MTE-II
corresponding to five times the myristoyl-CoA thioesterases activity
applied in lanes 1 and 6. The inset show the
same blot after exposure to x-ray film
overnight.
Figure 4:
Comparison of MTE-I with cytosolic and
peroxisomal isoenzymes. A, Western blot analysis was performed
on ACH2, (purified according to Yamada and co-workers (14)) and on a
peroxisomal acyl-CoA thioesterase that was partially purified.Lanes 1 and 4, 20 ng of MTE-I; lane 2,
a sample of ACH2 corresponding to 10 times the activity of MTE-I;
lane 3, a sample of peroxisomal acyl-CoA thioesterase that
corresponded to the activity of MTE-I. B, purified MTE-I,
ACH2, and partially purified peroxisomal acyl-CoA thioesterase were
analyzed for chain length specificity at a substrate concentration of
10 µM lauroyl-CoA, palmitoyl-CoA, and arachidoyl-CoA,
respectively. Open circles, MTE-I; closed circles,
ACH2; open triangles, peroxisomal acyl-CoA
thioesterase.
We prepared
10% homogenates of various rat organs from control and DEHP-treated
animals which were analyzed with anti-MTE-I by Western blot
(Fig. 5). Control rat livers were apparently devoid of MTE-I, but
heart, brown adipose tissue, and to a lesser degree kidney and brain,
contained a band of similar molecular mass as MTE-I. From rats treated
with DEHP, liver, heart, brown adipose tissue, and to a lesser degree
kidney and brain contained bands of identical molecular masses. Muscle
contained reactive protein bands of lower molecular masses.
Figure 5:
Western blot analysis of various rat
organs. Western blot analysis was performed on homogenates from various
organs from control and DEHP-treated rats. 10 µg of protein of each
homogenate was applied to each well. Odd numbers correspond to
control homogenates and even numbers to homogenates from
DEHP-treated rats. Lanes 1 and 2, liver; lanes 3 and 4, kidney; lanes 5 and 6, muscle;
lanes 7 and 8, heart; lanes 9 and
10, brain; lanes 11 and 12, testis;
lanes 13 and 14, brown adipose
tissue.
Thus,
the Western blot experiments demonstrated that the novel MTE-I was
indeed localized in mitochondria and that cytosol and peroxisomes of
DEHP-induced rat liver contained immunologically related acyl-CoA
thioesterases. Furthermore, the effect of DEHP on expression of MTE-I
and related enzymes is likely to be liver specific, since
immunologically related proteins are constitutively expressed at high
levels in tissues of heart and brown fat.
-oxidized in mitochondria and peroxisomes
or incorporated into triacylglycerols and phospholipids. A prerequisite
for these reactions to occur is the activation of the fatty acids to
their corresponding CoA-thioesters by acyl-CoA synthetases which are
present in several cellular compartments
(26) . Long chain
acyl-CoA thioesterase, in theory a counteracting enzyme activity, has
also been described in several cellular compartments and the regulation
of the activity by treatment of rats with peroxisome proliferators has
been studied in mitochondria
(8, 10, 27) ,
peroxisomes
(9, 11) , microsomes (28, 29), and
cytosol
(8, 13, 15) . Although only a few long
chain acyl-CoA thioesterases have been isolated and characterized, the
strong induction of some of these enzymes by peroxisome proliferators
suggests that their functions may be related to lipid metabolism.
values were similar for the thioesterases and dehydrogenases,
suggesting that they can compete for the same substrates. Such a
competition may be crucial to maintain maximal fatty acid oxidation
activity and capacity of the citric acid cycle in mitochondria. One
possible physiological function of long chain acyl-CoA thioesterases in
mitochondria may thus be regulation of
-oxidation at the substrate
level. This hypothesis is supported by the finding that MTE-I is
constitutively expressed in tissues that have very high capacity for
-oxidation of fatty acids, i.e. heart and brown adipose
tissue.
Figure 6:
Comparison of chain length specificities
of mitochondrial acyl-CoA thioesterases and acyl-CoA dehydrogenases.
Values for mitochondrial acyl-CoA dehydrogenases were recalculated from
Izai et al. (31) to correspond to the percentages of the
activity with palmitoyl-CoA (100%). The values for mitochondrial
acyl-CoA thioesterases were recalculated from Table II to correspond to
the percentages of the activities with palmitoyl-CoA. A,
acyl-CoA chain length specificities of the novel very long chain
acyl-CoA thioesterase (MTE-I) and very long chain acyl-CoA
dehydrogenase (VLCADH) at a fixed substrate concentration of
10 µM. B, comparison of the novel long chain
acyl-CoA thioesterase (MTE-II) and long chain acyl-CoA
dehydrogenase (LCADH) at a fixed substrate concentration of 10
µM.
Treatment of rodents with hypolipidemic agents has been
shown to be accompanied by a pronounced proliferation of peroxisomes
and induction of several peroxisomal enzymes involved in
-oxidation of fatty acids (32, 33) with variable effects on
mitochondria. Although the overall induction of
-oxidation in
mitochondria is only about 2-fold
(31) , compared to the
8-10-fold increase of
-oxidation in peroxisomes (34), the
degree of induction of separate mitochondrial
-oxidation enzymes
can be much higher, as exemplified by 10-fold inductions of the
recently described trifunctional
-oxidation enzyme
(35) and
-
-enoyl-CoA isomerase
(36) .
Treatment of rats with peroxisome proliferators also results in strong
increases in the levels of long chain acyl-CoA and CoASH
(27, 37) in total homogenates, which is due to increased levels in
the peroxisomal, mitochondrial, and cytosolic fractions of rat liver,
indicating that the lipid metabolism is strongly enhanced
(38) .
Although treatment of rats with peroxisome proliferating drugs mainly
induces peroxisomal
-oxidation enzymes, mitochondrial
-oxidation is still likely to be quantitatively most important in
the degradation of fatty acids. The increased levels of long chain
acyl-CoA in livers of rats treated with peroxisome proliferators
suggest an increased pressure on the mitochondrial
-oxidation
which may result in inhibition of mitochondrial functions
(39) ,
due to sequestration of intramitochondrial CoASH. Indeed, it has been
shown that ketogenesis is enhanced 2-5-fold in responce to
clofibrate treatment
(40, 41) .
Table:
Purification of mitochondrial very long chain
acyl-CoA thioesterase (MTE-I)
Table:
Calculated Vand
K
values for MTE-I and MTE-II
min
.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.