©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Very Long Chain and Long Chain Acyl-CoA Thioesterases in Rat Liver Mitochondria
IDENTIFICATION, PURIFICATION, CHARACTERIZATION, AND INDUCTION BY PEROXISOME PROLIFERATORS (*)

L. Thomas Svensson (1) (2), Stefan E. H. Alexson (2)(§), , J. Kalervo Hiltunen (3)(¶)

From the (1) Department of Metabolic Research, The Wenner-Gren Institute Arrhenius Laboratories F3, Stockholm University, S-106 91 Stockholm and the (2) Department of Medical Laboratory Sciences and Technology, Karolinska Institutet, Huddinge University Hospital, S-141 86 Huddinge, Sweden and the (3) Biocenter Oulu, Department of Medical Biochemistry, University of Oulu, FIN-90 220 Oulu and the Department of Biochemistry and Biotechnology, University of Kuopio, P. O. B. 1627, FIN-70211 Kuopio, Finland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 -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.

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.


EXPERIMENTAL PROCEDURES

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) .

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.


RESULTS

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%).

The activity in control mitochondria was increased from 12 to 18 nmol 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).

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.


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 Kvalues 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 Kvalues 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 Kvalues 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.


DISCUSSION

The fate of fatty acids in cellular metabolism depends on several physiological or pathological factors. Under normal conditions fatty acids are mainly -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.

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 Kvalues 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) .

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.

  
Table: Purification of mitochondrial very long chain acyl-CoA thioesterase (MTE-I)

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.


  
Table: Calculated Vand Kvalues for MTE-I and MTE-II

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 min.



FOOTNOTES

*
This work was supported by grants from Stiftelsen Lars Hiertas Minne, Tore Nilsons fond för medicinsk forskning, Sigrid Juselius Foundation, The Bank of Sweden Tercentenary Foundation, and the Swedish Natural Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Medical Laboratory Sciences and Technology, Karolinska Institutet, Huddinge University Hospital, S-141 86 Huddinge, Sweden. Tel.: +46-8-7461601; Fax: +46-8-7461698. E mail: alexson@metabol.su.se.

Supported under a research contract with the Medical Council of the Academy of Finland.

The abbreviations used are: DEHP, di(2-ethylhexyl)phthatlate; pCMB, p-chloromercuribenzoic acid; DFP, diisopropyl fluorophosphate; MTE-I, mitochondrial very long chain acyl-CoA thioesterase; MTE-II, mitochondrial long chain acyl-CoA thioesterase; ACH2, cytosolic acyl-CoA hydrolase 2.

S. Zaltash, L. T. Svensson, and S. E. H. Alexson, unpublished results.


ACKNOWLEDGEMENTS

We thank Susanna Engberg for skillful technical assistance and Shahparak Zaltash for the characterization of the peroxisomal isoenzyme.


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