Long-chain acyl-CoA Hydrolase Research Articles

Metabolic consequences of dehydroepiandrosterone in lean and obese adult Zucker rats.

Several previously reported studies have shown that in rodents exogenously administered dehydroepiandrosterone (DHEA) can cause numerous metabolic changes. In the present study some additional metabolic effects of DHEA are presented using old lean and obese Zucker rats. With this experimental model most of the effects were found to occur in the obese rat. These included decreases in serum insulin and cholesterol as well as a decrease in hepatic acyl-CoA cholesterol-acyl transferase activity. In both lean and obese rats an increase in hepatic long-chain fatty acyl-CoA hydrolase was found. This increase in activity may partially explain DHEA's effect on decreasing body weight in rodents.

The acyl-CoA thioesterase I is regulated by PPARα and HNF4α via a distal response element in the promoter

The cytosolic acyl-coenzyme A thioesterase I (Acot1) is an enzyme that hydrolyzes long-chain acyl-CoAs of C(12)-C(20)-CoA in chain length to the free fatty acid and CoA. Acot1 was shown previously to be strongly upregulated at the mRNA and protein level in rodents by fibrates. In this study, we show that Acot1 mRNA levels were increased by 90-fold in liver by treatment with Wy-14,643 and that Acot1 mRNA was also increased by 15-fold in the liver of hepatocyte nuclear factor 4alpha (HNF4alpha) knockout animals. Our study identified a direct repeat 1 (DR1) located in the Acot1 gene promoter in mouse, which binds the peroxisome proliferator-activated receptor alpha (PPARalpha) and HNF4alpha. Chromatin immunoprecipitation (ChIP) assay showed that the identified DR1 bound PPARalpha/retinoid X receptor alpha (RXRalpha) and HNF4alpha, whereas the binding in ChIP was abrogated in the PPARalpha and HNF4alpha knockout mouse models. Reporter gene assays showed activation of the Acot1 promoter in cells by the PPARalpha agonist Wy-14,643 after cotransfection with PPARalpha/RXRalpha. However, transfection with a plasmid containing HNF4alpha also resulted in an increase in promoter activity. Together, these data show that Acot1 is under regulation by an interplay between HNF4alpha and PPARalpha.

Nafamostat is hydrolysed by human liver cytosolic long-chain acyl-CoA hydrolase

Although the authors recently reported that nafamostat, a clinically used serine protease inhibitor, was mainly hydrolysed by carboxylesterase in human liver microsomes, the involvement of human liver cytosol has not been elucidated. The current study examined the in vitro metabolism of nafamostat with human liver cytosols. Kinetic analysis indicated that the Vmax and Km values in the liver cytosols were 9.82 nmol min−1 mg−1 protein and 197 µM for a liver sample HL-1, and 15.1 nmol min−1 mg−1 protein and 157 µM for HL-2, respectively. The Vmax/Km values in both cytosols were at least threefold higher than those in the corresponding microsomes. The liver cytosolic activity for nafamostat hydrolysis was inhibited by phenylmethylsulfonyl fluoride (PMSF) (43% inhibition at 100 µM), whereas diisopropyl fluorophosphate (DFP) and bis(p-nitrophenyl)phosphate (BNPP) failed to inhibit the activity. Furthermore, the hydrolytic activity was also reduced by palmitoyl-CoA (67% inhibition at 100 µM) but not by acetyl-CoA. Effects of PMSF, DFP and BNPP on cytosolic palmitoyl-CoA hydrolytic activity were comparable with those of the cytosolic nafamostat hydrolytic activity. In addition, the palmitoyl-CoA hydrolytic activity was competitively inhibited by nafamostat with the apparent Ki value of 164 µM for the liver cytosol from HL-2. These results suggest that an isoform of long-chain acyl-CoA hydrolase may be responsible for the nafamostat hydrolysis in human liver cytosol.

Causes and prevention of tamoxifen-induced accumulation of triacylglycerol in rat liver

Tamoxifen can induce hepatic steatosis in women. In this study, we wanted to elucidate the mechanism behind the tamoxifen-induced accumulation of triacylglycerol in liver in female rats, and we hoped to prevent this development by combination treatment with the modified fatty acid tetradecylthioacetic acid (TTA). The increased hepatic triacylglycerol level after tamoxifen treatment was accompanied by decreased acetyl-coenzyme A carboxylase (ACC) and FAS activities, increased glycerol-3-phosphate acyltransferase (GPAT) activity, and a tendency to increased diacylglycerol acyltransferase (DGAT) activity. The activities and mRNA levels of enzymes involved in beta-oxidation, ketogenesis, and uptake of lipids from liver were unaffected by tamoxifen, whereas the uptake of lipoproteins was unchanged and the uptake of fatty acids was decreased. Combination treatment with tamoxifen and TTA (Tam+TTA) normalized the hepatic triacylglycerol level and increased the activities of ACC, FAS, GPAT, and DGAT compared with tamoxifen-treated rats. The activities and mRNA levels of enzymes involved in beta-oxidation, ketogenesis, and uptake of lipids were increased after Tam+TTA treatment. In conclusion, tamoxifen increased the hepatic triacylglycerol level, probably as a result of increased triacylglycerol biosynthesis combined with unchanged beta-oxidation. The tamoxifen-induced accumulation of triacylglycerol was prevented by cotreatment with TTA, through mechanisms of increased mitochondrial and peroxisomal beta-oxidation.

Localization of a long-chain acyl-CoA hydrolase in spermatogenic cells in mice

Brain acyl-CoA hydrolase (BACH) hydrolyzes long-chain acyl-CoAs to free fatty acids and CoA-SH. BACH is highly distributed in brain and is localized in neurons, but not glial cells. This suggests that BACH plays a specific role in neurons. BACH is also detected in testis, although the expression profile of BACH is unknown in testis. In this study, developmental changes and cellular distribution of BACH were examined in mouse testis. Before postnatal day (P) 10, BACH was detected at very low levels by Western blotting. Then, BACH content rapidly increased from P14 and reached maximum levels at P21, remaining high until at least P70. The increase in BACH content corresponded to the appearance of pachytene spermatocytes, which was confirmed by immunohistochemistry. BACH was also detectable in spermatids, but not in spermatogonia, mature spermatozoa. These results suggest that BACH is expressed in a cell-specific manner and plays a role in spermatogenesis.

Long-chain acyl-CoA hydrolase in the brain

Long-chain acyl-CoA hydrolases are a group of enzymes that cleave acyl-CoAs into fatty acids and coenzyme A (CoA-SH). Because acyl-CoAs participate in numerous reactions encompassing lipid synthesis, energy metabolism and regulation, modulating intracellular levels of acyl-CoAs would affect cellular functions. Therefore, acyl-CoA synthetases have been intensively studied. In contrast, acyl-CoA hydrolases have been less investigated, especially in the brain despite the fact that its long-chain acyl-CoA hydrolyzing activity is much higher than that in any other organ in the body. However, recent studies have dissected the multiplicity of this class of enzymes on a genomic basis, and have allowed us to discuss their function. Here, we describe a cytosolic long-chain acyl-CoA hydrolase (referred to as BACH) that is constitutively expressed in the brain, comparing it with other acyl-CoA hydrolases found in peripheral organs that have a role in fatty acid oxidation.

Inducible expression of long-chain acyl-CoA hydrolase gene in cell cultures.

A long-chain acyl-CoA hydrolase, BACH, is markedly distributed in the brain and localized in neurons. However, the physiological significance of BACH is unclear. To study the gene function, we expressed the mouse BACH gene in C3H 10T1/2 fibroblastic cells using a mifepristone (RU486)-inducible gene expression system. A cell clone, 10T-S6/44, was generated by stable transfection of two plasmids encoding a mifepristone-dependent transactivator and an inducible transgene product, BACH with a C-terminal MYC-tag (BACH-MYC). The transgene expression in the 10T-S6/44 cells was tightly regulated by mifepristone. Induction of BACH-MYC and an increase in palmitoyl-CoA hydrolase activity were observed in the cells treated with 3 x 10(-11) M mifepristone and reached maximal levels at a concentration of 1 x 10(-9) M for 48 h. The growth rate of cells showing the maximal induction of BACH-MYC was reduced, whereas phospholipid synthesis was unchanged. These results suggested that BACH affects specific cellular systems and functions, but not all acyl-CoA-utilizing processes.

Human brain acyl-CoA hydrolase isoforms encoded by a single gene

Acyl-CoA hydrolases are a group of enzymes that catalyze the hydrolysis of acyl-CoA thioesters to free fatty acids and CoA-SH. The human brain acyl-CoA hydrolase (BACH) gene comprises 13 exons, generating several isoforms through the alternative use of exons. Four first exons (1a–1d) can be used, and three patterns of splicing occur at exon X located between exons 7 and 8 that contains an internal 3 ′-splice acceptor site and creates premature stop codons. When examined with green fluorescent protein-fusion constructs expressed in Neuro-2a cells, the nuclear localization signal encoded by exon 9 was functional by itself, whereas the whole structure was cytosolic, suggesting nuclear translocation of the enzyme. This was consistent with dual staining of the cytosol and nucleus in certain neurons by immunohistochemistry using anti-BACH antibody. The mitochondrial targeting signals encoded by exons 1b and 1c were also functional and directed mitochondrial localization of BACH isoforms with the signals. Although BACH mRNA containing the sequence derived from exon 1a, but not exon X, was exclusively expressed in human brain, these results suggest that the human BACH gene can express long-chain acyl-CoA hydrolase activity in multiple intracellular compartments by generating BACH isoforms with differential localization signals to affect various cellular functions that involve acyl-CoAs.

Purification, Molecular Cloning, and Genomic Organization of Human Brain Long-Chain Acyl-CoA Hydrolase

An acyl-CoA hydrolase, referred to as hBACH, was purified from human brain cytosol. The enzyme had a molecular mass of 100 kDa and 43-kDa subunits, and was highly active with long-chain acyl-CoAs, e.g. a maximal velocity of 295 micromol/min/mg and K(m) of 6.4 microM for palmitoyl-CoA. Acyl-CoAs with carbon chain lengths of C(8-18) were also good substrates. In human brain cytosol, 85% of palmitoyl-CoA hydrolase activity was titrated by an anti-BACH antibody, which accounted for over 75% of the enzyme activity found in the brain tissue. The cDNA isolated for hBACH, when expressed in Escherichia coli, directed the expression of palmitoyl-CoA hydrolase activity and a 44-kDa protein immunoreactive to the anti-BACH antibody, which in turn neutralized the hydrolase activity. The hBACH cDNA encoded a 338-amino acid sequence which was 95% identical to that of a rat homolog. The hBACH gene spanned about 130 kb and comprised 9 exons, and was mapped to 1p36.2 on the cytogenetic ideogram. These findings indicate that the long-chain acyl-CoA hydrolase present in the brain is well conserved between man and the rat, suggesting a conserved role for this enzyme in the mammalian brain, and enabling genetic studies on the functional analysis of acyl-CoA hydrolase.

CDNA Cloning and Genomic Organization of Peroxisome Proliferator-Inducible Long-Chain Acyl-CoA Hydrolase from Rat Liver Cytosol

The cDNA for a peroxisome proliferator-inducible long-chain acyl-CoA hydrolase from rat liver cytosol, referred to as rLACH2, was isolated and its genomic structure was determined. The cDNA encoded a 419-amino-acid polypeptide with a calculated molecular weight of 46,011. Sequence analysis identified an active-site serine motif (Gly-x-Ser-x-Gly) common to carboxylesterases and lipases. When expressed inEscherichia coli,the cDNA directed expression of a protein immunoreactive to an anti-rLACH2 antibody with a molecular mass of 47 kDa, identical to that of purified rLACH2. Northern blot analysis showed marked induction of rLACH2 mRNA in the liver after feeding rats with di(2-ethylhexyl)phthalate, a peroxisome proliferator. The rLACH2 gene spanned about 19 kb and comprised 3 exons, the intron/exon boundaries of which were consistent with the donor/acceptor splice rule. A putative peroxisome proliferator response element (AGGTCATGGTTCA) was identified in the 5′-flanking region, suggesting the involvement of peroxisome proliferator-activated receptors in the regulation of rLACH2 gene expression.

Neuron-specific distribution of long-chain acyl-CoA hydrolase in rat brain

Delayed activation of the rapid response team (RRT) is common and has been associated with adverse outcomes. However, little is known about the factors associated with delayed activation.This was an observational study from two hospitals in Ottawa, Canada, including adult inpatients with experiencing an activation of the RRT. Data was collected between May 1, 2012 and May 31, 2016 and groups were divided between those with activation within 1 h of meeting call criteria and those with >1 h (delayed activation). The primary outcome was in-hospital mortality. There were 6131 patients included in the study, of which 1441 (26.0%) experienced a delay. The reasons for RRT call were significantly different (P < 0.001) with respiratory distress (29.3% versus 24.8%), and hypotension (17.4% versus 13.2%) being more common in the delayed group, and dysrhythmias (15.9% versus 18.5%) and altered level of consciousness (13.5% versus 18.7%) being less common. RRT activation was more delayed on non-surgical services (P < 0.001). Delayed activation was associated with increased mortality (Adjusted odds ratio [OR] 1.23, 95% CI 1.07–1.41), ICU admission (Adjusted OR 1.72, 95% CI 1.51–1.96), and hospital length of stay (13 versus 15 days, P < 0.001).

Molecular Cloning and Expression of cDNAs Encoding Rat Brain and Liver Cytosolic Long-Chain Acyl-CoA Hydrolases

cDNAs encoding the long-chain acyl-CoA hydrolases (ACHs) from rat brain and liver, referred to as rBACH and rLACH1, respectively, were isolated and sequenced. The rBACH cDNA contained an open reading frame encoding a 338-amino acid polypeptide with a calculated molecular weight of 37,559, of which the deduced amino acid sequence matched partial amino acid sequences directly determined for peptides generated by tryptic digestion or CNBr cleavage of purified rBACH. The rLACH1 cDNA contained an open reading frame encoding a 343-amino acid polypeptide with a molecular weight of 38,240. When expressed inEscherichia coli,these cDNAs produced palmitoyl-CoA hydrolase activity and 44-kDa proteins with molecular masses similar to those of purified rBACH and rLACH1 (43 kDa). These expressed proteins and enzyme activity were immunoblotted and neutralized, respectively, by anti-rBACH or anti-rLACH1 antibodies. rLACH1 cDNA had 84 and 94% identity with rBACH cDNA at the nucleotide and amino acid levels, respectively. However, the 5′-end of the former cDNA which contained the N-terminal coding region of rLACH1 was entirely different from the corresponding region of rBACH cDNA, suggesting that these enzymes may be generated by alternative use of exons of the same gene. Northern blot analysis showed that ACH mRNA was expressed constitutively in the rat brain and testis, whereas its expression in the liver was inducible by treatment with the peroxisome proliferator. This study demonstrated the molecular diversity of ACH and suggested the presence of tissue-specific mechanisms to regulate the ACH gene expression.

Long-Chain Acyl-CoA Hydrolase from Rat Brain Cytosol: Purification, Characterization, and Immunohistochemical Localization

Long-chain acyl-CoA hydrolase (EC 3.1.2.2), which is found primarily in the brain in rats, catalyzes the hydrolysis of fatty acyl-CoA thioesters. We purified this enzyme, referred to as ACH, from the rat brain cytosol. The molecular masses of the native enzyme and the subunit were estimated to be 104 and 36 kDa, respectively. The enzyme showed high activity with long-chain acyl-CoAs, e.g., with maximal velocity of 262 μmol/min/mg andKmof 5.7 μMfor palmitoyl-CoA, but acyl-CoAs with carbon chain lengths of C8–18were also good substrates. The enzyme was refractory to the inhibitory effect of diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, but sensitive top-chloromercuribenzoate. In the rat brain cytosol, about 90% of palmitoyl-CoA hydrolase activity was titrated by anti-ACH antibody, which accounted for over 70% of the enzyme activity found in the brain tissue. Immunoblots of the cytosol prepared from rat brain regional blocks indicated the broad distribution of ACH over the brain, with a relatively high level in the pons and medulla. Immunohistochemically, ACH was localized to neurons. In addition to various nuclei, some neuronal cells, such as mitral cells in the olfactory bulb, pyramidal cells in the cerebral cortex, and Purkinje cells in the cerebellum, were also immunostained with anti-ACH antibody. Brain cytosols prepared from ten mammalian species including human contained a single polypeptide reactive to anti-ACH antibody with molecular masses of 34–36 kDa, together with high activities of palmitoyl-CoA hydrolase. These findings suggest the physiological significance of ACH in the brain, although its precise role remains to be determined.

Characterization of hepatic responses of rat to administration of perfluorooctanoic and perfluorodecanoic acids at low levels

Male rats were fed a diet that contained perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) at concentrations ranging from 0.0025–0.04% (w/w) and from 0.00125–0.01% (w/w), respectively, for 1 week. The hepatic responses of the rats to PFOA and PFDA were examined. Upon the administration of PFOA and PFDA, three peroxisome proliferator-responsive parameters, peroxisomal β3-oxidation, microsomal 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase and cytosolic long-chain acyl-CoA hydrolase, were induced in a dose-dependent manner. A multiple regression analysis of the three parameters revealed that the data from rats treated with PFOA and PFDA shared one common line, indicating a marked correlation among the inductions of the three parameters. The activities of glutathione (GSH) S-transferases towards 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) were depressed by PFOA and PFDA. Significant inverse correlations were found between activities of GSH S-transferases and peroxisomal β-oxidation. The administration of PFOA and PFDA significantly increased hepatic concentration of triacylglycerol. The perfluorocarboxylic acids at relatively high doses caused accumulation of cholesterol in liver. Electron microscopic studies showed that the administration of PFOA and PFDA caused an increase in cell size and proliferations of peroxisomes, and that the treatment of rats with PFDA at dietary concentration of 0.01% caused a marked increase in small lipid droplet in hepatocytes, indicative of hepatotoxic manifestations. The present results suggest that when PFOA and PFDA are administered at low levels, there are no differences between the properties of the perfluorocarboxylic acids as peroxisome proliferators, although the administration of PFDA at the doses exceeding a certain level becomes markedly toxic to hepatocytes.

Fatty acyl-CoA oxidase activity is induced before long-chain acyl-CoA hydrolase activity and acyl-CoA binding protein in liver of rat treated with peroxisome proliferating 3-thia fatty acids.

1. In this study we explored the relationship between specific acyl-CoA esters and induction of acyl-CoA binding protein (ACBP) and enzymes related to the proliferation of peroxisomes. Male Wistar rats were administered a single dose (150 mg/day/kg) of sulphur-substituted fatty acid analogues, and the effects of tetradecylthioacetic acid and 3-thiadicarboxylic acid, which both act as peroxisome proliferators, were compared with the effects of tetradecylthiopropionic acid and palmitic acid which do not induce peroxisome proliferation. 2. The hepatic level of total long-chain acyl-CoA was significantly increased within 12 h of feeding these fatty acids, except in rat fed tetradecylthioacetic acid. Hplc chromatograms of liver extracts prepared from rat fed tetradecylthioacetic acid showed that tetradecylthioacetyl-CoA ester accumulated in the liver 4 h after feeding and had disappeared after 24 h. In liver extracts of the tetradecylthiopropionic acid-treated rat tetradecylthiopropionyl-CoA was not observed, but the appearance of a new long-chain acyl-CoA ester, probably a metabolite of tetradecylthiopropionic acid, was detected. This new peak reached a maximum 4h after feeding. In rat fed tetradecylthioacetic acid and 3-thiadicarboxylic acid the hepatic level of fatty acyl-CoA oxidase mRNA increased 8 h after feeding, while the acyl-CoA oxidase activity had increased after 12 h. 3. The early accumulation of specific tetradecylthioacetyl-CoA suggests that this ester may be a possible mediator of the induction of fatty acyl-CoA oxidase. The level of hepatic acyl-CoA binding protein, long-chain acyl-CoA hydrolase activity and long-chain acyl-CoA synthetase activity did not change after a single dose of all four fatty acids. Prolonged administration of 3-thia fatty acids resulted, however, in a dose- and time-dependent increase in hepatic ACBP content and ACBP mRNA level. The amount of ACBP increased in parallel to the long-chain acyl-CoA hydrolase activity. The correlated induction of fatty acyl-CoA binding protein and long-chain acyl-CoA hydrolase seems to be dependent on a sustained accumulation of total long-chain acyl-CoA esters.

Existence of an Enzymatic Pathway Furnishing Arachidonic Acid for Prostaglandin Synthesis from Arachidonoyl CoA in Rabbit Kidney Medulla

We examined whether arachidonoyl CoA (AA-CoA) can be a possible supplier of arachidonic acid (AA) for prostaglandin (PG) synthesis in rabbit kidney medulla. AA-CoA was preincubated with or without the 105000 g supernatant (cytosol) fraction from the kidney medulla for 5 min at 37°C followed by the incubation with the microsomal fraction (0.5 mg protein) (a rich source of PG synthesizing enzymes) in the presence of reduced glutathione and hydroquinone for 5 min at 37°C, and the formed PGE2, F2α and D2 were measured by high-pressure liquid chromatography using 9-anthryldiazomethane for derivatization. The addition of AA-CoA (20 nmol) alone changed the total PG formation (the sum of PGE2, F2α and D2) from 0.14 to 1.55 ng. When the cytosolic fraction (10-100 μl) was added together with 20 nmol-AA-CoA in the preincubation mixture, total PG formation was further increased 2- to 3-fold compared to AA-CoA alone. Experiments utilizing AA, instead of AA-CoA and boiled cytosolic fraction revealed that the stimulatory action of the cytosolic fraction on the AA-CoA-induced PG synthesis may not be due to the effect on cyclooxygenase activity and due to long-chain acyl CoA hydrolase. These results show that long-chain acyl CoA hydrolase which hydrolyzes AA-CoA to AA is present in the cytosol of rabbit kidney medulla and suggest that this enzyme activity is a potential mediator of supply of AA for PG synthesis in this region.

Characterization of microsomal long-chain acyl-CoA hydrolase activity in the rat submandibular gland

1. 1. Long-chain fatty acyl-CoA hydrolase in submandibular gland microsomes was characterized and compared to that in liver ones. 2. 2. In rat submandibular gland, the microsomal long-chain acyl-CoA hydrolase showed a higher relative activity for polyunsaturated fatty acyl-CoAs than that of liver microsomes. 3. 3. It was suggested that the hydrolase in rat submandibular gland microsomes may play a role in modulation in the acyl-CoA pool size.

Purification and Properties of Long-Chain Acyl-CoA Hydrolases from the Liver Cytosol of Rats Treated with Peroxisome Proliferator

Two long-chain acyl-CoA hydrolases, referred to as ACH1 and ACH2, were purified from the liver cytosol of rats fed a diet containing di(2-ethylhexyl)phthalate, a peroxisome proliferator. The molecular mass of ACH1 was estimated to be 73 kDa by gel filtration, and that of the subunits, 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The corresponding values of ACH2 were 42 and 43 kDa, respectively. Both enzymes were active toward fatty acyl-CoAs with chain-lengths of C 12-16, but ACH1 had relatively broad specificity as acyl-CoAs with C 8-18 were good substrates. A marked difference in their catalytic properties was found in the maximal velocity; for palmitoyl-CoA, 553 and 4.23 μmol/min/mg with K m values of 5.9 and 5.4 μM for ACH1 and ACH2, respectively. ACH2 underwent severe substrate inhibition with high concentrations of long-chain acyl-CoAs, whereas ACH1 did not. Examination with various reagents including divalent cations, sulfhydryl-blocking reagent, nucleotides, and hypolipidemic drugs, characterized ACH1 and ACH2 with several properties distinct from those of mitochondrial and microsomal hydrolases. ACH1 and ACH2 were also discernible in that the former, but not the latter, was inhibited by ATP. In the liver cytosol of rats treated with di(2-ethylhexyl)phthalate, about 90% of palmitoyl-CoA hydrolase activity was titrated with anti-ACH1 and anti-ACH2 antibodies. Immunoblot analysis suggested the presence of the enzymes also in extrahepatic tissues, especially in the brain and testis (ACH1), and in the heart and kidney (ACH2).

Purification, characterization and modulation of a microsomal carboxylesterase in rat liver for the hydrolysis of acyl-CoA

A carboxylesterase containing long-chain acyl-CoA hydrolase activity was purified to apparent homogeneity from rat liver microsomes. Palmitoyl-CoA was the most preferred substrate, followed by stearoyl-CoA and oleoyl-CoA. Arachidonoyl-CoA, linoleoyl-CoA and acetyl-CoA were not hydrolysed by the enzyme. The purified enzyme had no activity on the hydrolysis of phospholipids and neutral lipids. The molecular mass of the enzyme was found to be 56 kDa by SDS/PAGE and 64 kDa by gel-filtration chromatography. On isoelectric focusing, the purified enzyme behaved like the ES-4 type, with a pI of 6.15. Determination of the amino acid sequence revealed that its N-terminal sequence is 100% homologous with the only other known N-terminal sequence for a rat carboxylesterase isoenzyme (ES-10). Enzyme activity was inhibited by lysophosphatidic acid and activated by lysophosphatidylcholine. The modulation of enzyme activity by these lysophospholipids might represent a plausible mechanism for the physiological control of acyl-CoA concentrations.

Relationship between translocation of long-chain acyl-CoA hydrolase, phosphatidate phosphohydrolase and CTP:phosphocholine cytidylyltransferase and the synthesis of triglycerides and phosphatidylcholine in rat liver.

Translocation of long-chain acyl-coenzyme A hydrolase from the microsomal fraction to the cytosolic fraction was promoted in cell-free extracts of rat liver by palmitic acid, oleic acid, tetradecylthioacetic acid, and tetradecylthiopropionic acid, and by their CoA esters. The CoA esters were more effective than the non-esterified acids in the translocation of the enzyme. Treatment of normolipidemic rats with sulfur-substituted non-beta-oxidizable fatty acid analogues resulted in a transitory increase in hepatic concentration of long-chain acyl-CoA. Longer feeding times almost normalized the hepatic long-chain acyl-CoA content. Microsomal long-chain acyl-CoA hydrolase activity was inhibited, whereas the activity of the cytosolic form was stimulated. The rise in enzyme activity coincided with a reduction in liver content of triglyceride and an increase in hepatic phospholipid content. The results suggest that the activity of long-chain acyl-CoA hydrolase in the cytosol may control the amount of acyl-CoA thioesters in the liver. Esterified and non-esterified fatty acids caused in vitro translocation of phosphatidate phosphohydrolase and cytidine 5'-triphosphate (CTP):phosphocholine cytidylyltransferase from the cytosolic fraction to the microsomal fraction. However, the translocation of these two enzyme systems was not obtained in vivo. The activity of phosphatidate phosphohydrolase decreased in microsomal and cytosolic fractions while the activity of cytidylyltransferase in these fractions increased. The activities of soluble phosphatidate phosphohydrolase and long-chain acyl-CoA hydrolase appeared to be inversely correlated.(ABSTRACT TRUNCATED AT 250 WORDS)