Far from Inert: Membrane Lipids Possess Intrinsic Reactivity That Has Consequences for Cell Biology.

In this article, it is hypothesized that a fundamental chemical reactivity exists between some non-lipid constituents of cellular membranes and ester-based lipids, the significance of which is not generally recognized. Many peptides and smaller organic molecules have now been shown to undergo lipidation reactions in model membranes in circumstances where direct reaction with the lipid is the only viable route for acyl transfer. Crucially, drugs like propranolol are lipidated in vivo with product profiles that are comparable to those produced in vitro. Some compounds have also been found to promote lipid hydrolysis. Drugs with high lytic activity in vivo tend to have higher toxicity in vitro. Deacylases and lipases are proposed as key enzymes that protect cells against the effects of intrinsic lipidation. The toxic effects of intrinsic lipidation are hypothesized to include a route by which nucleation can occur during the formation of amyloid fibrils.

Histone Deacetylase 11 Is a Fatty-Acid Deacylase.

Histone deacetylase 11 (HDAC11) is a sole member of the class IV HDAC subfamily with negligible intrinsic deacetylation activity. Here, we report in vitro profiling of HDAC11 deacylase activities, and our data unequivocally show that the enzyme efficiently removes acyl moieties spanning 8-18 carbons from the side chain nitrogen of the lysine residue of a peptidic substrate. Additionally, N-linked lipoic acid and biotin are removed by the enzyme, although with lower efficacy. Catalytic efficiencies toward dodecanoylated and myristoylated peptides were 77 700 and 149 000 M-1 s-1, respectively, making HDAC11 the most proficient fatty-acid deacylase of the HDAC family. Interestingly, HDAC11 is strongly inhibited by free myristic, palmitic, and stearic acids with inhibition constants of 6.5, 0.9, and 1.6 µM, respectively. At the same time, its deacylase activity is stimulated more than 2.5-fold by both palmitoyl-coenzyme A and myristoyl-coenzyme A, pointing toward metabolic control of the enzymatic activity by fatty-acid metabolites. Our data reveal novel enzymatic activity of HDAC11 that can, in turn, facilitate the uncovering of additional biological functions of the enzyme as well as the design of isoform-specific HDAC inhibitors.

Ach1 is involved in shuttling mitochondrial acetyl units for cytosolic C2 provision in Saccharomyces cerevisiae lacking pyruvate decarboxylase.

Acetyl-coenzyme A (acetyl-CoA) is not only an essential intermediate in central carbon metabolism, but also an important precursor metabolite for native or engineered pathways that can produce many products of commercial interest such as pharmaceuticals, chemicals or biofuels. In the yeast Saccharomyces cerevisiae, acetyl-CoA is compartmentalized in the cytosol, mitochondrion, peroxisome and nucleus, and cannot be directly transported between these compartments. With the acetyl-carnitine or glyoxylate shuttle, acetyl-CoA produced in peroxisomes or the cytoplasm can be transported into the cytoplasm or the mitochondria. However, whether acetyl-CoA generated in the mitochondria can be exported to the cytoplasm is still unclear. Here, we investigated whether the transfer of acetyl-CoA from the mitochondria to the cytoplasm can occur using a pyruvate decarboxylase negative, non-fermentative yeast strain. We found that mitochondrial Ach1 can convert acetyl-CoA in this compartment into acetate, which crosses the mitochondrial membrane before being converted into acetyl-CoA in the cytosol. Based on our finding we propose a model in which acetate can be used to exchange acetyl units between mitochondria and the cytosol. These results will increase our fundamental understanding of intracellular transport of acetyl units, and also help to develop microbial cell factories for many kinds of acetyl-CoA derived products.

NatB domain-containing CRA-1 antagonizes hydrolase ACER-1 linking acetyl-CoA metabolism to the initiation of recombination during C. elegans meiosis.

The formation of DNA double-strand breaks (DSBs) must take place during meiosis to ensure the formation of crossovers, which are required for accurate chromosome segregation, therefore avoiding aneuploidy. However, DSB formation must be tightly regulated to maintain genomic integrity. How this regulation operates in the context of different chromatin architectures and accessibility, and how it is linked to metabolic pathways, is not understood. We show here that global histone acetylation levels undergo changes throughout meiotic progression. Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans. We provide evidence that the regulation of histone acetylation requires CRA-1, a NatB domain-containing protein homologous to human NAA25, which controls the levels of acetyl-Coenzyme A (acetyl-CoA) by antagonizing ACER-1, a previously unknown and conserved acetyl-CoA hydrolase. CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein. We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation.

From arylamine N-acetyltransferase to folate-dependent acetyl CoA hydrolase: impact of folic acid on the activity of (HUMAN)NAT1 and its homologue (MOUSE)NAT2.

Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme's active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these enzymes and for improving chemotherapeutic approaches to pathological conditions including estrogen receptor-positive breast cancer.

Enzymatic and transcriptional regulation of the cytoplasmic acetyl-CoA hydrolase ACOT12.

Acyl-CoA thioesterase 12 (ACOT12) is the major enzyme known to hydrolyze the thioester bond of acetyl-CoA in the cytosol in the liver. ACOT12 contains a catalytic thioesterase domain at the N terminus and a steroidogenic acute regulatory protein-related lipid transfer (START) domain at the C terminus. We investigated the effects of lipids (phospholipids, sphingolipids, fatty acids, and sterols) on ACOT12 thioesterase activity and found that the activity was inhibited by phosphatidic acid (PA) in a noncompetitive manner. In contrast, the enzymatic activity of a mutant form of ACOT12 lacking the START domain was not inhibited by the lipids. These results suggest that the START domain is important for regulation of ACOT12 activity by PA. We also found that PA could bind to thioesterase domain, but not to the START domain, and had no effect on ACOT12 dissociation. ACOT12 is detectable in the liver but not in hepatic cell lines such as HepG2, Hepa-1, and Fa2N-4. ACOT12 mRNA and protein levels in rat primary hepatocytes decreased following treatment with insulin. These results suggest that cytosolic acetyl-CoA levels in the liver are controlled by lipid metabolites and hormones, which result in allosteric enzymatic and transcriptional regulation of ACOT12.

In vitro cultured human Sertoli cells secrete high amounts of acetate that is stimulated by 17ß-estradiol and suppressed by insulin deprivation.

BACKGROUND: Several important functions for a successful spermatogenesis are dependent on Sertoli cells (SCs). Besides their unique characteristics as support cells, they produce essential cofactors and metabolites, and are responsible for nurturing the developing germ cells. The continuous production of lipids, phospholipids and proteins by germ cells must require high amounts of metabolic precursors. Thus, we hypothesized that hSCs could produce acetate in a hormonally-regulated manner. METHODS: hSC-enriched primary cultures were maintained in the absence of insulin or in the presence of 17ß-estradiol (E2) or 5α-dihydrotestosterone (DHT). Acetate production was determined by 1H-NMR. mRNA gene expression levels of Acetyl CoA hydrolase (ACoA Hyd) and Acetyl CoA synthase (ACoA Synt) were determined by RT-PCR. RESULTS: hSCs produced high amounts of acetate suggesting that this metabolite should play a key role on the progression of spermatogenesis, namely as a metabolic precursor for the synthesis of cellular constituents. In addition, acetate metabolism proved to be under strict hormonal regulation. In the presence of E2 or DHT, hSCs produced different amounts of acetate. While E2 treatment increased acetate production, increasing ACoA Hyd gene transcript levels, DHT-treated cells showed decreased acetate production, differently modulating the ratio ACoA Hyd/ACoA Synt. Surprisingly, insulin-deprivation completely suppressed acetate production/export and significantly decreased the ACoA Hyd gene transcript levels. GENERAL SIGNIFICANCE: Taken together, these results suggest that, although hSCs are primarily described as lactate producers, the elevated production of acetate deserves special attention, in order to clarify the mechanisms behind its hormonal regulation and its role on a successful spermatogenesis.

ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma.

Insect stage trypanosomes use an "acetate shuttle" to transfer mitochondrial acetyl-CoA to the cytosol for the essential fatty acid biosynthesis. The mitochondrial acetate sources are acetate:succinate CoA-transferase (ASCT) and an unknown enzymatic activity. We have identified a gene encoding acetyl-CoA thioesterase (ACH) activity, which is shown to be the second acetate source. First, RNAi-mediated repression of ASCT in the ACH null background abolishes acetate production from glucose, as opposed to both single ASCT and ACH mutants. Second, incorporation of radiolabeled glucose into fatty acids is also abolished in this ACH/ASCT double mutant. ASCT is involved in ATP production, whereas ACH is not, because the ASCT null mutant is ∼1000 times more sensitive to oligomycin, a specific inhibitor of the mitochondrial F(0)/F(1)-ATP synthase, than wild-type cells or the ACH null mutant. This was confirmed by RNAi repression of the F(0)/F(1)-ATP synthase F(1)ß subunit, which is lethal when performed in the ASCT null background but not in the wild-type cells or the ACH null background. We concluded that acetate is produced from both ASCT and ACH; however, only ASCT is responsible, together with the F(0)/F(1)-ATP synthase, for ATP production in the mitochondrion.

Carnitine palmitoyltransferase I control of acetogenesis, the major pathway of fatty acid {beta}-oxidation in liver of neonatal swine.

To examine the regulation of hepatic acetogenesis in neonatal swine, carnitine palmitoyltransferase I (CPT I) activity was measured in the presence of varying palmitoyl-CoA (substrate) and malonyl-CoA (inhibitor) concentrations, and [1-(14)C]-palmitate oxidation was simultaneously measured. Accumulation rates of (14)C-labeled acetate, ketone bodies, and citric acid cycle intermediates within the acid-soluble products were determined using radio-HPLC. Measurements were conducted in mitochondria isolated from newborn, 24-h (fed or fasted), and 5-mo-old pigs. Acetate rather than ketone bodies was the predominant radiolabeled product, and its production increased twofold with increasing fatty acid oxidation during the first 24-h suckling period. The rate of acetogenesis was directly proportional to CPT I activity. The high activity of CPT I in 24-h-suckling piglets was not attributable to an increase in CPT I gene expression, but rather to a large decrease in the sensitivity of CPT I to malonyl-CoA inhibition, which offset a developmental decrease in affinity of CPT I for palmitoyl-CoA. Specifically, the IC(50) for malonyl-CoA inhibition and K(m) value for palmitoyl-CoA measured in 24-h-suckling pigs were 1.8- and 2.7-fold higher than measured in newborn pigs. The addition of anaplerotic carbon from malate (10 mM) significantly reduced (14)C accumulation in acetate (P < 0.003); moreover, the reduction was much greater in newborn (80%) than in 24-h-fed (72%) and 5-mo-old pigs (55%). The results demonstrate that acetate is the primary product of hepatic mitochondrial beta-oxidation in Sus scrofa and that regulation during early development is mediated primarily via kinetic modulation of CPT I.

Re-characterisation of Saccharomyces cerevisiae Ach1p: fungal CoA-transferases are involved in acetic acid detoxification.

Saccharomyces cerevisiae and Neurospora crassa mutants defective in the so-called acetyl-CoA hydrolases Ach1p and Acu-8, respectively, display a severe growth defect on acetate, which is most strongly pronounced under acidic conditions. Acetyl-CoA hydrolysis is an energy wasting process and therefore denoted as a biochemical conundrum. Acetyl-CoA hydrolases show high sequence identity to the CoA-transferase CoaT from Aspergillus nidulans. Therefore, we extensively re-characterised the yeast enzyme. Ach1p showed highest specific activity for the CoASH transfer from succinyl-CoA to acetate and only a minor acetyl-CoA-hydrolase activity. Complementation of an ach1 mutant with the coaT gene reversed the growth defect on acetate confirming the in vivo function of Ach1p as a CoA-transferase. Our results imply that Ach1p is involved in mitochondrial acetate detoxification by a CoASH transfer from succinyl-CoA to acetate. Thereby, Ach1p does not perform the energy wasting hydrolysis of acetyl-CoA but conserves energy by the detoxification of mitochondrial acetate.

Role of acetyl coenzyme A synthesis and breakdown in alternative carbon source utilization in Candida albicans.

Acetyl coenzyme A (acetyl-CoA) is the central intermediate of the pathways required to metabolize nonfermentable carbon sources. Three such pathways, i.e., gluconeogenesis, the glyoxylate cycle, and beta-oxidation, are required for full virulence in the fungal pathogen Candida albicans. These processes are compartmentalized in the cytosol, mitochondria, and peroxosomes, necessitating transport of intermediates across intracellular membranes. Acetyl-CoA is trafficked in the form of acetate by the carnitine shuttle, and we hypothesized that the enzymes that convert acetyl-CoA to/from acetate, i.e., acetyl-CoA hydrolase (ACH1) and acetyl-CoA synthetase (ACS1 and ACS2), would regulate alternative carbon utilization and virulence. We show that C. albicans strains depleted for ACS2 are unviable in the presence of most carbon sources, including glucose, acetate, and ethanol; these strains metabolize only fatty acids and glycerol, a substantially more severe phenotype than that of Saccharomyces cerevisiae acs2 mutants. In contrast, deletion of ACS1 confers no phenotype, though it is highly induced in the presence of fatty acids, perhaps explaining why acs2 mutants can utilize fatty acids. Strains lacking ACH1 have a mild growth defect on some carbon sources but are fully virulent in a mouse model of disseminated candidiasis. Both ACH1 and ACS2 complement mutations in their S. cerevisiae homolog. Together, these results show that acetyl-CoA metabolism and transport are critical for growth of C. albicans on a wide variety of nutrients. Furthermore, the phenotypic differences between mutations in these highly conserved genes in S. cerevisiae and C. albicans support recent findings that significant functional divergence exists even in fundamental metabolic pathways between these related yeasts.

Enzymes involved in arachidonic acid release in adrenal and Leydig cells.

Stimulation of receptors and subsequent signal transduction results in the activation of arachidonic acid (AA) release. Once AA is released from phospholipids or others esters, it may be metabolized via the cycloxygenase or the lipoxygenase pathways. How the cells drive AA to these pathways is not elucidated yet. It is reasonable to speculate that each pathway will have different sources of free AA triggered by different signal transduction pathways. Several reports have shown that AA and its lipoxygenase-catalyzed metabolites play essential roles in the regulation of steroidogenesis by influencing cholesterol transport from the outer to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Signals that stimulate steroidogenesis also cause the release of AA from phospholipids or other esters by mechanisms that are not fully understood. This review focuses on the enzymes of AA release that impact on steroidogenesis.

Molecular cloning and functional expression of human cytosolic acetyl-CoA hydrolase.

A cDNA encoding human cytosolic acetyl-CoA hydrolase (CACH) was isolated from a human liver cDNA library, sequenced and functionally expressed in insect cells. The human CACH cDNA encodes a 555-amino-acid sequence that is 81.4%/78.7% identical to those of the mouse/rat homologue, suggesting a conserved role for this enzyme in the human and rodent livers. Bioinformatical study further reveals a high degree of similarity among the human and rodent CACHs as follows: First, the gene is composed of 15 exons ranging in size from 56 to 157 bp. Second, the protein consists of two thioesterase regions and a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. Third, the promoter region is GC-rich and contains GC boxes, but lacks both TATA and CCAAT boxes, the typical criteria of housekeeping genes. A consensus peroxisome proliferator responsive element (PPRE) present in the rodent CACH promoter regions supports marked CACH induction in rat liver by peroxisome proliferator (PP).

Acetate generation in rat liver mitochondria; acetyl-CoA hydrolase activity is demonstrated by 3-ketoacyl-CoA thiolase.

Acetate has been found as an endogenous metabolite of beta-oxidation of fatty acids in liver. In order to investigate the regulation of acetate generation in liver mitochondria, we attempted to purify a mitochondrial acetyl-CoA hydrolase in rat liver. This acetyl-CoA-hydrolyzing activity in isolated mitochondria was induced by the treatment of rats with di(2-ehtylhexyl)phthalate (DEHP), a peroxisome proliferator which induces expression of several peroxisomal and mitochondrial enzymes involved in beta-oxidation of fatty acids. The purified enzyme was 43-kDa in molecular mass by SDS/PAGE. Internal amino acid sequencing of this enzyme revealed that it was identical with mitochondrial 3-ketoacyl-CoA thiolase, suggesting that this enzyme has two kinds of activities, 3-ketoacyl-CoA thiolase and acetyl-CoA hydrolase activities. Kinetic studies clearly indicated that this enzyme had the both activities and each activity was inhibited by the substrates of the other activity, that is, 3-ketoacyl-CoA thiolase activity was inhibited by acetyl-CoA, on the other hand, acetyl-CoA hydrolase activity was inhibited by acetoacetyl-CoA in a competitive manner. These findings suggested that acetate generation in liver mitochondria is a side reaction of this known enzyme, 3-ketoacyl-CoA thiolase, and this enzyme may regulate its activities depending on each substrate level.

Expression of a yeast acetyl CoA hydrolase in the mitochondrion of tobacco plants inhibits growth and restricts photosynthesis.

Acetyl Coenzyme A (acetyl CoA) is required in the mitochondria to fuel the operation of the Krebs cycle and within the cytosolic, peroxisomal and plastidial compartments wherein it acts as the immediate precursor for a wide range of anabolic functions. Since this metabolite is impermeable to membranes it follows that discrete pathways both for its synthesis and for its utilization must be present in each of these organelles and that the size of the various compartmented pools are independently regulated. To determine the specific role of acetyl CoA in the mitochondria we exploited a transgenic approach to introduce a yeast acetyl CoA hydrolase (EC 3.1.2.1.) into this compartment in tobacco plants. Despite the facts that the introduced enzyme was correctly targeted and that there were marked reductions in the levels of citrate and malate and an increase in the acetate content of the transformants, the transgenic plants surprisingly exhibited increased acetyl CoA levels. The lines were further characterised by a severe growth retardation, abnormal leaf colouration and a dramatic reduction in photosynthetic activity correlated with a marked reduction in the levels of transcripts of photosynthesis and in the content of photosynthetic pigments. The altered rate of photosynthesis in the transgenics was also reflected by a modified carbon partitioning in leaves of these lines, however, further studies revealed that this was most likely caused by a decreased source to sink transport of carbohydrate. In summary these results suggest that the content of acetyl CoA is under tight control and that alterations in the level of this central metabolite have severe metabolic and developmental consequences in tobacco.

Brassica juncea 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase 1: expression and characterization of recombinant wild-type and mutant enzymes.

3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS; EC 2.3.3.10) is the second enzyme in the cytoplasmic mevalonate pathway of isoprenoid biosynthesis, and catalyses the condensation of acetyl-CoA with acetoacetyl-CoA (AcAc-CoA) to yield S-HMG-CoA. In this study, we have first characterized in detail a plant HMGS, Brassica juncea HMGS1 (BjHMGS1), as a His6-tagged protein from Escherichia coli. Native gel electrophoresis analysis showed that the enzyme behaves as a homodimer with a calculated mass of 105.8 kDa. It is activated by 5 mM dithioerythreitol and is inhibited by F-244 which is specific for HMGS enzymes. It has a pH optimum of 8.5 and a temperature optimum of 35 degrees C, with an energy of activation of 62.5 J x mol(-1). Unlike cytosolic HMGS from chicken and cockroach, cations like Mg2+, Mn2+, Zn2+ and Co2+ did not stimulate His6-BjHMGS1 activity in vitro; instead all except Mg2+ were inhibitory. His6-BjHMGS1 has an apparent K(m-acetyl-CoA) of 43 microM and a V(max) of 0.47 micromol x mg(-1) x min(-1), and was inhibited by one of the substrates (AcAc-CoA) and by both products (HMG-CoA and HS-CoA). Site-directed mutagenesis of conserved amino acid residues in BjHMGS1 revealed that substitutions R157A, H188N and C212S resulted in a decreased V(max), indicating some involvement of these residues in catalytic capacity. Unlike His6-BjHMGS1 and its soluble purified mutant derivatives, the H188N mutant did not display substrate inhibition by AcAc-CoA. Substitution S359A resulted in a 10-fold increased specific activity. Based on these kinetic analyses, we generated a novel double mutation H188N/S359A, which resulted in a 10-fold increased specific activity, but still lacking inhibition by AcAc-CoA, strongly suggesting that His-188 is involved in conferring substrate inhibition on His6-BjHMGS1. Substitution of an aminoacyl residue resulting in loss of substrate inhibition has never been previously reported for any HMGS.

Functional characterization and localization of acetyl-CoA hydrolase, Ach1p, in Saccharomyces cerevisiae.

Acetyl-CoA hydrolase (Ach1p), catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating intracellular acetyl-CoA or CoASH pools; however, its intracellular functions and distribution remain to be established. Using site-directed mutagenesis analysis, we demonstrated that the enzymatic activity of Ach1p is dependent upon its putative acetyl-CoA binding sites. The ach1 mutant causes a growth defect in acetate but not in other non-fermentable carbon sources, suggesting that Ach1p is not involved in mitochondrial biogenesis. Overexpression of Ach1p, but not constructs containing acetyl-CoA binding site mutations, in ach1-1 complemented the defect of acetate utilization. By subcellular fractionation, most of the Ach1p in yeast was distributed with mitochondria and little Ach1p in the cytoplasm. By immunofluorescence microscopy, we show that Ach1p and acetyl-CoA binding site-mutated constructs, but not its N-terminal deleted construct, are localized in mitochondria. Moreover, the onset of pseudohyphal development in homozygote ach1-1 diploids was abolished. We infer that Ach1p may be involved in a novel acetyl-CoA biogenesis and/or acetate utilization in mitochondria and thereby indirectly affect pseudohyphal development in yeast.

Mouse cytosolic acetyl-CoA hydrolase, a novel candidate for a key enzyme involved in fat metabolism: cDNA cloning, sequencing and functional expression.

A cytosolic acetyl-CoA hydrolase (CACH) cDNA has been isolated from mouse liver cDNA library and sequenced. Recombinant expression of the cDNA in insect cells resulted in overproduction of active acetyl-CoA hydrolyzing enzyme protein. The mouse CACH cDNA encoded a 556-amino-acid sequence that was 93.5% identical to rat CACH, suggesting a conserved role for this enzyme in the mammalian liver. Database searching shows no homology to other known proteins, but reveals homological cDNA sequences showing two single-nucleotide polymorphisms (SNPs) in the CACH coding region. The discovery of mouse CACH cDNA is an important step towards genetic studies on the functional analysis of this enzyme by gene-knockout and transgenic approaches.

Production of acetate in the liver and its utilization in peripheral tissues.

In experimental rat liver perfusion we observed net production of free acetate accompanied by accelerated ketogenesis with long-chain fatty acids. Mitochondrial acetyl-CoA hydrolase, responsible for the production of free acetate, was found to be inhibited by the free form of CoA in a competitive manner and activated by reduced nicotinamide adenine dinucleotide (NADH). The conditions under which the ketogenesis was accelerated favored activation of the hydrolase by dropping free CoA and elevating NADH levels. Free acetate was barely metabolized in the liver because of low affinity, high K(m), of acetyl coenzyme A (acetyl-CoA) synthetase for acetate. Therefore, infused ethanol was oxidized only to acetate, which was entirely excreted into the perfusate. The acetyl-CoA synthetase in the heart mitochondria was much lower in K(m) than it was in the liver, thus the heart mitochondria was capable of oxidizing free acetate as fast as other respiratory substrates, such as succinate. These results indicate that rat liver produces free acetate as a byproduct of ketogenesis and may supply free acetate, as in the case of ketone bodies, to extrahepatic tissues as fuel.

Molecular cloning and functional expression of rat liver cytosolic acetyl-CoA hydrolase.

A cytosolic acetyl-CoA hydrolase (CACH) was purified from rat liver to homogeneity by a new method using Triton X-100 as a stabilizer. We digested the purified enzyme with an endopeptidase and determined the N-terminal amino-acid sequences of the two proteolytic fragments. From the sequence data, we designed probes for RT-PCR, and amplified CACH cDNA from rat liver mRNA. The CACH cDNA contains a 1668-bp ORF encoding a protein of 556 amino-acid residues (62 017 Da). Recombinant expression of the cDNA in insect cells resulted in overproduction of functional acetyl-CoA hydrolase with comparable acyl-CoA chain-length specificity and Michaelis constant for acetyl-CoA to those of the native CACH. Database searching shows no homology to other known proteins, but reveals high similarities to two mouse expressed sequence tags (91% and 93% homology) and human mRNA for KIAA0707 hypothetical protein (50% homology) of unknown function.