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.     

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).     

Rat ATP citrate-lyase. Molecular cloning and sequence analysis of a full-length cDNA and mRNA abundance as a function of diet, organ, and age

ATP citrate-lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. We have isolated a full-length cDNA copy of 4.3 kilobase pairs encoding the ATP-citrate lyase mRNA by screening rat liver cDNA library using oligonucleotide probes designed from peptide sequences obtained from the purified rat enzyme. Expression of this cDNA in bacteria, followed by immunoblotting with antibody directed against the ATP citrate-lyase, further demonstrated the identity of this clone. Nucleic acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1100 amino acids in length with a calculated molecular weight of 121,293. RNA blot analysis indicated an mRNA species of about 4.3 kilobase pairs in livers of chow-fed rats. Rats maintained on low fat, high carbohydrate diets exhibited a striking increase (50-fold) in the level of liver ATP citrate-lyase mRNA as compared with the control animals maintained on a normal diet. The tissue distribution of this mRNA in chow-fed animals revealed a relatively high abundance of the message in liver and adrenal, moderate levels were found in lung, brain, and large intestine with only trace amounts of the message in small intestine, stomach, testis, spleen, pancreas, kidney, and heart. During rat development, the ATP citrate-lyase mRNA was relatively high in the liver at parturition, followed by a reduction in its level during suckling. Higher amounts of the mRNA were detected again in adult animals. The isolation and characterization of the mRNA for ATP citrate-lyase will allow further studies on the reaction mechanism and metabolic regulation of this key enzyme in lipogenesis and cholesterogenesis.     

Mouse liver NAD(P)H:quinone acceptor oxidoreductase: protein sequence analysis by tandem mass spectrometry, cDNA cloning, expression in Escherichia coli, and enzyme activity analysis.

The amino acid sequence of mouse liver NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) has been determined by tandem mass spectrometry and deduced from the nucleotide sequence of the cDNA encoding for the enzyme. The electrospray mass spectral analyses revealed, as previously reported (Prochaska HJ, Talalay P, 1986, J Biol Chem 261:1372-1378), that the 2 forms--the hydrophilic and hydrophobic forms--of the mouse liver quinone reductase have the same molecular weight. No amino acid sequence differences were found by tandem mass spectral analyses of tryptic peptides of the 2 forms. Moreover, the amino-termini of the mouse enzymes are acetylated as determined by tandem mass spectrometry. Further, only 1 cDNA species encoding for the quinone reductase was found. These results suggest that the 2 forms of the mouse quinone reductase have the same primary sequences, and that any difference between the 2 forms may be attributed to a labile posttranslational modification. Analysis of the mouse quinone reductase cDNA revealed that the enzyme is 273 amino acids long and has a sequence homologous to those of rat and human quinone reductases. In this study, the mouse quinone reductase cDNA was also ligated into a prokaryotic expression plasmid pKK233.2, and the constructed plasmid was used to transform Escherichia coli strain JM109. The E. coli-expressed mouse quinone reductase was purified and characterized. Although mouse quinone reductase has an amino acid sequence similar to those of the rat and human enzymes, the mouse enzyme has a higher NAD(P)H-menadione reductase activity and is less sensitive to flavones and dicoumarol, 2 known inhibitors of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification, characterization, and ontogeny of acetyl-CoA carboxylase isozyme of chick embryo brain.

Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of fatty acids. Because fatty acids are required during myelination in the developing brain, it was proposed that the level of acetyl-CoA carboxylase may be highest in embryonic brain. The presence of acetyl-CoA carboxylase activity was detected in chick embryo brain. Its activity varied with age, showing a peak in the 17-18-day-old embryo and decreasing thereafter. The enzyme, affinity-purified from 18-day-old chick embryo brain, appeared as a major protein band on polyacrylamide electrophoresis gels in the presence of sodium dodecyl sulfate (Mr 265,000), indistinguishable from the 265 kDa isozyme of liver acetyl-CoA carboxylase. It had significant activity (Sp act = 1.1 mumol/min per mg protein) in the absence of citrate. There was a maximum stimulation of only 25% in the presence of citrate. Dephosphorylation using [acetyl-CoA carboxylase] phosphatase 2 did not result in activation of the enzyme. Palmitoyl-CoA (0.1 mM) and malonyl-CoA (1 mM) inhibited the activity to 95% and 71%, respectively. Palmitoylcarnitine, however, did not show significant inhibition. The enzyme was inhibited (greater than 95%) by avidin; however, avidin did not show significant inhibition in the presence of excess biotin. The enzyme was also inhibited (greater than 90%) by antibodies against liver acetyl-CoA carboxylase. An immunoblot or avidin-blot detected only one protein band (Mr 265,000) in preparations from chick embryo brain or adult liver. These observations suggest that acetyl-CoA carboxylase is present in embryonic brain and that the enzyme appears to be similar to the 265 kDa isozyme of liver.     

Molecular cloning of cDNA for rat mitochondrial 3-oxoacyl-CoA thiolase

Messenger RNA of rat 3-oxoacyl-CoA thiolase (acetyl-CoA acyltransferase), a mitochondrial matrix enzyme involved in fatty acid beta-oxidation, was enriched by immunoprecipitation of rat liver free polysomes and recombinant plasmids were prepared from the enriched mRNA by a modification of the vector-primer method of Okayama and Berg. The transformants were initially screened for 3-oxoacyl-CoA thiolase cDNA sequences by differential colony hybridization with [32P]cDNAs, synthesized from the immunopurified and unpurified mRNAs. The cDNA clones for 3-oxoacyl-CoA thiolase were identified by hybrid-arrested translation and hybrid-selected translation. One of the clones, designated pT1-1, contained a 700-base insert and hybridized to a mRNA species of 1.6 X 10(3) bases in rat liver. The transformants were rescreened using the cDNA insert of pT1-1 as a hybridization probe and a clone (pT1-19) with a 1.5 X 10(3)-base insert was obtained. Activity and concentration of 3-oxoacyl-CoA thiolase mRNA were quantified by in vitro translation and dot-blot analysis using the cDNA insert as a hybridization probe. The level of translatable and hybridizable mRNA in rat liver was increased about 5.1-fold and 4.6-fold, respectively, after administration of di-(2-ethylhexyl)phthalate, a potent inducer of the enzyme. The 3-oxoacyl-CoA thiolase mRNA levels thus determined correlated closely with levels of the activity and amount of this enzyme.     

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.     

Human brown fat inducible thioesterase variant 2 cellular localization and catalytic function

The mammalian brown fat inducible thioesterase variant 2 (BFIT2), also known as ACOT11, is a multimodular protein containing two consecutive hotdog-fold domains and a C-terminal steroidogenic acute regulatory protein-related lipid transfer domain (StarD14). In this study, we demonstrate that the N-terminal region of human BFIT2 (hBFIT2) constitutes a mitochondrial location signal sequence, which undergoes mitochondrion-dependent posttranslational cleavage. The mature hBFIT2 is shown to be located in the mitochondrial matrix, whereas the paralog "cytoplasmic acetyl-CoA hydrolase" (CACH, also known as ACOT12) was found in the cytoplasm. In vitro activity analysis of full-length hBFIT2 isolated from stably transfected HEK293 cells demonstrates selective thioesterase activity directed toward long chain fatty acyl-CoA thioesters, thus distinguishing the catalytic function of BFIT2 from that of CACH. The results from a protein-lipid overlay test indicate that the hBFIT2 StarD14 domain binds phosphatidylinositol 4-phosphate.     

The purification and properties of a protein kinase and the partial purification of a phosphoprotein phosphatase that inactivate and activate acetyl-CoA carboxylase

the occurrence of a soluble fraction from rat liver that inactivates acetyl-CoA carboxylase was previously reported by this laboratory (1). The purification of this fraction is now reported, and we show that it behaves as a cAMP-independent kinase that inactivates acetyl-CoA carboxylase by phosphorylation. The kinase has a molecular weight of 160,000 and it requires ATP and Mg²⁺ for activity. A partial purification from rat liver cytosol of a Mg²⁺-requiring phosphoprotein phosphatase of high molecular weight (greater than 200,000) which dephosphorylates phosphorylated acetyl-CoA carboxylase with the regeneration of enzyme activity is also reported. The kinase, phosphatase, and acetyl-CoA carboxylase are separable from each other by a combination of ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration.     

Mouse uroporphyrinogen decarboxylase: CDNA cloning,expression, and mapping

Uroporphyrinogen decarboxylase (URO-decarboxylase; EC 4.1.1.37), the heme biosynthetic enzyme responsible for the conversion of uroporphyrinogen III to coproporphyrinogen III, is the enzymatic defect in porphyria cutanea tarda, the most common porphyria. The mouse URO-decarboxylase cDNA was isolated from a mouse adult liver cDNA library. The longest clone of 1.5 kb, designated pmUROD-1, had 5′ and 3′ untranslated sequences of 281 and 97 bp, respectively, and an open reading frame of 1104 bp encoding a 367-amino acid polypeptide with a predicted molecular mass of 40,595 Da. The mouse and human coding sequences had 87.8% and 90.0% nucleotide and amino acid identity, respectively. The authenticity of the mouse cDNA was established by expression of the active enzyme in Escherichia coli. In addition, the analysis of two sets of multilocus genetic crosses localized the mouse gene, Urod, on Chromosome (Chr) 4, consistent with the map location of the human gene to a position of conserved synteny on Chr 1. The availability of the mouse URO-decarboxylase should facilitate studies of the structure and organization of the mouse genomic sequence and the development of a mouse model of this inherited porphyria. Received: 27 November 1995 / Accepted: 17 January 1996     

Ethylmalonyl-CoA decarboxylase, a new enzyme involved in metabolite proofreading

A limited number of enzymes are known that play a role analogous to DNA proofreading by eliminating non-classical metabolites formed by side activities of enzymes of intermediary metabolism. Because few such "metabolite proofreading enzymes" are known, our purpose was to search for an enzyme able to degrade ethylmalonyl-CoA, a potentially toxic metabolite formed at a low rate from butyryl-CoA by acetyl-CoA carboxylase and propionyl-CoA carboxylase, two major enzymes of lipid metabolism. We show that mammalian tissues contain a previously unknown enzyme that decarboxylates ethylmalonyl-CoA and, at lower rates, methylmalonyl-CoA but that does not act on malonyl-CoA. Ethylmalonyl-CoA decarboxylase is particularly abundant in brown adipose tissue, liver, and kidney in mice, and is essentially cytosolic. Because Escherichia coli methylmalonyl-CoA decarboxylase belongs to the family of enoyl-CoA hydratase (ECH), we searched mammalian databases for proteins of uncharacterized function belonging to the ECH family. Combining this database search approach with sequencing data obtained on a partially purified enzyme preparation, we identified ethylmalonyl-CoA decarboxylase as ECHDC1. We confirmed this identification by showing that recombinant mouse ECHDC1 has a substantial ethylmalonyl-CoA decarboxylase activity and a lower methylmalonyl-CoA decarboxylase activity but no malonyl-CoA decarboxylase or enoyl-CoA hydratase activity. Furthermore, ECHDC1-specific siRNAs decreased the ethylmalonyl-CoA decarboxylase activity in human cells and increased the formation of ethylmalonate, most particularly in cells incubated with butyrate. These findings indicate that ethylmalonyl-CoA decarboxylase may correct a side activity of acetyl-CoA carboxylase and suggest that its mutation may be involved in the development of certain forms of ethylmalonic aciduria.     

Diurnal variations of lipogenic enzymes, their substrate and effector levels, and lipogenesis from tritiated water in rat liver

The activities of glucose-6-phosphate dehydrogenase, malic enzyme, fatty acid synthetase and acetyl-CoA carboxylase (extracted with or without phosphatase inhibitor) in rat liver did not vary significantly during 24 h. The hepatic levels of glucose 6-phosphate and malate increased coordinately 3-6 h after the beginning (1900 h) of food intake and were high until morning, whereas the levels of acetyl-CoA and citrate peaked at 1900 h and then decreased. However, it is remarkable that the in vivo incorporation of 3H from tritiated water into fatty acids in liver increased with the level of malonyl-CoA after food intake. Comparing the substrate and effector levels with the Km and Ka values for the enzymes, the levels of acetyl-CoA, malonyl-CoA and citrate appear to limit the enzyme activities. It is suggested that, after food intake, the physiological activity of acetyl-CoA carboxylase was increased with the substrate increase and/or with the catalytic activation with citrate, and consequently, the fatty acid synthetase activity was also increased, whereas the enzyme activities measured under optimum conditions were not.     

Enzymatically inactive forms of acetyl-CoA carboxylase in rat liver mitochondria.

Biotinyl proteins were labelled by incubation of SDS-denatured preparations of subcellular fractions of rat liver with [14C]methylavidin before polyacrylamide-gel electrophoresis. Fluorographic analysis showed that mitochondria contained two forms of acetyl-CoA carboxylase [acetyl-CoA:carbon dioxide ligase (ADP-forming) EC 6.4.1.2], both of which were precipitated by antibody to the enzyme. When both forms were considered, almost three-quarters of the total liver acetyl-CoA carboxylase was found in the mitochondrial fraction of liver from fed rats while only 3.5% was associated with the microsomal fraction. The remainder was present in cytosol, either as the intact active enzyme or as a degradation product. The actual specific activity of the cytosolic enzyme was approx. 2 units/mg of acetyl-CoA carboxylase protein while that of the mitochondrial enzyme was about 20-fold lower, indicating that mitochondrial acetyl-CoA carboxylase was relatively inactive. Fractionation of mitochondria with digitonin showed that acetyl-CoA carboxylase was associated with the outer mitochondrial membrane. The available evidence suggests that mitochondrial acetyl-CoA carboxylase represents a reservoir of enzyme which can be released and activated under lipogenic conditions.     

Molecular cloning, expression and tissue distribution of hamster diacetyl reductase. Identity with L-xylulose reductase

Using rapid amplification of cDNA ends PCR, a cDNA species for diacetyl reductase (EC 1.1.1.5) was isolated from hamster liver. The encoded protein consisted of 244 amino acids, and showed high sequence identity to mouse lung carbonyl reductase and hamster sperm P26h protein, which belong to the short-chain dehydrogenase/reductase family. The enzyme efficiently reduced L-xylulose as well as diacetyl, and slowly oxidized xylitol. The K(m) values for L-xylulose and xylitol were similar to those reported for L-xylulose reductase (EC 1.1.1.10) of guinea pig liver. The identity of diacetyl reductase with L-xylulose reductase was demonstrated by co-purification of the two enzyme activities from hamster liver and their proportional distribution in other tissues.     

Molecular cloning of cDNA for acetyl-coenzyme A carboxylase

Poly(A)+ RNA from lactating rat mammary glands was size-fractionated to enrich the relative amount of acetyl-CoA carboxylase mRNA. The enriched mRNA was used to generate a lambda gt11 cDNA library. Initial screening with polyclonal antiserum to acetyl-CoA carboxylase produced three positive clones. Western blot analysis revealed that two clones, lambda DH3 and lambda KH18, synthesized 165,000-dalton proteins that were recognized by antibodies to acetyl-CoA carboxylase and beta-galactosidase, indicating that acetyl-CoA carboxylase/beta-galactosidase fusion proteins were produced. Competition experiments with purified acetyl-CoA carboxylase further demonstrated that the fusion proteins contained acetyl-CoA carboxylase protein segments. Antibodies which are specific to the fusion proteins were isolated. These antibodies cross-reacted only with acetyl-CoA carboxylase in a preparation of partially purified acetyl-CoA carboxylase. In addition, the antibodies immunoprecipitated enzyme activity from a crude liver homogenate. Northern blot analysis of total RNA revealed two RNA species: one 10 kilobases and the other 3.0 kilobases. The levels of these RNA species increased when starved animals were fed a fat-free diet, indicating that they are coordinately regulated.     

Mechanism of glucagon inhibition of liver acetyl-CoA carboxylase. Interrelationship of the effects of phosphorylation, polymer-protomer transition, and citrate on enzyme activity

The short-term regulation of rat liver acetyl-CoA carboxylase by glucagon has been studied in hepatocytes from rats that had been fasted and refed a fat-free diet. Glucagon inhibition of the activity of this enzyme can be accounted for by a direct correlation between phosphorylation, polymer-protomer ratio, and activity. Glucagon rapidly inactivates acetyl-CoA carboxylase with an accompanying 4-fold increase in the phosphorylation of the enzyme and 3-fold increase in the protomer-polymer ratio of enzyme protein. Citrate, an allosteric activator of acetyl-CoA carboxylase required for enzyme activity, has no effect on these phenomena, indicating a mechanism that is independent of citrate concentration within the cell. The observation of these effects of glucagon on acetyl-CoA carboxylase activity is absolutely dependent upon the minimization of proteolytic degradation of the enzyme after cell lysis. Therefore, for the first time, an interrelationship has been demonstrated between phosphorylation, protomer-polymer ratio, and citrate for the inactivation of acetyl-CoA carboxylase by glucagon.     

Formation of malonyl coenzyme A in rat heart. Identification and purification of an isozyme of A carboxylase from rat heart

Acetyl-CoA carboxylase is thought to be absent in the heart since the latter is highly catabolic and nonlipogenic. It has been suggested that the high level of malonyl-CoA that is found in the heart is derived from mitochondrial propionyl-CoA carboxylase, which also uses acetyl-CoA. In the present study, acetyl-CoA carboxylase was identified and purified from homogenates of rat heart. The isolated enzyme had little activity in the absence of citrate (specific activity, less than 0.1 units/mg); however, citrate stimulated its activity (specific activity, 1.8 units/mg in the presence of 10 mM citrate). Avidin inhibited greater than 95% of activity, and addition of biotin reversed this inhibition. Further, malonyl-CoA (1 mM) and palmitoyl-CoA (100 microM) inhibited greater than 90% of carboxylase activity. Similar to acetyl-CoA carboxylase of lipogenic tissues, the heart enzyme could be activated greater than 6-fold by preincubation with liver (acetyl-CoA carboxylase)-phosphatase 2. The activation was accompanied by a decrease in the K0.5 for citrate to 0.68 mM. These observations suggest that the activity in preparations from heart is due to authentic acetyl-CoA carboxylase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparation from heart showed the presence of one major protein band (Mr 280,000) and a minor band (Mr 265,000) while that from liver gave a major protein band (Mr 265,000). A Western blot probed with avidin-peroxidase suggested that both the 280- and 265-kDa species contained biotin. Antibodies to liver acetyl-CoA carboxylase, which inhibited greater than 95% of liver carboxylase activity, inhibited only 35% of heart enzyme activity. In an immunoblot (using antibodies to liver enzyme) the 265-kDa species, and not the major 280-kDa species, in the heart preparation was specifically stained. These observations suggest the presence of two isoenzymes of acetyl-CoA carboxylase that are immunologically distinct, the 265-kDa species being predominant in the liver and the 280-kDa species being predominant in the heart.