Inhibition of peroxisomal fatty acyl-CoA oxidase by antimycin A.

Peroxisomal fatty acyl-CoA oxidase was inhibited by micromolar concentrations of antimycin A, an inhibitor of mitochondrial respiration. The inhibition was observed with all three substrates tested, i.e. palmitoyl-CoA, trihydroxycoprostanoyl-CoA and hexadecanedioyl-CoA. The peroxisomal D-amino acid oxidase was also inhibited by antimycin, but the peroxisomal L-alpha-hydroxyacid oxidase and uric acid oxidase and the mitochondrial monoamine oxidase were not. The degree of inhibition of acyl-CoA oxidase by antimycin was strongly dependent on the amount of cellular protein present in the assay mixture: at a fixed antimycin concentration, the inhibition was gradually lost with increasing protein concentrations. At a fixed cellular protein concentration in the assay mixtures, the mitochondrial oxidation of glutamate or palmitoylcarnitine was inhibited at antimycin concentrations that were much lower than those required for the inhibition of fatty acyl-CoA oxidase. Our results, nevertheless, demonstrate that antimycin A must be used with caution, when it is added to homogenates or subcellular fractions in order to distinguish between mitochondrial and peroxisomal fatty acid oxidation.     

Fluorometric assay for rat liver peroxisomal fatty acyl-coenzyme A oxidase activity

These studies report the development of a simple, specific, and highly sensitive fluorometric assay for rat liver peroxisomal fatty acyl-CoA oxidase activity. In this in vitro procedure fatty acyl-CoA-dependent H2O2 production was coupled in a peroxidase-catalyzed reaction to the oxidation of scopoletin (6-methoxy-7-hydroxycoumarin), a highly fluorescent compound, to a nonfluorescent product. Enzyme-catalyzed reaction rates as low as 5 pmol of H2O2 produced per minute could readily be detected. The reaction was studied in liver homogenates from normal rats with respect to absolute activity, time course, protein concentration dependence, substrate concentration dependence, pH optimum, substrate specificity, and cofactor requirements. The properties of the enzyme activity as assessed by the fluorometric assay agree well with those determined by other investigators using other assay methods. After subcellular fractionation of liver homogenates by differential centrifugation, the fatty acyl-CoA oxidase activity distributed like known peroxisomal marker enzymes. These results demonstrate that the fluorometric assay of fatty acyl-CoA oxidase should be useful in studying the distribution, properties, and subcellular localization of the enzyme, particularly in enzyme sources of low activity or in situations when only small amounts of material are available.     

A sensitive spectrophotometric assay for peroxisomal acyl-CoA oxidase.

A simple spectrophotometric assay was developed for peroxisomal fatty acyl-CoA oxidase activity. The assay, based on the H2O2-dependent oxidation of leuco-dichlorofluorescein catalysed by exogenous peroxidase, is more sensitive than methods previously described. By using mouse liver samples, cofactor requirements were assessed and a linear relationship was demonstrated between dye oxidation and enzyme concentration. By using this assay on subcellular fractions, palmitoyl-CoA oxidase activity was localized for the first time in microperoxisomes of rat intestine. The assay was also adapted to measure D-amino acid oxidase activity, demonstrating the versatility of this method for measuring activity of other H2O2-producing oxidases.     

Fluorometric assay of peroxisomal oxidases

The present paper deals with the adaptation of the fluorometric measurement of H2O2 originally described by Guilbault et al. (1967, Anal. Chem. 39, 271) for the assay of the peroxisomal oxidation of D-amino acids, L-alpha-hydroxyacids, uric acid, and acyl-CoA esters. The present work essentially covers three facets: (i) the general kinetics of the assay of peroxisomal oxidases and the influence of each component of the assay medium on these kinetics; (ii) the measurement of peroxisomal oxidase activities in subcellular fractions and tissues from human and untreated and clofibrate-treated rodents; and (iii) the comparison between the oxidase activities measured by the fluorometric and spectrophotometric methods.     

Evidence that peroxisomal acyl-CoA synthetase is located at the cytoplasmic side of the peroxisomal membrane.

1. Subfractionation by isopycnic density-gradient centrifugation in self-generating Percoll gradients of peroxisome-rich fractions prepared by differential centrifugation confirmed the presence of acyl-CoA synthetase in peroxisomes. Peroxisomes did not contain nicotinamide or adenine nucleotides other than CoA. 2. The gradient fractions most enriched in peroxisomes were pooled and the peroxisomes sedimented by centrifugation, resulting in a 50-fold-purified peroxisomal preparation as revealed by marker enzyme analysis. 3. Palmitate oxidation by intact purified peroxisomes was CoA-dependent, whereas palmitoyl-CoA oxidation was not, demonstrating that the peroxisomal CoA was available for the thiolase reaction, located in the peroxisomal matrix, but not for acyl-CoA synthetase. This suggests that the latter enzyme is located at the cytoplasmic side of the peroxisomal membrane. 4. Additional evidence for this location of peroxisomal acyl-CoA synthetase was as follows. Mechanical disruption of purified peroxisomes resulted in the release of catalase from the broken organelles, but not of acyl-CoA synthetase, indicating that the enzyme was membrane-bound. Acyl-CoA synthetase was not latent, despite the fact that at least one of its substrates appears to have a limited membrane permeability, as evidenced by the presence of CoA in purified peroxisomes. Finally, Pronase, a proteinase that does not penetrate the peroxisomal membrane, almost completely inactivated the acyl-CoA synthetase of intact peroxisomes.     

Optimal detection of cholinesterase activity in biological samples: Modifications to the standard Ellman’s assay

Ellman’s assay is the most commonly used method to measure cholinesterase activity. It is cheap, fast, and reliable, but it has limitations when used for biological samples. The problems arise from 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), which is unstable, interacts with free sulfhydryl groups in the sample, and may affect cholinesterase activity. We report that DTNB is more stable in 0.09 M Hepes with 0.05 M sodium phosphate buffer than in 0.1 M sodium phosphate buffer, thereby notably reducing background. Using enzyme-linked immunosorbent assay (ELISA) to enrich tissue homogenates for cholinesterase while depleting the sample of sulfhydryl groups eliminates unwanted interactions with DTNB, making it possible to measure low cholinesterase activity in biological samples. To eliminate possible interference of DTNB with enzyme hydrolysis, we introduce a modification of the standard Ellman’s assay. First, thioesters are hydrolyzed by cholinesterase to produce thiocholine in the absence of DTNB. Then, the reaction is stopped by a cholinesterase inhibitor and the produced thiocholine is revealed by DTNB and quantified at 412 nm. Indeed, this modification of Ellman’s method increases butyrylcholinesterase activity by 20 to 25%. Moreover, high stability of thiocholine enables separation of the two reactions of the Ellman’s method into two successive steps that may be convenient for some applications.     

Determination of peroxisomal fatty acyl-CoA oxidase activity using a lauroyl-CoA-based fluorometric assay

A simple, sensitive fluorometric method for the determination of peroxisomal fatty acyl-CoA oxidase (EC 1.3.99.3) activity has been developed. Studies of enzyme activity relative to subcellular distribution and to clofibrate induction indicate that this assay is specific for peroxisomal fatty acyl-CoA oxidase. The lauroyl-CoA-dependent production of H2O2 is quantitated by measuring the oxidation of 4-hydroxyphenyl-acetic acid to a fluorescent product in a horseradish peroxidase-coupled assay. Assays can be performed in either a fixed time or continuous mode. In either mode, H2O2 production is related to a change in fluorescence intensity through use of a standard curve generated with known amounts of H2O2. The use of lauroyl-CoA (12:0), rather than the more generally used substrate palmitoyl-CoA (16:0), provides significant advantages. Much of the substrate inhibition problem associated with palmitoyl-CoA has been avoided, and a greater than 4.5-fold higher specific activity has been achieved compared with a palmitoyl-CoA-based assay. In the fixed-time mode, linearity relative to time and to the amount of enzyme added has been established without resorting to the use of bovine serum albumin as a substrate binding medium. Sensitivity is estimated to be at least equal to that of the most sensitive methods reported, while reliability, versatility and range have been improved. Use of this method should greatly facilitate the study of peroxisomal beta-oxidation regulatory mechanisms in hepatocyte cell culture systems as well as in other circumstances where low activities or small samples must be assayed.     

Expression and purification of his-tagged rat peroxisomal acyl-CoA oxidase I wild-type and E421 mutant proteins

Rat peroxisomal acyl-CoA oxidase I is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patients has been previously reported. We cloned the gene of rat peroxisomal acyl-CoA oxidase I into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5' end of the gene. The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel HiTrap chelating metal-affinity column in 90% yield to apparent homogeneity. The specific activity of the purified His-tagged rat peroxisomal acyl-CoA oxidase I was 1.5 micromol/min/mg. It has been proposed that Glu421 is a catalytic residue responsible for deprotonation of alpha-proton of acyl-CoA substrate. We constructed four mutant expression plasmids of the enzyme, pACO(E421D), pACO(E421A), pACO(E421Q), and pACO(E421G) using site-directed mutagenesis. Mutant proteins were overexpressed in E. coli and purified with a nickel metal-affinity column. Kinetic studies of wild-type and mutant proteins were carried out, and the result confirmed that Glu421 is a catalytic residue of rat peroxisomal acyl-CoA oxidase I. Our overexpression in E. coli and one-step purification of the highly active N-terminal His-tagged rat peroxisomal acyl-CoA oxidase I greatly facilitated our further investigation of this enzyme, and our result from site-directed mutagenesis increased our understanding of the mechanism for the reaction catalyzed by peroxisomal acyl-CoA oxidase I.     

Intrinsic enoyl-CoA isomerase activity of rat acyl-CoA oxidase I

Rat peroxisomal acyl-CoA oxidase I is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patient has been previously reported. It was found that rat acyl-CoA oxidase I has intrinsic enoyl-CoA isomerase activity, which was confirmed using incubation followed with HPLC analysis in this study. Various 3-enoyl-CoA substrates with cis or trans configuration were synthesized and used in the study of enzyme substrate specificity. The isomerase activity of the enzyme was characterized through studies of kinetics, pH dependence, and enzyme inhibition. Most k(cat)/K(M) values of rat peroxisomal acyl-CoA oxidase I for isomerization reaction are comparable with those of authentic rat liver peroxisomal Delta(3)-Delta(2)-enoyl-CoA isomerase and rat liver peroxisomal multifunctional enzyme 1 when hexenoyl-CoA and octenoyl-CoA with cis- or trans-configuration were used as substrate. Glu421 was found to be the catalytic residue for both oxidase and isomerase activities of the enzyme. The isomerase activity of rat peroxisomal acyl-CoA oxidase I is probably due to a spontaneous process driven by thermodynamic equilibrium with formation of a conjugated structure after deprotonation of substrate alpha-proton. The energy level of transition state may be lowered by a stable dienolate intermediate, which gain further stabilization via charge transfer with electron-deficient FAD cofactor of the enzyme.     

Acyl-CoA oxidase 1 from Arabidopsis thaliana. Structure of a key enzyme in plant lipid metabolism

The peroxisomal acyl-CoA oxidase family plays an essential role in lipid metabolism by catalyzing the conversion of acyl-CoA into trans-2-enoyl-CoA during fatty acid beta-oxidation. Here, we report the X-ray structure of the FAD-containing Arabidopsis thaliana acyl-CoA oxidase 1 (ACX1), the first three-dimensional structure of a plant acyl-CoA oxidase. Like other acyl-CoA oxidases, the enzyme is a dimer and it has a fold resembling that of mammalian acyl-CoA oxidase. A comparative analysis including mammalian acyl-CoA oxidase and the related tetrameric mitochondrial acyl-CoA dehydrogenases reveals a substrate-binding architecture that explains the observed preference for long-chained, mono-unsaturated substrates in ACX1. Two anions are found at the ACX1 dimer interface and for the first time the presence of a disulfide bridge in a peroxisomal protein has been observed. The functional differences between the peroxisomal acyl-CoA oxidases and the mitochondrial acyl-CoA dehydrogenases are attributed to structural differences in the FAD environments.     

Peroxisomal beta-oxidation: insights from comparative biochemistry

The activities of fatty acyl-CoA oxidase (FAO) and carnitine palmitoyl transferase (CPT), indices of the capacities of peroxisomal beta-oxidation and mitochondrial beta-oxidation, respectively, were determined in livers of several vertebrate species notable for differences in dietary fatty acid composition. In suckling rats FAO activities were half that in adult rats and CPT/FAO ratios twice that of adult rats. As their milk diet is dominated by medium chain fatty acids, this observation is consistent with current ideas about the role of peroxisomal beta-oxidation in rat liver in oxidation of long chain unsaturated fatty acids. In nectar-feeding hummingbirds (fatty acids synthesized de novo) FAO activities were 50% greater than adult rats and CPT/FAO ratios one-third less than adult rats, suggesting that peroxisomal beta-oxidation is relatively more important in this species, despite a fatty-acid-poor diet. In marine fish (herring, dogfish shark, hagfish) FAO activities were all less than 15% that of rats and undetectable in hagfish. CPT/FAO ratios were greater in herring (8.1) and hagfish (greater than 30) than adult rats (3.1), suggesting that peroxisomal beta-oxidation is relatively less important in these species despite a natural diet containing high levels of long chain polyunsaturated fatty acids. These data are discussed in relation to current ideas about the role of peroxisomes in beta-oxidation of fatty acids.     

cDNA cloning and analysis of tissue-specific expression of mouse peroxisomal straight-chain acyl-CoA oxidase.

Straight-chain acyl-CoA oxidase is the first and rate limiting enzyme in the peroxisomal beta-oxidation pathway catalysing the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs, thereby producing H2O2. To study peroxisomal beta-oxidation we cloned and characterized the cDNA of mouse peroxisomal acyl-CoA oxidase. It consists of 3778 bp, including a 1983-bp ORF encoding a polypeptide of 661 amino-acid residues. Like the rat and human homologue the C-terminus contains an SKL motif, an import signal present in several peroxisomal matrix proteins. Sequence analysis revealed high amino-acid homology with rat (96%) and human (87%) acyl-CoA oxidase in addition to minor homology ( approximately 40%) with other related proteins, such as rabbit trihydroxy-cholestanoyl-CoA oxidase, human branched chain acyl-CoA oxidase and rat trihydroxycoprostanoyl-CoA oxidase. Acyl-CoA oxidase mRNA and protein expression were most abundant in liver followed by kidney, brain and adipose tissue. During mouse brain development acyl-CoA oxidase mRNA expression was highest during the suckling period indicating that peroxisomal beta-oxidation is most critical during this developmental stage. Comparing tissue mRNA levels of peroxisome proliferator-activated receptor alpha and acyl-CoA oxidase, we noticed a constant relationship in all tissues investigated, except heart and adipose tissue in which much more, and respectively, much less, peroxisome proliferator-activated receptor alpha mRNA in proportion to acyl-CoA oxidase mRNA was found. Our data show that acyl-CoA oxidase is an evolutionary highly conserved enzyme with a distinct pattern of expression and indicate an important role in lipid metabolism.     

Enzymic and morphological studies on catalase positive particles from brown fat of cold adapted rats.

Brown adipose tissue of normal and cold-adapted adult rats has been investigated morphologically and cytochemically. In thin-sections catalase-positive particles appear as circular, oval or elongated profiles lying either as single particles or forming groups. Biochemical studies on peroxisomal enzymes show an increase of catalase activity to the tenfold amount after cold adaptation. The tissue is devoid of D-aminoacid oxidase and glycolate oxidase, while low activities of middle-chain-alpha-hydroxyacid oxidases could be detected. The catalase-positive particles were purified by differential and is lower than that of the liver peroxisomes. Enzymic investigations of the fractions render it probably that particles contain carnitine acetyltransferase, whereas they are lacking NAD-dependent glycerophosphate dehydrogenase. The pellets derived from the gradient centrifugation have been checked morphologically for purity. After performing DAB-cytochemistry for identification of the peroxidatic activity of catalase, most of the particles were shown to be structurally intact and homogeneously filled with reaction product.     

Peroxisomal fatty acyl-CoA oxidase is not regulated by triiodothyronine

In studies using primary cultures of adult rat hepatocytes in serum-free medium, peroxisomal fatty acyl-CoA oxidase activity was not altered by the presence of 3,5,3'-triiodothyronine, whereas time- and dose-dependent increases in the thyroid hormone-responsive enzyme mitochondrial glycero-3-phosphate dehydrogenase were seen. Activity of peroxisomal oxidase was stimulated with clofibric acid in the absence of 3,5,3'-triiodothyronine. The results demonstrate that hepatic peroxisomal fatty acyl-CoA oxidase activity is not directly regulated by 3,5,3'-triiodothyronine and that stimulation of peroxisomal fatty acyl-CoA oxidase activity by clofibric acid does not require thyroid hormone.     

Peroxisomal acyl-CoA synthetases

Peroxisomes carry out many essential lipid metabolic functions. Nearly all of these functions require that an acyl group-either a fatty acid or the acyl side chain of a steroid derivative-be thioesterified to coenzyme A (CoA) for subsequent reactions to proceed. This thioesterification, or "activation", reaction, catalyzed by enzymes belonging to the acyl-CoA synthetase family, is thus central to cellular lipid metabolism. However, despite our rather thorough understanding of peroxisomal metabolic pathways, surprisingly little is known about the specific peroxisomal acyl-CoA synthetases that participate in these pathways. Of the 26 acyl-CoA synthetases encoded by the human and mouse genomes, only a few have been reported to be peroxisomal, including ACSL4, SLC27A2, and SLC27A4. In this review, we briefly describe the primary peroxisomal lipid metabolic pathways in which fatty acyl-CoAs participate. Then, we examine the evidence for presence and functions of acyl-CoA synthetases in peroxisomes, much of which was obtained before the existence of multiple acyl-CoA synthetase isoenzymes was known. Finally, we discuss the role(s) of peroxisome-specific acyl-CoA synthetase isoforms in lipid metabolism. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.     

Acyl-CoA synthetase and the peroxisomal enzymes of beta-oxidation in human liver. Quantitative analysis of their subcellular localization.

The presence of acyl-CoA synthetase (EC 6.2.1.3) in peroxisomes and the subcellular distribution of beta-oxidation enzymes in human liver were investigated by using a single-step fractionation method of whole liver homogenates in metrizamide continuous density gradients and a novel procedure of computer analysis of results. Peroxisomes were found to contain 16% of the liver palmitoyl-CoA synthetase activity, and 21% and 60% of the enzyme activity was localized in mitochondria and microsomal fractions respectively. Fatty acyl-CoA oxidase was localized exclusively in peroxisomes, confirming previous results. Human liver peroxisomes were found to contribute 13%, 17% and 11% of the liver activities of crotonase, beta-hydroxyacyl-CoA dehydrogenase and thiolase respectively. The absolute activities found in peroxisomes for the enzymes investigated suggest that in human liver fatty acyl-CoA oxidase is the rate-limiting enzyme of the peroxisomal beta-oxidation pathway, when palmitic acid is the substrate.     

The soluble carnitine palmitoyltransferase from bovine liver. A comparison with the enzymes from peroxisomes and from the mitochondrial inner membrane.

The properties of two carnitine acyltransferases (CPT) purified from bovine liver are compared to confirm that they are different proteins. The soluble CPT and the inner CPT from mitochondria differ in subunit Mr, native Mr, pI and reactivity with thiol reagents. All eight free thiol groups in soluble CPT react with 5,5'-dithiobis-(2-nitrobenzoate) in the absence of any unfolding reagent, and activity is gradually lost. The inner CPT activity is completely stable in the presence of 5,5'-dithiobis-(2-nitrobenzoate), and only one thiol group per molecule of subunit is modified in the native enzyme. Antisera to each enzyme inhibit that enzyme, but do not cross-react. CPT activity in subcellular fractions can now be identified by titration with these antibodies. The soluble CPT from bovine liver is probably peroxisomal in origin, but, although antigenically similar, it differs from the peroxisomal carnitine octanoyltransferase found in rat and mouse liver in its specificity for the longer-chain acyl-CoA substrates.     

Chlorpromazine and carnitine-dependency of rat liver peroxisomal beta-oxidation of long-chain fatty acids.

The enzyme targets for chlorpromazine inhibition of rat liver peroxisomal and mitochondrial oxidations of fatty acids were studied. Effects of chlorpromazine on total fatty acyl-CoA synthetase activity, on both the first and the third steps of peroxisomal beta-oxidation, on the entry of fatty acyl-CoA esters into the peroxisome and on catalase activity, which allows breakdown of the H2O2 generated during the acyl-CoA oxidase step, were analysed. On all these metabolic processes, chlorpromazine was found to have no inhibitory action. Conversely, peroxisomal carnitine octanoyltransferase activity was depressed by 0.2-1 mM-chlorpromazine, which also inhibits mitochondrial carnitine palmitoyltransferase activity in all conditions in which these enzyme reactions are assayed. Different patterns of inhibition by the drug were, however, demonstrated for both these enzyme activities. Inhibitory effects of chlorpromazine on mitochondrial cytochrome c oxidase activity were also described. Inhibitions of both cytochrome c oxidase and carnitine palmitoyltransferase are proposed to explain the decreased mitochondrial fatty acid oxidation with 0.4-1.0 mM-chlorpromazine reported by Leighton, Persico & Necochea [(1984) Biochem. Biophys. Res. Commun. 120, 505-511], whereas depression by the drug of carnitine octanoyltransferase activity is presented as the factor responsible for the decreased peroxisomal beta-oxidizing activity described by the above workers.     

Fatty acyl-CoA oxidase in rat kidney and liver after application of thyroxine

Male rats were fed a standard diet containing 2.5 mg% L-thyroxine. After 10 and 20 days, in postnuclear fractions of the kidney the specific activity of fatty acyl-CoA oxidase, the enzyme responsible for the rate-limiting first step of peroxisomal fatty acid beta-oxidation, was increased by 100 and 160%, respectively. A similar effect was found in the liver. It is suggested that thyroxine essentially affects only this step of the fatty acid beta-oxidation sequence. Presumably the elevation of fatty acyl-CoA oxidase is one reason for the beneficial action of thyroid hormones in toxic lesions of the kidney.     

Detection of peroxisomal fatty acyl-coenzyme A oxidase activity.

It has been postulated that the peroxisomal fatty acid-oxidizing system [Lazarow & de Duve (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2043--2046; Lazarow (1978) J. Biol. Chem. 253, 1522--1528] resembles that of mitochondria, except for the first oxidative reaction. In this step, O2 would be directly reduced to H2O2 by an oxidase. Two specific procedures developed to detect the activity of the characteristic enzyme fatty acyl-CoA oxidase are presented, namely polarographic detection of palmitoyl-CoA-dependent cyanide-insensitive O2 consumption and palmitoyl-CoA-dependent H2O2 generation coupled to the peroxidation of methanol in an antimycin A-insensitive reaction. Fatty acyl-CoA oxidase activity is stimulated by FAD, which supports the flavoprotein nature postulated for this enzyme. Its activity increases 7-fold per g wet wt. of liver in rats treated with nafenopin, a hypolipidaemic drug. Subcellular fractionation of livers from normal and nafenopin-treated animals provides evidence for its peroxisomal localization. The stoicheiometry for palmitoyl-CoA-dependent O2 consumption, H2O2 generation and NAD+ reduction is 1 : 1 : 1. This suggests that fatty acyl-CoA oxidase is the rate-limiting enzyme of the peroxisomal fatty acid-oxidizing system.