Futile cycling of intermediates of fatty acid biosynthesis toward peroxisomal beta-oxidation in Saccharomyces cerevisiae

The flux of fatty acids toward beta-oxidation was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate synthesis in the peroxisome from the polymerization, by a bacterial polyhydroxyalkanoate synthase, of the beta-oxidation intermediates 3-hydroxyacyl-CoAs. Synthesis of polyhydroxyalkanoate was dependent on the beta-oxidation enzymes acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase multifunctional protein, which are involved in generating 3-hydroxyacyl-CoAs, and on the peroxin PEX5, which is involved in the import of proteins into the peroxisome. In wild type cells grown in media containing fatty acids, the polyhydroxyalkanoate monomer composition was largely influenced by the nature of the external fatty acid, such that even-chain monomers are generated from oleic acid and odd-chain monomers are generated from heptadecenoic acid. In contrast, polyhydroxyalkanoate containing predominantly 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate was synthesized in a mutant deficient in the peroxisomal 3-ketothiolase (fox3 Delta 0) growing either on oleic acid or heptadecenoic acid as well as in wild type and fox3 Delta 0 mutants grown on glucose or raffinose, indicating that 3-hydroxyacyl-CoAs used for polyhydroxyalkanoate synthesis were generated from the degradation of intracellular short- and medium-chain fatty acids by the beta-oxidation cycle. Inhibition of fatty acid biosynthesis with cerulenin blocked the synthesis of polyhydroxyalkanoate from intracellular fatty acids but still enabled the use of extracellular fatty acids for polymer production. Mutants affected in the synthesis of lipoic acid showed normal polyhydroxyalkanoate synthesis capacity. Together, these results uncovered the existence of a substantial futile cycle whereby short- and medium-chain intermediates of the cytoplasmic fatty acid biosynthetic pathway are directed toward the peroxisomal beta-oxidation pathway.     

Analysis of the beta-oxidation of trans-unsaturated fatty acid in recombinant Saccharomyces cerevisiae expressing a peroxisomal PHA synthase reveals the involvement of a reductase-dependent pathway

The degradation of fatty acids having cis- or trans-unsaturated bond at an even carbon was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanaote is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxy-acyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The synthesis of polyhydroxyalkanoate in cells grown in media containing 10-cis-heptadecenoic acid was dependent on the presence of 2,4-dienoyl-CoA reductase activity as well as on Delta3,Delta2-enoyl-CoA isomerase activity. The synthesis of polyhydroxyalkanoate from 10-trans-heptadecenoic acid in mutants devoid of 2,4-dienoyl-CoA reductase revealed degradation of the trans fatty acid directly via the enoyl-CoA hydratase II activity of the multifunctional enzyme (MFE), although the level of polyhydroxyalkanoate was 10-25% to that of wild type cells. Polyhydroxyalkanoate produced from 10-trans-heptadecenoic acid in wild type cells showed substantial carbon flux through both a reductase-dependent and a direct MFE-dependent pathway. Flux through beta-oxidation was more severely reduced in mutants devoid of Delta3,Delta2-enoyl-CoA isomerase compared to mutants devoid of 2,4-dienoyl-CoA reductase. It is concluded that the intermediate 2-trans,4-trans-dienoyl-CoA is metabolized in vivo in yeast by both the enoyl-CoA hydratase II activity of the multifunctional protein and the 2,4-dienoyl-CoA reductase, and that the synthesis of the intermediate 3-trans-enoyl-CoA in the absence of the Delta3,Delta2-enoyl-CoA isomerase leads to the blockage of the direct MFE-dependent pathway in vivo.     

Molecular identification and characterization of the Arabidopsis delta(3,5),delta(2,4)-dienoyl-coenzyme A isomerase, a peroxisomal enzyme participating in the beta-oxidation cycle of unsaturated fatty acids

Degradation of unsaturated fatty acids through the peroxisomal beta-oxidation pathway requires the participation of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle. The auxiliary enzyme delta(3,5),delta(2,4)-dienoyl-coenzyme A (CoA) isomerase has been well studied in yeast (Saccharomyces cerevisiae) and mammals, but no plant homolog had been identified and characterized at the biochemical or molecular level. A candidate gene (At5g43280) was identified in Arabidopsis (Arabidopsis thaliana) encoding a protein showing homology to the rat (Rattus norvegicus) delta(3,5),delta(2,4)-dienoyl-CoA isomerase, and possessing an enoyl-CoA hydratase/isomerase fingerprint as well as aspartic and glutamic residues shown to be important for catalytic activity of the mammalian enzyme. The protein, named AtDCI1, contains a peroxisome targeting sequence at the C terminus, and fusion of a fluorescent protein to AtDCI1 directed the chimeric protein to the peroxisome in onion (Allium cepa) cells. AtDCI1 expressed in Escherichia coli was shown to have delta(3,5),delta(2,4)-dienoyl-CoA isomerase activity in vitro. Furthermore, using the synthesis of polyhydroxyalkanoate in yeast peroxisomes as an analytical tool to study the beta-oxidation cycle, expression of AtDCI1 was shown to complement the yeast mutant deficient in the delta(3,5),delta(2,4)-dienoyl-CoA isomerase, thus showing that AtDCI1 is also appropriately targeted to the peroxisome in yeast and has delta(3,5),delta(2,4)-dienoyl-CoA isomerase activity in vivo. The AtDCI1 gene is expressed constitutively in several tissues, but expression is particularly induced during seed germination. Proteins showing high homology with AtDCI1 are found in gymnosperms as well as angiosperms belonging to the Monocotyledon or Dicotyledon classes.     

The membrane of peroxisomes in Saccharomyces cerevisiae is impermeable to NAD(H) and acetyl-CoA under in vivo conditions.

We investigated how NADH generated during peroxisomal beta-oxidation is reoxidized to NAD+ and how the end product of beta-oxidation, acetyl-CoA, is transported from peroxisomes to mitochondria in Saccharomyces cerevisiae. Disruption of the peroxisomal malate dehydrogenase 3 gene (MDH3) resulted in impaired beta-oxidation capacity as measured in intact cells, whereas beta-oxidation was perfectly normal in cell lysates. In addition, mdh3-disrupted cells were unable to grow on oleate whereas growth on other non-fermentable carbon sources was normal, suggesting that MDH3 is involved in the reoxidation of NADH generated during fatty acid beta-oxidation rather than functioning as part of the glyoxylate cycle. To study the transport of acetyl units from peroxisomes, we disrupted the peroxisomal citrate synthase gene (CIT2). The lack of phenotype of the cit2 mutant indicated the presence of an alternative pathway for transport of acetyl units, formed by the carnitine acetyltransferase protein (YCAT). Disruption of both the CIT2 and YCAT gene blocked the beta-oxidation in intact cells, but not in lysates. Our data strongly suggest that the peroxisomal membrane is impermeable to NAD(H) and acetyl-CoA in vivo, and predict the existence of metabolite carriers in the peroxisomal membrane to shuttle metabolites from peroxisomes to cytoplasm and vice versa.     

Pex11p plays a primary role in medium-chain fatty acid oxidation, a process that affects peroxisome number and size in Saccharomyces cerevisiae

The Saccharomyces cerevisiae peroxisomal membrane protein Pex11p has previously been implicated in peroxisome proliferation based on morphological observations of PEX11 mutant cells. Pex11p-deficient cells fail to increase peroxisome number in response to growth on fatty acids and instead accumulate a few giant peroxisomes. We report that mutants deficient in genes required for medium-chain fatty acid (MCFA) beta-oxidation display the same phenotype as Pex11p-deficient cells. Upon closer inspection, we found that Pex11p is required for MCFA beta-oxidation. Disruption of the PEX11 gene results in impaired formation of MCFA-CoA esters as measured in intact cells, whereas their formation is normal in cell lysates. The sole S. cerevisiae MCFA-CoA synthetase (Faa2p) remains properly localized to the inner leaflet of the peroxisomal membrane in PEX11 mutant cells. Therefore, the in vivo latency of MCFA activation observed in Pex11p-deficient cells suggests that Pex11p provides Faa2p with substrate. When PEX11 mutant cells are shifted from glucose to oleate-containing medium, we observed an immediate deficiency in beta-oxidation of MCFAs whereas giant peroxisomes and a failure to increase peroxisome abundance only became apparent much later. Our observations suggest that the MCFA oxidation pathway regulates the level of a signaling molecule that modulates the number of peroxisomal structures in a cell.     

Channeling of 3-hydroxy-4-trans-decenoyl coenzyme A on the bifunctional beta-oxidation enzyme from rat liver peroxisomes and on the large subunit of the fatty acid oxidation complex from Escherichia coli

Rates of the NAD+-dependent oxidation of 2-trans,4-trans-decadienoyl-CoA, a metabolite of trans-omega-6-unsaturated fatty acids, catalyzed by the mitochondrial enoyl-CoA hydratase plus 3-hydroxyacyl-CoA dehydrogenase and by the corresponding enzymes from peroxisomes, as well as Escherichia coli, were compared. The study of the mitochondrial system revealed that the conventional kinetic theory of coupled enzyme reactions cannot be applied to systems in which the primary reaction has a small equilibrium constant, and/or the concentration of coupling enzyme is higher than 0.01 Km for the intermediate and higher than the steady-state concentration of the intermediate. In contrast to the results obtained with the mitochondrial beta-oxidation system of unlinked enzymes, the steady-state velocities of 2-trans,4-trans-decadienoyl-CoA degradation catalyzed by either the peroxisomal bifunctional enzyme or by the E. coli fatty acid oxidation complex were found to be equal to the activities of enoyl-CoA hydratase even though the concentration of coupling enzyme was equal to that of the primary enzyme, and the quotient of Vmax/Km for the dehydration of 3-hydroxy-4-trans-decenoyl-CoA is much larger than the Vmax/Km for its dehydrogenation. The extraordinarily high efficiencies of these two multifunctional proteins in catalyzing the degradation of 2-trans,4-trans-decadienoyl-CoA is best explained by the direct transfer of the 3-hydroxy-4-trans-decenoyl-CoA intermediate from the active site of enoyl-CoA hydratase to that of 3-hydroxyacyl-CoA dehydrogenase. The discovery of an intermediate channeling mechanism on the peroxisomal bifunctional enzyme explains on the molecular level why the peroxisomal beta-oxidation system is well suited for the degradation of trans-fatty acids.     

Peroxisomal beta-oxidation enzyme proteins in the Zellweger syndrome

The absence of peroxisomes in patients with the cerebro-hepato-renal (Zellweger) syndrome is accompanied by a number of biochemical abnormalities, including an accumulation of very long-chain fatty acids. We show by immunoblotting that there is a marked deficiency in livers from patients with the Zellweger syndrome of the peroxisomal beta-oxidation enzyme proteins acyl-CoA oxidase, the bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase. Using anti-(acyl-CoA oxidase), increased amounts of cross-reactive material of low Mr were seen in the patients. With anti-(oxoacyl-CoA thiolase), high Mr cross-reactive material, presumably representing precursor forms of 3-oxoacyl-CoA thiolase, was detected in the patients. Catalase protein was not deficient, in accordance with the finding that catalase activity is not diminished in the patients. Thus in contrast to the situation with catalase functional peroxisomes are required for the stability and normal activity of peroxisomal beta-oxidation enzymes.     

D-3-hydroxyacyl coenzyme A dehydratase from rat liver peroxisomes. Purification and characterization of a novel enzyme necessary for the epimerization of 3-hydroxyacyl-CoA thioesters

Rat liver peroxisomal D-3-hydroxyacyl-CoA dehydratase, which in combination with enoyl-CoA hydratase catalyzes the epimerization of 3-hydroxyacyl-CoA, was purified by a five-step procedure to yield a highly purified preparation as judged by gel electrophoresis of the native and denatured enzyme. Since the molecular mass of the native dehydratase was estimated to be twice that of its 44-kDa subunit, the enzyme seems to be composed of two, possibly identical subunits. This dehydratase catalyzes the reversible dehydration of D-3-hydroxyacyl-CoA to 2-trans-enoyl-CoA, but, in contrast to enoyl-CoA hydratase, does not act on 2-cis-enoyl-CoA. The dehydratase is virtually inactive toward crotonyl-CoA, but exhibits high activity with 2-trans-hexenoyl-CoA as a substrate and acts with decreasing efficiency on all 2-enoyl-CoAs tested from 2-hexenoyl-CoA to 2-hexadecenoyl-CoA. The pH optimum of the enzyme is close to 8. Equilibrium ratios of 3-hydroxyoctanoyl-CoA/2-trans-octenoyl-CoA and 3-hydroxyoctanoyl-CoA/2-cis-octenoyl-CoA were found to be close to 3 and 137, respectively. It is suggested that 2-cis-enoyl-CoA intermediates formed during the beta-oxidation of polyunsaturated fatty acids in peroxisomes are hydrated by enoyl-CoA hydratase to D-3-hydroxyacyl-CoAs which are epimerized to their L-isomers by the sequential actions of D-3-hydroxyacyl-CoA dehydratase and enoyl-CoA hydratase.     

Analysis of the alternative pathways for the beta-oxidation of unsaturated fatty acids using transgenic plants synthesizing polyhydroxyalkanoates in peroxisomes

Degradation of fatty acids having cis-double bonds on even-numbered carbons requires the presence of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle. Two alternative pathways have been described to degrade these fatty acids. One pathway involves the participation of the enzymes 2, 4-dienoyl-coenzyme A (CoA) reductase and Delta(3)-Delta(2)-enoyl-CoA isomerase, whereas the second involves the epimerization of R-3-hydroxyacyl-CoA via a 3-hydroxyacyl-CoA epimerase or the action of two stereo-specific enoyl-CoA hydratases. Although degradation of these fatty acids in bacteria and mammalian peroxisomes was shown to involve mainly the reductase-isomerase pathway, previous analysis of the relative activity of the enoyl-CoA hydratase II (also called R-3-hydroxyacyl-CoA hydro-lyase) and 2,4-dienoyl-CoA reductase in plants indicated that degradation occurred mainly through the epimerase pathway. We have examined the implication of both pathways in transgenic Arabidopsis expressing the polyhydroxyalkanoate synthase from Pseudomonas aeruginosa in peroxisomes and producing polyhydroxyalkanoate from the 3-hydroxyacyl-CoA intermediates of the beta-oxidation cycle. Analysis of the polyhydroxyalkanoate synthesized in plants grown in media containing cis-10-heptadecenoic or cis-10-pentadecenoic acids revealed a significant contribution of both the reductase-isomerase and epimerase pathways to the degradation of these fatty acids.     

Mitochondrial beta-oxidation in Aspergillus nidulans

Beta-oxidation (beta-ox) occurs exclusively in the peroxisomes of Saccharomyces cerevisiae and other yeasts, leading to the supposition that fungi lack mitochondrial beta-ox. Here we present unequivocal evidence that the filamentous fungus Aspergillus nidulans houses both peroxisomal and mitochondrial beta-ox. While growth of a peroxisomal beta-ox disruption mutant (DeltafoxA) was eliminated on a very long-chain fatty acid (C(22:1)), growth was only partially impeded on a long-chain fatty acid (C(18:1)) and was not affected at all on short chain (C4-C6) fatty acids. In contrast, growth of a putative enoyl-CoA hydratase mutant (DeltaechA) was abolished on short-chain and severely restricted on long- and very long-chain fatty acids. Furthermore fatty acids inhibited growth of the DeltaechA mutant but not the DeltafoxA mutant in the presence of an alternate carbon source (lactose). Disruption of echA led to a 28-fold reduction in 2-butenoyl-CoA hydratase activity in a preparation of organelles. EchA was also required for growth on isoleucine and valine. The subcellular localization of the FoxA and EchA proteins was confirmed through the use of red and green fluorescent protein fusions.     

Peroxisomal multifunctional beta-oxidation protein of Saccharomyces cerevisiae. Molecular analysis of the fox2 gene and gene product.

The gene encoding the multifunctional protein (MFP) of peroxisomal beta-oxidation in Saccharomyces cerevisiae was isolated from a genomic library via functional complementation of a fox2 mutant strain. The open reading frame consists of 2700 base pairs encoding a protein of 900 amino acids. The predicted molecular weight (98,759) is in close agreement with that of the isolated polypeptide (96,000). Analysis of the deduced amino acid sequence revealed similarity to the MFPs of two other fungi but not to that of rat peroxisomes or the multifunctional subunit of the Escherichia coli beta-oxidation complex. The FOX2 gene was overexpressed from a multicopy vector (YEp352) in S. cerevisiae and the gene product purified to apparent homogeneity. A truncated version of MFP lacking 271 carboxyl-terminal amino acids was also overexpressed and purified. Experiments to study the enzymatic properties of the wild-type MFP demonstrated an absence of activities originally assigned to an MFP of S. cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA). The truncated form contained only the D-3-hydroxyacyl-CoA dehydrogenase activity. These results clearly demonstrate that the beta-oxidation of fatty acids in S. cerevisiae follows a previously unknown stereochemical course, namely it occurs via a D-3-hydroxyacyl-CoA intermediate.     

Metabolic aspects of peroxisomal beta-oxidation

In the course of the last decade peroxisomal beta-oxidation has emerged as a metabolic process indispensable to normal physiology. Peroxisomes beta-oxidize fatty acids, dicarboxylic acids, prostaglandins and various fatty acid analogues. Other compounds possessing an alkyl-group of six to eight carbon atoms (many substituted fatty acids) are initially omega-oxidized in endoplasmic reticulum. The resulting carboxyalkyl-groups are subsequently chain-shortened by beta-oxidation in peroxisomes. Peroxisomal beta-oxidation is therefore, in contrast to mitochondrial beta-oxidation, characterized by a very broad substrate-specificity. Acyl-CoA oxidases initiate the cycle of beta-oxidation of acyl-CoA esters. The next steps involve the bi(tri)functional enzyme, which possesses active sites for enoyl-CoA hydratase-, beta-hydroxyacyl-CoA dehydrogenase- and for delta 2, delta 5 enoyl-CoA isomerase activity. The beta-oxidation sequence is completed by a beta-ketoacyl-CoA thiolase. The peroxisomes also contain a 2,4-dienoyl-CoA reductase, which is required for beta-oxidation of unsaturated fatty acids. The peroxisomal beta-hydroxyacyl-CoA epimerase activity is due to the combined action of two enoyl-CoA hydratases. (For a recent review of the enzymology of beta-oxidation enzymes see Ref. 225.) The broad specificity of peroxisomal beta-oxidation is in part due to the presence of at least two acyl-CoA oxidases, one of which, the trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) oxidase, is responsible for the initial dehydrogenation of the omega-oxidized cholesterol side-chain, initially hydroxylated in mitochondria. Shortening of this side-chain results in formation of bile acids and of propionyl-CoA. In relation to its mitochondrial counterpart, peroxisomal beta-oxidation in rat liver is characterized by a high extent of induction following exposure of rats to a variety of amphipathic compounds possessing a carboxylic-, or sulphonic acid group. In rats some high fat diets cause induction of peroxisomal fatty acid beta-oxidation and of trihydroxy-5 beta-cholestanoyl-CoA oxidase. Induction involves increased rates of synthesis of the appropriate mRNA molecules. Increased half-lives of mRNA- and enzyme molecules may also be involved. Recent findings of the involvement of a member of the steroid hormone receptor superfamily during induction, suggest that induction of peroxisomal beta-oxidation represents another regulatory phenomenon controlled by nuclear receptor proteins. This will likely be an area of intense future research. Chain-shortening of fatty acids, rather than their complete beta-oxidation, is the prominent feature of peroxisomal beta-oxidation.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Arabidopsis thaliana multifunctional protein gene (MFP2) of peroxisomal beta-oxidation is essential for seedling establishment

The multifunctional protein (MFP) of peroxisomal beta-oxidation catalyses four separate reactions, two of which (2-trans enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase) are core activities required for the catabolism of all fatty acids. We have isolated and characterized five Arabidopsis thaliana mutants in the MFP2 gene that is expressed predominantly in germinating seeds. Seedlings of mfp2 require an exogenous supply of sucrose for seedling establishment to occur. Analysis of mfp2-1 seedlings revealed that seed storage lipid was catabolized more slowly, long-chain acyl-CoA substrates accumulated and there was an increase in peroxisome size. Despite a reduction in the rate of beta-oxidation, mfp2 seedlings are not resistant to the herbicide 2,4-dichlorophenoxybutyric acid, which is catabolized to the auxin 2,4-dichlorophenoxyacetic acid by beta-oxidation. Acyl-CoA feeding experiments show that the MFP2 2-trans enoyl-CoA hydratase only exhibits activity against long chain (C18:0) substrates, whereas the MFP2 L-3-hydroxyacyl-CoA dehydrogenase is active on C6:0, C12:0 and C18:0 substrates. A mutation in the abnormal inflorescence meristem gene AIM1, the only homologue of MFP2, results in an abnormal inflorescence meristem phenotype in mature plants (Richmond and Bleecker, Plant Cell 11, 1999, 1911) demonstrating that the role of these genes is very different. The mfp2-1 aim1double mutant aborted during the early stages of embryo development showing that these two proteins share a common function that is essential for this key stage in the life cycle.     

The Multifunctional Protein in Peroxisomal β-Oxidation: STRUCTURE AND SUBSTRATE SPECIFICITY OF THE ARABIDOPSIS THALIANA PROTEIN MFP2*

Plant fatty acids can be completely degraded within the peroxisomes. Fatty acid degradation plays a role in several plant processes including plant hormone synthesis and seed germination. Two multifunctional peroxisomal isozymes, MFP2 and AIM1, both with 2-trans-enoyl-CoA hydratase and l-3-hydroxyacyl-CoA dehydrogenase activities, function in mouse ear cress (Arabidopsis thaliana) peroxisomal β-oxidation, where fatty acids are degraded by the sequential removal of two carbon units. A deficiency in either of the two isozymes gives rise to a different phenotype; the biochemical and molecular background for these differences is not known. Structure determination of Arabidopsis MFP2 revealed that plant peroxisomal MFPs can be grouped into two families, as defined by a specific pattern of amino acid residues in the flexible loop of the acyl-binding pocket of the 2-trans-enoyl-CoA hydratase domain. This could explain the differences in substrate preferences and specific biological functions of the two isozymes. The in vitro substrate preference profiles illustrate that the Arabidopsis AIM1 hydratase has a preference for short chain acyl-CoAs compared with the Arabidopsis MFP2 hydratase. Remarkably, neither of the two was able to catabolize enoyl-CoA substrates longer than 14 carbon atoms efficiently, suggesting the existence of an uncharacterized long chain enoyl-CoA hydratase in Arabidopsis peroxisomes.     

Induction, immunochemical identity and immunofluorescence localization of an 80000-molecular-weight peroxisome-proliferation-associated polypeptide (polypeptide PPA-80) and peroxisomal enoyl-CoA hydratase of mouse liver and renal cortex

The hypolipidaemic drugs methyl clofenapate, BR-931, Wy-14643 and procetofen induced a marked proliferation of peroxisomes in the parenchymal cells of liver and the proximal-convoluted-tubular epithelium of mouse kidney. The proliferation of peroxisomes was associated with 6–12-fold increase in the peroxisomal palmitoyl-CoA oxidizing capacity of the mouse liver. Enhanced activity of the peroxisomal palmitoyl-CoA oxidation system was also found in the renal-cortical homogenates of hypolipidaemic-drug-treated mice. The activity of enoyl-CoA hydratase in the mouse liver increased 30–50-fold and in the kidney cortex 3–5-fold with hypolipidaemic-drug-induced peroxisome proliferation in these tissues, and over 95% of this induced activity was found to be heat-labile peroxisomal enzyme in both organs. Sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic analysis of large-particle and microsomal fractions obtained from the liver and kidney cortex of mice treated with hypolipidaemic peroxisome proliferators demonstrated a substantial increase in the quantity of an 80000-mol.wt. peroxisome-proliferation-associated polypeptide (polypeptide PPA-80). The heat-labile peroxisomal enoyl-CoA hydratase was purified from the livers of mice treated with the hypolipidaemic drug methyl clofenapate; the antibodies raised against this electrophoretically homogeneous protein yielded a single immunoprecipitin band with purified mouse liver enoyl-CoA hydratase and with liver and kidney cortical extracts of normal and hypolipidaemic-drug-treated mice. These anti-(mouse liver enoyl-CoA hydratase) antibodies also cross-reacted with purified rat liver enoyl-CoA hydratase and with the polypeptide PPA-80 obtained from rat and mouse liver. Immunofluorescence studies with anti-(polypeptide PPA-80) and anti-(peroxisomal enoyl-CoA hydratase) provided visual evidence for the localization and induction of polypeptide PPA-80 and peroxisomal enoyl-CoA hydratase in the liver and kidney respectively of normal and hypolipidaemic-drug-treated mice. In the kidney, the distribution of these two proteins is identical and limited exclusively to the cytoplasm of proximal-convoluted-tubular epithelium. The immunofluorescence studies clearly complement the biochemical and ultrastructural observations of peroxisome induction in the liver and kidney cortex of mice fed on hypolipidaemic drugs. In addition, preliminary ultrastructural studies with the protein-A–gold-complex technique demonstrate that the heat-labile hepatic enoyl-CoA hydratase is localized in the peroxisome matrix.     

Immunochemical identity of peroxisomal enoyl-CoA hydratase with the peroxisome-proliferation -associated 80,000 mol wt polypeptide in rat liver

Peroxisome proliferators, which induce proliferation of hepatic peroxisomes, have been shown previously to cause a marked increase in an 80,000 mol wt polypeptide predominantly in the light mitochondrial and microsomal fractions of liver of rodents. We now present evidence to show that this hepatic peroxisome-proliferation-associated polypeptide, referred to as polypeptide PPA-80, is immunochemically identical with the multifunctional peroxisome protein displaying heat-labile enoyl-CoA hydratase activity. This conclusion is based on the following observations: (a) the purified polypeptide PPA-80 and the heat- labile enoyl-CoA hydratase from livers of rats treated with the peroxisome proliferators Wy-14,643 {[4-chloro-6(2,3-xylidino)-2-pyrimidinylthio]acetic acid} exhibit identical minimum molecular weights of approximately 80,000 on SDS polyacrylamide gel electrophoresis; (b) these two proteins are immunochemically identical on the basis of ouchterlony double diffusion, immunotitration, rocket immunoelectrophoresis, and crossed immunoelectrophoresis analysis; and (c) the immunoprecipitates formed by antibodies to polypeptide PPA-80 when dissociated on a sephadex G-200 column yield enoyl-CoA hydratase activity. Whether the polypeptide PPA-80 exhibits the activity of other enzyme(s) of the peroxisomal β-oxidation system such as fatty acyl-CoA oxidase activity or displays immunochemical identity with such enzymes remains to be determined. The availability of antibodies to polypeptide PPA-80 and enoyl-CoA hydratase facilitated immunofluorescent and immunocytochemical localization of the polypeptide PPA- 80 and enoyl-CoA hydratase in the rat liver. The indirect immunofluorescent studies with these antibodies provided direct visual evidence for the marked induction of polypeptide PPA-80 and enoyl-CoA hydratase in the livers of rats treated with Wy-14,643. The present studies also provide immunocytochemical evidence for the localization of polypeptide PPA- 80 and the heat-labile enoyl-CoA hydratase in the peroxisome, but not in the mitochondria, of hepatic parenchymal cells. These studies, therefore, provide morphological evidence for the existence of fatty acyl-CoA oxidizing system in peroxisomes. An increase of polypeptide PPA-80 on SDS polyacrylamide gel electrophoretic analysis of the subcellular fractions of liver of rodents treated with lipid-lowering drugs should serve as a reliable and sensitive indicator of enhanced peroxisomal β- oxidation system.     

Enzymes of beta-Oxidation in Different Types of Algal Microbodies

The algae Mougeotia and Eremosphaera were used for isolation of microbodies with the characteristics of leaf peroxisomes and unspecialized peroxisomes, respectively. In both types of organelles, the following enzymes of the β-oxidation pathway were determined: acyl-CoA oxido-reductase, enoyl-CoA hydratase, and 3-hydroxyacyl-CoA dehydrogenase. There are indications that the peroxisomal oxidoreductase of both algae is a H₂O₂-forming oxidase rather than a dehydrogenase.

The enzymes enoyl-CoA hydratase and acyl-CoA oxidoreductase are located also in the mitochondria from Eremosphaera but not from Mougeotia. The mitochondrial acyl-CoA oxidizing enzyme was found to be a dehydrogenase. The specific activities of acyl-CoA oxidase and enoyl-CoA hydratase are lower than in spinach leaf peroxisomes. However, the activity of 3-hydroxyacyl-CoA dehydrogenase in the peroxisomes of both algae is almost 2-fold higher. The capability for degradation of fatty acids is a common feature of all different types of peroxisomes from algae.

     

Overexpression of peroxisome proliferator-activated receptor-alpha (PPARalpha)-regulated genes in liver in the absence of peroxisome proliferation in mice deficient in both L- and D-forms of enoyl-CoA hydratase/dehydrogenase enzymes of peroxisomal beta-oxidation system

Peroxisomal beta-oxidation system consists of peroxisome proliferator-activated receptor alpha (PPARalpha)-inducible pathway capable of catalyzing straight-chain acyl-CoAs and a second noninducible pathway catalyzing the oxidation of 2-methyl-branched fatty acyl-CoAs. Disruption of the inducible beta-oxidation pathway in mice at the level of fatty acyl-CoA oxidase (AOX), the first and rate-limiting enzyme, results in spontaneous peroxisome proliferation and sustained activation of PPARalpha, leading to the development of liver tumors, whereas disruptions at the level of the second enzyme of this classical pathway or of the noninducible system had no such discernible effects. We now show that mice with complete inactivation of peroxisomal beta-oxidation at the level of the second enzyme, enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase (L-PBE) of the inducible pathway and D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase (D-PBE) of the noninducible pathway (L-PBE-/-D-PBE-/-), exhibit severe growth retardation and postnatal mortality with none surviving beyond weaning. L-PBE-/-D-PBE-/- mice that survived exceptionally beyond the age of 3 weeks exhibited overexpression of PPARalpha-regulated genes in liver, despite the absence of morphological evidence of hepatic peroxisome proliferation. These studies establish that peroxisome proliferation in rodent liver is highly correlatable with the induction mostly of the L- and D-PBE genes. We conclude that disruption of peroxisomal fatty acid beta-oxidation at the level of second enzyme in mice leads to the induction of many of the PPARalpha target genes independently of peroxisome proliferation in hepatocytes, raising the possibility that intermediate metabolites of very long-chain fatty acids and peroxisomal beta-oxidation act as ligands for PPARalpha.     

Effects of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate on peroxisomal activities and enzyme activities involved in lipid metabolism in rat liver

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on hepatic lipids and key enzymes involved in esterification, hydrolysis and oxidation of long-chain fatty acids at increasing doses were investigated in rats. TPA administration tended to decrease the mitochondrial activities of palmitoyl-CoA synthetase and carnitine palmitoyltransferase. The microsomal palmitoyl-CoA synthetase activity was increased. TPA administration was also associated with a dose-dependent increase of glycerophosphate acyltransferase activity both in the mitochondrial and microsomal fractions in particular. The data are consistent with a decreased catabolism of long-chain fatty acids at the mitochondrial level, and an increased capacity for esterification of fatty acids in the microsomal fraction. Peroxisomal beta-oxidation was increased about 2-fold in the peroxisome-enriched fraction of TPA-treated rats while the catalase and urate oxidase activities were only marginally affected. TPA administration revealed elevated capacity for hydrolysis of palmitoyl-CoA and palmitoyl-L-carnitine in the microsomal fraction. Neither increased cytosolic palmitoyl-CoA hydrolase activity nor increased hydroxylation of lauric acid nor changes of the hepatic content of cytochrome P-450 isoenzymic forms were observed in the TPA-treated animals. There was no induction of the protein content of the bifunctional enoyl-CoA hydratase. Thus, TPA behaves more like choline-deficient diet and ethionine treatment than well-known peroxisome proliferators. It seems possible that TPA selectively stimulated the peroxisomal activities, i.e., peroxisomal beta-oxidation rather than evoking a peroxisome proliferation capacity.