Beta-oxidation Enzymes Research Articles

Biochemical and Immunocytochemical Properties of Peroxisomes and Mitochondria in Bovine Chromaffin Cells.

The biochemical and immunocytochemical properties of peroxisomal and mitochondrial beta-oxidation enzymes in bovine adrenal chromaffin cells were investigated. Peroxisomes were detectable by immunofluorescence staining using antibodies against acyl-CoA oxidase, peroxisomal 3-ketoacyl-CoA thiolase and catalase. The mitochondria were abundantly stained with antibody against mitochondrial 3-ketoacyl-CoA thiolase. The biosynthesis and intracellular processing of acyl-CoA oxidase and the peroxisomal 3-ketoacyl-CoA thiolase was slower than that in fibroblasts. The peroxisomal beta-oxidation activities shown by [1-14C] lignoceric acid oxidation were slightly lower than those in fibroblasts, whereas the mitochondrial beta-oxidation activities shown by [1-14C] palmitic acid oxidation were almost identical to those in fibroblasts. Adrenal chromaffin cells are useful materials for investigating the peroxisomal and mitochondrial metabolism of autonomic neurons and may contribute to the clarification of neuronal dysfunction in peroxisomal and mitochondrial disorders.

Peroxisomal disorders: clinical aspects.

Peroxisomal disorders are divided into two groups from a clinical point of view. Diseases in the first group, peroxisome-deficient disorders (PDD), Zellweger-like syndrome, and isolated deficiencies of peroxisomal beta-oxidation enzymes, are characterized by common clinical features including psychomotor retardation, hypotonia, hepatic dysfunction and visual disturbance. The second group includes diseases with a unique manifestation, such as X-linked adrenoleukodystrophy, hyperoxaluria type I and rhizomelic chondrodysplasia punctata. We investigated clinical aspects and the genetic basis of PDD, and the significance of peroxisomes in the development of human brain. Neuroradiological and neurophysiological studies revealed that thick cortex, colpocephaly and multifocal spikes were characteristic findings of PDD patients in the early infantile period. Cytogenetic studies elucidated the presence of eleven complementation groups among PDD, indicating the presence of eleven pathogenic genes for PDD. Molecular studies elucidated two of these genes, PAF-1 and PXR-1. Immunohistochemical studies clarified that the catalase-positive neurons appeared in the basal ganglia, thalamus, and cerebellum at 28 weeks of gestation, and in the cortex at 35 weeks. Immunopositive glial cells appeared from the deep to superficial white matter with increasing gestational age. These results suggest the important role of peroxisomes in neuronal maturation and myelinogenesis.

Induction of beta-oxidation enzymes and microbody proliferation in Aspergillus nidulans.

Aspergillus nidulans is able to grow on oleic acid as sole carbon source. Characterization of the oleate-induced beta-oxidation pathway showed the presence of the two enzyme activities involved in the first step of this catabolic system: acyl-CoA oxidase and acyl-CoA dehydrogenase. After isopicnic centrifugation in a linear sucrose gradient, microbodies (peroxisomes) housing the beta-oxidation enzymes, isocitrate lyase and catalase were clearly resolved from the mitochondrial fraction, which contained fumarase. Growth on oleic acid was associated with the development of many microbodies that were scattered throughout the cytoplasm of the cells. These microbodies (peroxisomes) were round to elongated, made up 6% of the cytoplasmic volume, and were characterized by the presence of catalase. The beta-oxidation pathway was also induced in acetate-grown cells, although at lower levels; these cells lacked acyl-CoA oxidase activity. Nevertheless, growth on acetate did not cause a massive proliferation of microbodies in A. nidulans.

Dietary docosahexaenoic acid has little effect on peroxisomes in healthy mice.

NMRI mice were fed diets supplemented with 0.05, 0.2, or 2% (w/w) docosahexaenoic acid (DHA), a polyunsaturated fatty acid present in fish oil, for 3 d, 3 wk, or 3 mon. The doses of DHA were chosen to supply the mice with concentrations of DHA which approximate those that have been reported to be beneficial to patients with peroxisomal disease. Diets containing 0.05 or 0.2% DHA did not change hepatic, myocardial, and renal catalase (EC 1.11.1.6) activity except for a slight but significant increase (to 120%) in myocardial catalase activity in mice treated with the 0.05% DHA diet for 3 mon. A diet with 2% DHA induced myocardial catalase activity to 150% after both 3 d and 3 wk of administration. In the liver of mice fed this diet for 3 wk, hepatic catalase activity was increased to 140% while no induction of palmitoyl-CoA oxidase (EC 1.3.99.3), urate oxidase (EC 1.7.3.3), and L-alpha-hydroxyisovalerate oxidase (EC 1.1.3.a) was observed. With the light microscope, no changes in peroxisomal morphology were visually evaluated in catalase stained sections of liver, myocardium, and kidney of mice fed either diet. Our results show that in healthy mice a low dietary DHA dose (< 0.2%; this corresponds to a dose prescribed to peroxisomal patients) has no effect on several hepatic peroxisomal H2O2-producing enzymes, including the rate-limiting enzyme of the peroxisomal fatty acid beta-oxidation. This may indicate that such a DHA dose will not add a strong load on the often disturbed fatty acid metabolism in the liver of patients with peroxisomal disorders.

Transcriptional control of a nuclear gene encoding a mitochondrial fatty acid oxidation enzyme in transgenic mice: role for nuclear receptors in cardiac and brown adipose expression.

Expression of the gene encoding medium-chain acyl coenzyme A dehydrogenase (MCAD), a nuclearly encoded mitochondrial fatty acid beta-oxidation enzyme, is regulated in parallel with fatty acid oxidation rates among tissues and during development. We have shown previously that the human MCAD gene promoter contains a pleiotropic element (nuclear receptor response element [NRRE-1]) that confers transcriptional activation or repression by members of the nuclear receptor superfamily. Mice transgenic for human MCAD gene promoter fragments fused to a chloramphenicol acetyltransferase gene reporter were produced and characterized to evaluate the role of NRRE-1 and other promoter elements in the transcriptional control of the MCAD gene in vivo. Expression of the full-length MCAD promoter-chloramphenicol acetyltransferase transgene (MCADCAT.371) paralleled the known tissue-specific differences in mitochondrial beta-oxidation rates and MCAD expression. MCADCAT.371 transcripts were abundant in heart tissue and brown adipose tissue, tissues with high-level MCAD expression. During perinatal cardiac developmental stages, expression of the MCADCAT.371 transgene paralleled mouse MCAD mRNA levels. In contrast, expression of a mutant MCADCAT transgene, which lacked NRRE-1 (MCADCATdeltaNRRE-1), was not enriched in heart or brown adipose tissue and did not exhibit appropriate postnatal induction in the developing heart. Transient-transfection studies with MCAD promoter-luciferase constructs containing normal or mutant NRRE-1 sequences demonstrated that the nuclear receptor binding sequences within NRRE-1 are necessary for high-level transcriptional activity in primary rat cardiocytes. Electrophoretic mobility shift assays demonstrated that NRRE-1 was bound by several cardiac and brown adipose nuclear proteins and that these interactions required the NRRE-1 receptor binding hexamer sequences. Antibody supershift studies identified the orphan nuclear receptor COUP-TF as one of the endogenous cardiac proteins which bound NRRE-1. These results dictate an important role for nuclear receptors in the transcriptional control of a nuclear gene encoding a mitochondrial fatty acid oxidation enzyme and identify a gene regulatory pathway involved in cardiac energy metabolism.

Intermediate Channeling on the Trifunctional β-Oxidation Complex from Pig Heart Mitochondria

The kinetic properties of the purified trifunctional beta-oxidation complex (TOC) from pig heart mitochondria were analyzed with the aim of elucidating the functional consequence of having three sequentially acting enzymes of beta-oxidation associated in one complex. The kinetic parameters of TOC and of the component enzymes of TOC, long-chain enoyl-CoA hydratase, long-chain 3-hydroxyacyl-CoA dehydrogenase, and long-chain 3-ketoacyl-CoA thiolase, were determined with substrates having acyl chains with 16 carbon atoms. Quantification by high performance liquid chromatography of intermediates formed during the degradation of 2-trans-hexadecanoyl-CoA to myristoyl-CoA and acetyl-CoA by TOC revealed the accumulation of 3-hydroxyhexadecanoyl-CoA, whereas 3-ketohexadecanoyl-CoA was undetectable. The observed rates of NADH and acetyl-CoA formation were higher than the theoretical rates calculated by use of the kinetic parameters and measured concentrations of intermediates. When the sequence of reactions catalyzed by TOC was inhibited by acetyl-CoA, the steady-state concentration of the 3-hydroxyacyl-CoA intermediate was not affected, whereas a small amount of 3-ketohexadecanoyl-CoA was detected. The differences between observed and predicted reaction rates and between measured and expected concentrations of intermediates are best explained by the operation of a channeling mechanism. As a consequence of intermediate channeling between the active sites on the complex, more coenzyme A is available in the mitochondrial matrix and metabolites like 3-ketoacyl-CoA thioesters, which are strong inhibitors of several beta-oxidation enzymes, do not accumulate.

Contributions of the immunogold technique to investigation of the biology of peroxisomes.

The immunogold labeling technique has been extremely useful in investigation of the structure and function of peroxisomes. In this report a few examples of the application of this technique with significant implications in the field are briefly reviewed. The problem of extra-peroxisomal catalase, the subject of long controversy between the biochemists and cytochemists, was settled with the immunogold technique, which unequivocally revealed the presence of that enzyme not only in the cytoplasm, but also in the euchromatin region of nucleus, in addition to peroxisomes. On the other hand, lactate dehydrogenase, a typical cytoplasmic protein, has also been shown recently to be present in peroxisomes and to be involved in the reoxidation of NADH produced by the peroxisomal beta-oxidation system. The immunogold technique has revealed several distinct compartments in the matrix of mammalian peroxisomes: urate oxidase in the crystalline cores, alpha-hydroxy acid oxidase B in the marginal plates and D-amino acid oxidase in a non-crystalline condensed region of matrix. The specific alterations of peroxisomal proteins are reflected in their immunolabeling density with gold particles. Quantitation of gold-label by automatic image analysis has revealed that the induction of lipid beta-oxidation enzyme proteins by diverse hypolipidemic drugs is initiated and more pronounced in the pericentral regions of the liver lobule. Finally, immunogold labeling with an antibody to 70 kDa peroxisomal membrane protein has identified a novel class of small peroxisomes that initially incorporate radioactive amino acids more efficiently than regular peroxisomes and thus may represent early stages in the biogenesis of peroxisomes.

Pip2p: a transcriptional regulator of peroxisome proliferation in the yeast Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, peroxisomes are the exclusive site for the degradation of fatty acids. Upon growth with the fatty acid oleic acid as sole carbon source, not only are the enzymes of beta-oxidation and catalase A induced, but also the peroxisomal compartment as a whole increases in volume and the number of organelles per cell rises. We previously identified a cis-acting DNA sequence [oleate response element (ORE)] involved in induction of genes encoding peroxisomal proteins. The aim of our investigation was to test whether a single mechanism acting via the ORE coordinates the events necessary for the proliferation of an entire organelle. Here we report the cloning and characterization of the oleate-specific transcriptional activator protein Pip2p (pip: peroxisome induction pathway). Pip2p contains a typical Zn(2)-Cys(6) cluster domain and binds to OREs. A pip2 deletion strain is impaired in growth on oleate as sole carbon source and the induction of beta-oxidation enzymes is abolished. Moreover, only a few, small peroxisomes per cell can be detected. These results indicate that fatty acids activate Pip2p, which in turn activates the transcription of genes encoding beta-oxidation components and acts as the crucial activator of peroxisomes.

Urinary bile acids and peroxisomal bifunctional enzyme deficiency

The biosynthesis of normal bile acids involves beta-oxidation of the 8-carbon side-chain of cholesterol, in addition to numerous modifications of the sterol nucleus. Because beta-oxidation of the sterol side-chain has been localized to the peroxisome, bile acid analysis has been suggested to be useful in the diagnostic evaluation of individuals suspected of having peroxisomal disorders. Although data from subjects with generalized peroxisomal disorders support this, few data exist regarding the bile acids in individuals having single peroxisomal beta-oxidation enzyme disorders. In this study, we analyzed the urinary bile acids from 12 patients with peroxisomal bifunctional protein deficiency using continuous flow fast atom bombardment mass spectrometry. All 12 patients had abnormal spectra, although their ion profiles and rank order of intensity of ions varied considerably. Ten of 12 individuals had abnormal spectra with presence of taurine-conjugated tetrahydroxycholestenoates, allowing a definite diagnosis of a peroxisomal beta-oxidation defect and a presumptive diagnosis of bifunctional protein deficiency; the other two cases were nondiagnostically abnormal. The strengths and limitations of urinary bile acid analysis for the diagnosis of peroxisomal beta-oxidation disorders are discussed.

Urinary bile acids and peroxisomal bifunctional enzyme deficiency

The biosynthesis of normal bile acids involves beta-oxidation of the 8-carbon side-chain of cholesterol, in addition to numerous modifications of the sterol nucleus. Because beta-oxidation of the sterol side-chain has been localized to the peroxisome, bile acid analysis has been suggested to be useful in the diagnostic evaluation of individuals suspected of having peroxisomal disorders. Although data from subjects with generalized peroxisomal disorders support this, few data exist regarding the bile acids in individuals having single peroxisomal beta-oxidation enzyme disorders. In this study, we analyzed the urinary bile acids from 12 patients with peroxisomal bifunctional protein deficiency using continuous flow fast atom bombardment mass spectrometry. All 12 patients had abnormal spectral, although their iron profiles and rank order of intensity of ions varied considerably. Ten of 12 individuals had abnormal spectra with presence of taurine-conjugated tetrahydroxycholestenoates, allowing a definite diagnosis of a peroxisomal beta-oxidation defect and a presumptive diagnosis of bifunctional protein deficiency; the other two cases were nondiagnostically abnormal. The strengths and limitations of urinary bile acid analysis for the diagnosis of peroxisomal beta-oxidation disorders are discussed. © 1996 Wiley-Liss, Inc.

Activation of a novel metabolic gene regulatory pathway by chronic stimulation of skeletal muscle.

To determine whether expression of a nuclear gene encoding a mitochondrial fatty acid oxidation enzyme is regulated in parallel with skeletal muscle fibre-type-specific energy substrate preference, expression of the gene encoding medium-chain acyl-CoA dehydrogenase (MCAD) was delineated in canine latissimus dorsi muscle subjected to chronic motor nerve stimulation. In predominantly fast-twitch canine latissimus dorsi muscle, MCAD mRNA levels were regulated by chronic stimulation in a biphasic pattern. During the 1st wk of stimulation, steady-state MCAD mRNA levels decreased to 50% of unstimulated levels. MCAD mRNA levels began to increase during the 3rd wk of stimulation to reach a level 3.0-fold higher than levels in unstimulated contralateral control muscle by day 70. Immunodetectable MCAD mRNA levels throughout the stimulation period. The temporal pattern and magnitude of MCAD mRNA accumulation in response to muscle stimulation was distinct from that of mRNAs encoding other enzymes known to be regulated by this stimulus, including glyceraldehyde phosphate dehydrogenase, citrate synthase, and sarcoplasmic reticulum Ca-ATPase, but paralleled the protein levels of the peroxisome proliferator-activated receptor (PPAR), an orphan member of the nuclear hormone receptor superfamily known to regulate genes encoding fatty acid oxidation enzymes in liver. The skeletal muscle expression pattern of PPAR was also similar to that of MCAD in unstimulated rat skeletal muscles with distinct fiber-type compositions. These results demonstrate that a nuclear gene encoding a mitochondrial beta-oxidation enzyme is dynamically regulated in a pattern that parallels skeletal muscle fiber-type-specific energy substrate utilization and implicate an orphan nuclear receptor transcription factor as a candidate transducer of this response.

PRENATAL DIAGNOSIS OF PEROXISOMAL DISORDERS. † 869

Peroxisomal disorders are genetic disease caused either by a defect in peroxisome biogenesis resulting in deficiencies of multiple peroxisomal enzymes or by a deficiency of one peroxisomal enzyme. Seventeen different peroxisomal disorders are recognized nowadays, with progressive neurological deficits and early death in most. Both in peroxisome deficiency disorders(PDD) like Zellweger syndrome, neonatal Adrenoleukodystrophy (n-ALD) and infantile Refsum disease, in classic rhizomelic Chondrodysplasia punctata(RCDP) and disorders with a defect in peroxisomal beta-oxidation of fatty acids (including X-linked ALD) no effective treatment is available and early prenatal detection indicated. For PDD this is done by us by in vitro measurement of acylCoA:dihydroxyacetonephosphate acyltransferase(DHAPAT) activity and immuno(western)blotting with antisera raised against catalase, acylCoA oxidase and peroxisomal thiolase(Thio) resp. in uncultured chorionic villi(CV) homogenates. Since 1992 seventeen affected PDS fetusses were recognized by us by this procedure in sixty one at risk pregnancies in the 10th or 11th gestational week. Moreover, an additional thirteen affected PDS fetusses were found by analyzing de novo plasmalogen biosynthesis and very longchain fatty acids(VLCFA) in cultured CV-fibroblasts or amniocytes. Seven RCDP fetusses were diagnosed by the finding of abnormal immunoblots with anti-thiolase (absence of 41kDa band for mature thiolase protein) and deficient plasmalogens in CV. We also diagnosed isolated DHAPAT deficiency in a fetus by finding a deficiency of plasmalogens and DHAPAT, but normal Thio-blot in CV. Fifteen fetusses with a deficiency of one of the enzymes of peroxisomal beta-oxidation, including X-linked ALD, were diagnosed prenatally in 41 at risk pregnancies by the finding of elevated VLCFA and deficient activity of peroxisomal beta-oxidation in cultured CV-fibroblasts or amniocytes. No false positive or negative cases were found with these procedures.

L-Lactate Dehydrogenase A4- and A3B Isoforms Are Bona Fide Peroxisomal Enzymes in Rat Liver: EVIDENCE FOR INVOLVEMENT IN INTRAPEROXISOMAL NADH REOXIDATION

The subcellular localization of l-lactate dehydrogenase (LDH) in rat hepatocytes has been studied by analytical subcellular fractionation combined with the immunodetection of LDH in isolated subcellular fractions and liver sections by immunoblotting and immunoelectron microscopy. The results clearly demonstrate the presence of LDH in the matrix of peroxisomes in addition to the cytosol. Both cytosolic and peroxisomal LDH subunits have the same molecular mass (35.0 kDa) and show comparable cross-reactivity with an anti-cytosolic LDH antibody. As revealed by activity staining or immunoblotting after isoelectric focussing, both intracellular compartments contain the same liver-specific LDH-isoforms (LDH-A4 > LDH-A3B) with the peroxisomes comprising relatively more LDH-A3B than the cytosol. Selective KCl extraction as well as resistance to proteinase K and immunoelectron microscopy revealed that at least 80% of the LDH activity measured in highly purified peroxisomal fractions is due to LDH as a bona fide peroxisomal matrix enzyme. In combination with the data of cell fractionation, this implies that at least 0.5% of the total LDH activity in hepatocytes is present in peroxisomes. Since no other enzymes of the glycolytic pathway (such as phosphoglucomutase, phosphoglucoisomerase, and glyceraldehyde-3-phosphate dehydrogenase) were found in highly purified peroxisomal fractions, it does not seem that LDH in peroxisomes participates in glycolysis. Instead, the marked elevation of LDH in peroxisomes of rats treated with the hypolipidemic drug bezafibrate, concomitantly to the induction of the peroxisomal beta-oxidation enzymes, strongly suggests that intraperoxisomal LDH may be involved in the reoxidation of NADH generated by the beta-oxidation pathway. The interaction of LDH and the peroxisomal palmitoyl-CoA beta-oxidation system could be verified in a modified beta-oxidation assay by adding increasing amounts of pyruvate to the standard assay mixture and recording the change of NADH production rates. A dose-dependent decrease of NADH produced was simulated with the lowest NADH value found at maximal LDH activity. The addition of oxamic acid, a specific inhibitor of LDH, to the system or inhibition of LDH by high pyruvate levels (up to 20 mm) restored the NADH values to control levels. A direct effect of pyruvate on palmitoyl-CoA oxidase and enoyl-CoA hydratase was excluded by measuring those enzymes individually in separate assays. An LDH-based shuttle across the peroxisomal membrane should provide an efficient system to regulate intraperoxisomal NAD+/NADH levels and maintain the flux of fatty acids through the peroxisomal beta-oxidation spiral.

Identification of a Peroxisome Proliferator-responsive Element Upstream of the Human Peroxisomal Fatty Acyl Coenzyme A Oxidase Gene

Peroxisome proliferators cause a rapid and coordinated transcriptional activation of genes encoding the enzymes of the peroxisomal beta-oxidation pathway in rats and mice. Cis-acting peroxisome proliferator responsive elements (PPREs) have been identified in the 5'-flanking region of H202-producing rat acyl-CoA oxidase (ACOX) gene and in other genes inducible by peroxisome proliferators. To gain more insight into the purported nonresponsiveness of human liver cells to peroxisome volume density and in the activity of the beta-oxidation enzyme system, we have previously cloned the human ACOX gene, the first and rate-limiting enzyme of the peroxisomal beta-oxidation system. We now present information on a regulatory element for the peroxidase proliferator-activated receptor (PPAR)/retinoid X receptor (RXR) heterodimers. The PPRE, consists of AGGTCA C TGGTCA, which is a direct repeat of hexamer half-sites interspaced by a single nucleotide (DR1 motif). It is located at -1918 to -1906 base pairs upstream of the transcription initiation site of this human ACOX gene. This PPRE specifically binds to baculovirus-expressed recombinant rat PPAR alpha/RXR alpha heterodimers. In transient transfection experiments, the maximum induction of luciferase expression by ciprofibrate and/or 9-cis-retinoic acid is dependent upon cotransfection of expression plasmids for PPAR alpha and RXR alpha. The functionally of this human ACOX promoter was further demonstrated by linking it to a beta-galactosidase reporter gene or to a rat urate oxidase cDNA and establishing stably transfected African green monkey kidney (CV1) cell lines expressing reporter protein. The human ACOX promoter has been found to be responsive to peroxisome proliferators in CV1 cells stably expressing PPAR alpha, whereas only a basal level of promoter activity is detected in stably transfected cells lacking PPAR alpha. The presence of a PPRE in the promoter of this human peroxisomal ACOX gene and its responsiveness to peroxisome proliferators suggests that factors other than the PPRE in the 5'-flanking sequence of the human ACOX gene may account for differences, if any, in the pleiotropic responses of humans to peroxisome proliferators.

Induction of peroxisomal beta-oxidation and P-450 4A-dependent activities by pivalic and trichloroacetic acid in rat liver and kidney.

The influence of pivalic acid (PIV), a compound often used to make pro-drugs, and of the structurally related trichloroacetic acid (TCA), on several hepatic and renal enzymes was investigated in Sprague-Dawley rats, following a 4-day treatment period. The PIV and TCA treatments resulted in a similar and selective induction (2-3 times) of peroxisomal palmitoyl-CoA oxidase and the cytochrome P-450 4A dependent microsomal (omega)- and (omega-1)-lauric acid activities, both in liver and kidney. Western blot analysis of liver and kidney microsomes from PIV- and TCA-treated rats, using antibody to the P-450 4A1, revealed induction of members of the P-450 4A subfamily. These results suggest that PIV, like TCA, is a renal and hepatic peroxisome proliferator in rats, and further support the previously indicated close association between the peroxisomal fatty acid beta-oxidation enzymes and microsomal P-450 4A sub-family enzymes.

Thyroid hormone (T3) inhibits ciprofibrate-induced transcription of genes encoding beta-oxidation enzymes: cross talk between peroxisome proliferator and T3 signaling pathways.

Peroxisome proliferators cause rapid and coordinated transcriptional activation of genes encoding peroxisomal beta-oxidation system enzymes by activating peroxisome proliferator-activated receptor (PPAR) isoform(s). Since the thyroid hormone (T3; 3,3',5-triiodothyronine) receptor (TR), another member of the nuclear hormone receptor superfamily, regulates a subset of fatty acid metabolism genes shared with PPAR, we examined the possibility of interplay between peroxisome proliferator and T3 signaling pathways. T3 inhibited ciprofibrate-induced luciferase activity as well as the endogenous peroxisomal beta-oxidation enzymes in transgenic mice carrying a 3.2-kb 5'-flanking region of the rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase gene fused to the coding region of luciferase. Transfection assays in hepatoma H4-II-E-C3 and CV-1 cells indicated that this inhibition is mediated by TR in a ligand-dependent fashion. Gel shift assays revealed that modulation of PPAR action by TR occurs through titration of limiting amounts of retinoid X receptor (RXR) required for PPAR activation. Increasing amounts of RXR partially reversed the inhibition in a reciprocal manner; PPAR also inhibited TR activation. Results with heterodimerization-deficient TR and PPAR mutants further confirmed that interaction between PPAR and TR signaling systems is indirect. These results suggest that a convergence of the peroxisome proliferator and T3 signaling pathways occurs through their common interaction with the heterodimeric partner RXR.

Purification and Properties of 3-Hydroxyacyl Coenzyme A Dehydrogenase-Binding Protein from Rat Liver Mitochondria

Mitochondria isolated from rat liver were freeze-thawed and washed with 0.1 M potassium phosphate, pH 7.4. Most of the 3-hydroxyacyl coenzyme A (CoA) dehydrogenase activities were removed and this mitochondrial membrane fraction could bind exogenous 3-hydroxyacyl-CoA dehydrogenase. 3-Hydroxyacyl-CoA dehydrogenase-binding protein was extracted from the washed membrane fraction with a buffer containing 2% Triton X-100 and 2% sodium taurodeoxycholate. The binding protein was purified by Ultrogel AcA 34 gel chromatography, calcium phosphate gel-cellulose chromatography and 3-hydroxyacyl-CoA dehydrogenase affinity chromatography. The molecular mass of the purified binding protein was estimated to be 140 kDa by gel filtration and its subunit molecular mass was determined as 60 kDa by SDS-PAGE suggesting that the protein is a homodimer. The binding protein and 3-hydroxyacyl-CoA dehydrogenase formed a complex at low ionic strength and the stoichiometry revealed that 1 mol of the binding protein bound 2 mol of 3-hydroxyacyl-CoA dehydrogenase. Purified 3-hydroxyacyl-CoA dehydrogenase-binding protein interacted with 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase but did not bind other mitochondrial beta-oxidation enzymes. The pH optimum of the binding activity was from pH 6 to 7 and the binding activity was diminished by increasing the concentration of salt in the medium.

Distinction between peroxisomal bifunctional enzyme and acyl‐CoA oxidase deficiencies

The clinical distinction between patients with a disorder of peroxisome assembly (e.g., Zellweger syndrome) and those with a defect in a peroxisomal fatty acid beta-oxidation enzyme can be difficult. We studied 29 patients suspected of belonging to the latter group. Using complementation analysis, 24 were found to be deficient in enoylcoenzyme A hydratase/3-hydroxyacylcoenzyme A dehydrogenase bifunctional enzyme and 5 were deficient in acyl-CoA oxidase. Elevated plasma very long-chain fatty acids (VLCFA), impaired fibroblast VLCFA beta-oxidation, decreased fibroblast phytanic acid oxidation, normal plasmalogen synthesis, normal plasma L-pipecolic acid level, and normal subcellular catalase distribution were characteristic findings in both disorders. The elevation in plasma VLCFA levels and impairment in fibroblast VLCFA beta-oxidation were more severe in bifunctional-deficient than in oxidase-deficient patients. The clinical course in bifunctional deficiency (profound hypotonia, neonatal seizures, dysmorphic features, age at death approximately 9 months) was more severe than in oxidase deficiency (moderate hypotonia without dysmorphic features, development of a leukodystrophy, age at death approximately 4 yr). Based on these findings, accurate early diagnosis of these deficiencies of peroxisomal beta-oxidation enzymes is possible.

Adrenoleucodystrophy and BSERA

Complementation studies, using fused cell lines from patients with peroxisomal disorders, have shown correction of defective plasmalogen synthesis and phytanic acid oxidation as wll as an increase in the number of peroxisomes. At least six complementation groups have been reported. We demostrate here that complementing cell lines also acquire the ability to oxidize very long chain fatty acids (VLCFA), and that complementation groups defined with this technique are identical to those reported previously when plasmalogen synthesis was used as the criterion for complementation. This VLCFA complementation technique is of particular value in the study of patients in whom defective VLCFA is the only or major enzymatic defect, and we show complementation between cell lines from two patients each with an isolated defect in one of the peroxisomal fatty acid beta-oxidation enzymes.

Peroxisome mosaicism in the livers of peroxisomal deficiency patients

Peroxisomal deficiency disorders, which are genetically transmitted, are assumed to be expressed in all cells, and the use of cultured skin fibroblasts for diagnosis and research is based on this assumption. We describe three patients with clinical, biochemical, and microscopic evidence of a peroxisomal disorder. However, their liver displays mosaicism, i.e., parenchymal cells with peroxisomes are adjacent to cells without peroxisomes. Ten percent (volume), 8%, and less than 1% of the parenchyma possessed peroxisomes that can be identified in immunocytochemical tests for six matrix and membrane proteins performed by light and electron microscopy. In the bulk of the parenchyma, catalase is localized in the cytoplasm, and in such cells no peroxisomes are evident by electron microscopy and immunolabeling for the 43-kd peroxisomal membrane protein (PMP) in two patients; in the third case, peroxisomal membrane ghosts are present. Immunoblots of peroxisomal beta-oxidation enzymes show a pattern similar to that from patients with a generalized peroxisomal deficiency. In contrast to the clinical and biochemical signs of peroxisomal dysfunction and hepatic histopathology, cultured fibroblasts from two patients demonstrate normal peroxisomal functions, including very-long-chain fatty acid oxidation and plasmalogen synthesis.