Peroxisomal Fractions Research Articles

Peroxisomes from pepper fruits ( Capsicum annuum L.): purification, characterisation and antioxidant activity

Pepper is a vegetable of importance in human nutrition. Currently, one of the most interesting properties of natural products is their antioxidant content. In this work, the purification and characterisation of peroxisomes from fruits of a higher plant was carried out, and their antioxidative enzymatic and non-enzymatic content was investigated. Green and red pepper fruits (Capsicum annuum L., type Lamuyo) were used in this study. The analysis by electron microscopy showed that peroxisomes from both types of fruits contained crystalline cores which varied in shape and size, and the presence of chloroplasts and chromoplasts in green and red pepper fruits, respectively, was confirmed. Peroxisomes were purified by differential and sucrose density-gradient centrifugations. In the peroxisomal fractions, the activity of the photorespiration, beta-oxidation and glyoxylate cycle enzymes, and the ROS-related enzymes catalase, superoxide dismutase, xanthine oxidase, glutathione reductase and NADP(+)-dehydrogenases, was determined. Most enzymes studied had higher specific activity and protein content in green than in red fruits. By native PAGE and western blot analysis, the localisation of a Mn-SOD in fruit peroxisomes was demonstrated. The ascorbate and glutathione levels were also determined in crude extracts and in peroxisomes purified from both green and red peppers. The total ascorbate content (200-220 mg per 100 g FW) was similar in crude extracts from the two types of fruits, but higher in peroxisomes from red peppers. The glutathione concentration was 2-fold greater in green pepper crude extracts than in red fruits, whereas peroxisomes from both tissues showed similar values. The presence in pepper peroxisomes of different antioxidative enzymes and their corresponding metabolites implies that these organelles might be an important pool of antioxidants in fruit cells, where these enzymes could also act as modulators of signal molecules (O2*-, H202) during fruit maturation.

Hepatic enzymatic synthesis and hydrolysis of CoA esters of solvent-derived oxa acids.

Many ethylene glycol-derived solvents are oxidized to xenobiotic alkoxyacetic acids (3-oxa acids) by hepatic enzymes. The toxicity of these ubiquitous solvents has been associated with their oxa acid metabolites. For many xenobiotic carboxylic acids, the toxicity is associated with the CoA ester of the acid. In this study, related alkoxyacetic acids were evaluated as potential substrates for acyl-CoA synthetases found in mitochondrial, peroxisomal, and microsomal fractions isolated from rat liver. Likewise, chemically synthesized oxa acyl-CoAs were used as substrates for acyl-CoA hydrolases associated with the same rat liver fractions. Activities of the xenobiotic oxygen-substituted substrates were compared with analogous physiologic aliphatic substrates by UV-vis spectrophotometric methods. All of the solvent-derived oxa acids were reasonable substrates for the acyl-CoA synthetases, although their activity was usually less than the corresponding physiologic acid. Acyl-CoA hydrolase activities were decreased compared with acyl-CoA synthetase activities for all substrates, especially for the oxa acyl-CoAs. These studies suggest that these xenobiotic carboxylic acids may be converted to reactive acyl-CoA moieties which will persist in areas of the cell proximal to lipid synthesis, beta-oxidation, protein acylation, and amino acid conjugation. The interaction of these xenobiotic acyl-CoAs with those processes may be important to their toxicity and/or detoxification.

Approaching complete peroxisome characterization by gas-phase fractionation.

We examined the utility of gas-phase fractionation (GPF) in the m/z dimension to increase proteome coverage and reproducibility of peptide ion selection by direct microliquid chromatography/electrospray ionization-tandem mass spectrometry (microLC/ESI-MS/MS) analysis of the peptides produced by proteolytic digestion of unfractionated proteins from a yeast whole-cell lysate and in a peroxisomal membrane protein fraction derived from isolated yeast peroxisomes. We also investigated GPF in the relative ion intensity dimension and propose denoting the two types of GPF as GPF(m/z) and GPF(RI). Comparison of results of direct nuLC/ESI-MS/MS analysis of the unfractionated mixture of peptides from proteolysis of a yeast whole cell lysate by DD ion selection from 400-1800 m/z in triplicate and GPF(m/z) from 400-800, 800-1200 and 1200-1800 produced the following results: (i) 1.3 x more proteins were identified by GPF(m/z) for an equal amount of effort (i.e., 3 microLC/ESI-MS/MS) and (ii) proteins identified by GPF(m/z) had a lower average codon bias value. Use of GPF(RI) identified more proteins per m/z unit scanned than GPF(m/z) or triplicate analysis over a wide m/z range. After tryptic digestion of all the proteins from a discontinuous Nycodenz gradient fraction known to be enriched with yeast peroxisomal membrane proteins we detected 93% (38/41) of known peroxisomal proteins using GPF(m/z), but only 73% using a standard wide m/z range survey scan.

Rat liver acyl-CoA synthetase 4 is a peripheral-membrane protein located in two distinct subcellular organelles, peroxisomes, and mitochondrial-associated membrane

Obesity and non-insulin-dependent diabetes favor storage of fatty acids in triacylglycerol over oxidation. Recently, individual acyl-CoA synthetase (ACS) isoforms have been implicated in the channeling of fatty acids either toward lipid synthesis or toward oxidation. Although ACS1 had been localized to three different subcellular regions in rat liver, endoplasmic reticulum, mitochondria, and peroxisomes, the study had used an antibody raised against the full-length ACS1 protein which cross-reacts with other isoforms, probably because all ACS family members contain highly conserved amino acid sequences. Therefore, we examined the subcellular location of ACS1, ACS4, and ACS5 in rat liver to determine which isoform was present in peroxisomes, whether the ACSs were intrinsic membrane proteins, and which ACS isoforms were up-regulated by PPARα ligands. Non-cross-reacting ACS1, ACS4, and ACS5 peptide antibodies showed that ACS4 was the only ACS isoform present in peroxisomes isolated from livers of gemfibrozil-treated rats. ACS4 was also present in fractions identified as mitochondria-associated membrane (MAM). ACS1 was present in endoplasmic reticulum fractions and ACS5 was present in mitochondrial fractions. Incubation with troglitazone, a specific inhibitor of ACS4, decreased ACS activity in the MAM fractions 30–45% and in the peroxisomal fractions about 30%. Because the signal for ACS4 protein in peroxisomes was so strong compared to the MAM fraction, we examined ACS4 mRNA abundance in livers of rats treated with the PPARα agonist GW9578. Treatment with GW9578 increased ACS4 mRNA abundance 40% and ACS1 mRNA 25%. Although we had originally proposed that ACS4 is linked to triacylglycerol synthesis, it now appears that ACS4 may also be important in activating fatty acids destined for peroxisomal oxidation. We also determined that, unlike ACS1 and 5, ACS4 is not an intrinsic membrane protein. This suggests that ACS4 is probably targeted and linked to MAM and peroxisomes by interactions with other proteins.

Monomeric alcohol oxidase is preferentially digested by a novel protease from Candida boidinii

A protease activity has been partially purified from peroxisomal matrix fractions of the methylotrophic yeast Candida boidinii. The enzyme migrates as a single peak on a sucrose velocity gradient with an apparent native molecular mass of ∼80–90 kDa. Activity can be recovered from nonreducing sodium dodecyl sulfate gels as a ∼20 kDa species, suggesting it is an oligomer. The protein exhibits chymotrypsin-like activity and cleaves the model compound suc-L-L-V-Y-AMC. Additionally, monomers of alcohol oxidase (AO), an abundant protein of C. boidinii peroxisomes, generated in vitro or in pulse-radiolabeled cells, are preferentially sensitive to degradation by the protease. Sensitivity is lost over time in vivo as AO folds and matures into octamers, suggesting that the protease may be involved in these processes.

Identification and Biochemical Characterization ofArabidopsis thaliana Sulfite Oxidase: A NEW PLAYER IN PLANT SULFUR METABOLISM

In mammals and birds, sulfite oxidase (SO) is a homodimeric molybdenum enzyme consisting of an N-terminal heme domain and a C-terminal molybdenum domain (EC ). In plants, the existence of SO has not yet been demonstrated, while sulfite reductase as part of sulfur assimilation is well characterized. Here we report the cloning of a plant sulfite oxidase gene from Arabidopsis thaliana and the biochemical characterization of the encoded protein (At-SO). At-SO is a molybdenum enzyme with molybdopterin as an organic component of the molybdenum cofactor. In contrast to homologous animal enzymes, At-SO lacks the heme domain, which is evident both from the amino acid sequence and from its enzymological and spectral properties. Thus, among eukaryotes, At-SO is the only molybdenum enzyme yet described possessing no redox-active centers other than the molybdenum. UV-visible and EPR spectra as well as apparent K(m) values are presented and compared with the hepatic enzyme. Subcellular analysis of crude cell extracts showed that SO was mostly found in the peroxisomal fraction. In molybdenum cofactor mutants, the activity of SO was strongly reduced. Using antibodies directed against At-SO, we show that a cross-reacting protein of similar size occurs in a wide range of plant species, including both herbacious and woody plants.

D-Aspartate oxidase and D-amino acid oxidase are localised in the peroxisomes of terrestrial gastropods

D-Aspartate oxidase and D-amino acid oxidase were found in high activity in the tissues of representative species of terrestrial gastropods. Analytical subcellular fractionation demonstrated that both of these oxidases co-localised with the peroxisome markers, acyl-CoA oxidase and catalase, in the digestive gland homogenate. Electron microscopy of peak peroxisome fractions showed particles of uniform size with generally well preserved variably electron-dense matrices bounded by an apparently single limiting membrane. Many of the particles exhibited a core region of enhanced electron density. Catalase cytochemistry of peak fractions confirmed the peroxisome identity of the organelles. Peroxisome-enriched subcellular fractions were used to investigate the properties of gastropod D-aspartate oxidase and D-amino acid oxidase activities. The substrate and inhibitor specificities of the two activities demonstrated that two distinct enzymes were present analogous to, but not identical to, the equivalent mammalian peroxisomal enzymes.

Antioxidative defense to lead stress in subcellular compartments of pea root cells.

Lead, similar to other heavy metals and abiotic factors, causes many unfavorable changes at the subcellular and molecular levels in plant cells. An increased level of superoxide anion in Pisum sativum root cells treated with 1 mM Pb(NO3)2 evidenced oxidative stress conditions. We found increased activities of enzymatic components of the antioxidative system (catalase and superoxide dismutase) in the cytosol, mitochondrial and peroxisomal fractions isolated from root cells of Pisum sativum grown in modified Hoagland medium in the presence of lead ions (0.5 or 1 mM). Two isoenzyme forms of superoxide dismutase (Cu,Zn-SOD and Mn-SOD) found in different subcellular compartments of pea roots were more active in Pb-treated plants than in control. Increased amount of alternative oxidase accompanied by an increased activity of this enzyme was found in mitochondria isolated from lead-treated roots. These results show that plants storing excessive amounts of lead in roots defend themselves against the harmful oxidative stress caused by this heavy metal.

Purification of brain peroxisomes and localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol biosynthesis. Because proper CNS development depends on de novo cholesterol biosynthesis, peroxisomes must play a critical functional role in this process. Surprisingly, no information is available on the peroxisomal isoprenoid/cholesterol biosynthesis pathway in normal brain tissue or on the compartmentalization of isoprene metabolism in the CNS. This has been due mainly to the lack of a well-defined isolation procedure for brain tissue, and also to the presence of myelin in brain tissue, which results in significant contamination of subcellular fractions. As a first step in characterizing the peroxisomal isoprenoid pathway in the CNS, we have established a purification procedure to isolate peroxisomes and other cellular organelles from the brain stem, cerebellum and spinal cord of the mouse brain. We demonstrate by use of marker enzymes and immunoblotting with antibodies against organelle specific proteins that the isolated peroxisomes are highly purified and well separated from the ER and mitochondria, and are free of myelin contamination. The isolated peroxisomal fraction was purified at least 40-fold over the original homogenate. In addition, we show by analytical subcellular fractionation and immunoelectron microscopy that HMG-CoA reductase protein and activity are localized both in the ER and peroxisomes in the CNS.

Antioxidant System within Yeast Peroxisome: BIOCHEMICAL AND PHYSIOLOGICAL CHARACTERIZATION OF CbPmp20 IN THE METHYLOTROPHIC YEAST CANDIDA BOIDINII

Candida boidinii Pmp20 (CbPmp20), a protein associated with the inner side of peroxisomal membrane, belongs to a recently identified protein family of antioxidant enzymes, the peroxiredoxins, which contain one cysteine residue. Pmp20 homologs containing the putative peroxisome targeting signal type 1 have also been identified in mammals and lower eukaryotes. However, the physiological function of these Pmp20 family proteins has been unclear. In this study, we investigated the biochemical and physiological functions of recombinant CbPmp20 protein in methanol-induced peroxisomes of C. boidinii using the PMP20-deleted strain of C. boidinii (pmp20Delta strain). The His(6)-tagged CbPmp20 fusion protein was found to have glutathione peroxidase activity in vitro toward alkyl hydroperoxides and H(2)O(2). Catalytic activity and dimerization of His(6)-CbPmp20 depended on the only cysteine residue corresponding to Cys(53). The pmp20Delta strain was found to have lost growth ability on methanol as a carbon and energy source. The pmp20Delta growth defect was rescued by CbPmp20, but neither CbPmp20 lacking the peroxisome targeting signal type 1 sequence nor CbPmp20 haboring the C53S mutation retrieved the growth defect. Interestingly, the pmp20Delta strain had a more severe growth defect than the cta1Delta strain, which lacks catalase, another antioxidant enzyme within the peroxisome. During incubation of these strains in methanol medium, the cta1Delta strain accumulated H(2)O(2), whereas the pmp20Delta strain did not. Therefore, it is speculated to be the main function of CbPmp20 is to decompose reactive oxygen species generated at peroxisomal membrane surface, e.g. lipid hydroperoxides, rather than to decompose H(2)O(2). In addition, we detected a physiological level of reduced glutathione in peroxisomal fraction of C. boidinii. These results may indicate a physiological role for CbPmp20 as an antioxidant enzyme within peroxisomes rich in reactive oxygen species.

Induction of a peroxisomal malate dehydrogenase isoform in liver of starved rats.

The influence of starvation on malate dehydrogenase (MDH) in rat liver was investigated. Native electrophoresis revealed two MDH isoforms in non-starved rats and three isoenzymes in starved rats. After sucrose density gradient centrifugation of cell organelles from liver, MDH activity was detected in the mitochondrial and cytosolic fractions from non-starved rats. However, additional activity was found in the peroxisomal fraction from starved rats. The latter was identified as the electrophoretically new isoform in starved animals. The three isoforms of malate dehydrogenase from hepatocytes were separated and partially purified by chromatography on DEAE-Toyopearl. Several kinetic and regulatory properties of the three isoforms were rather similar. It is suggested that the newly expressed isoform of MDH operates in the glyoxylate cycle of liver peroxisomes of food-starved animals.

Hepatic microsomal and peroxisomal docosahexaenoate biosynthesis during piglet development.

The roles of peroxisomes and microsomes on the biosynthetic pathway for docosahexaenoic acid (DHA) from alpha-linolenic acid (ALA) were investigated. Microsomes and peroxisomes were prepared from livers of fetal and neonatal piglets by a combination of differential and gradient layer centrifugation. Microsomes, peroxisomes, and combined cell fractions were incubated with [13C-U]18:3n-3. The [M] and [M + 18] isotopomers of the fatty acids in the long-chain polyunsaturated fatty acid (LCPUFA) n-3 pathway were detected by gas chromatography-mass spectrometry. The quantity of each fatty acid was determined by gas chromatography, and synthesis of each fatty acid was calculated for a 30-min period. Synthesis of DHA was not detected in combined fetal liver fractions. The data suggest that DHA in the fetus is probably supplied from maternal sources through the placenta. In either singly incubated microsomal or peroxisomal preparations from neonatal livers, no DHA synthesis was detected. After combination of the microsomal and peroxisomal fractions, DHA synthesis was evident and increased rapidly between birth and 2 wk of age. This is the first demonstration of the entire biosynthetic LCPUFA n-3 pathway in subcellular organelles starting from isotopically labeled ALA to the final product, DHA, with all the intermediates present and isotopically labeled. The primary importance of the data is that it unequivocally demonstrates that peroxisomes are required for biosynthesis of DHA from ALA.

Characterization of Peroxisomal Pex5p from Rat Liver: Pex5p IN THE Pex5p-Pex14p MEMBRANE COMPLEX IS A TRANSMEMBRANE PROTEIN

Pex5p is the receptor for the vast majority of peroxisomal matrix proteins. Here, we show that about 15% of rat liver Pex5p is found in the peroxisomal fraction representing 0.06% of total peroxisomal protein. This population of Pex5p displays all the characteristics of an intrinsic membrane protein. Protease protection assays indicate that this pool of Pex5p has domains exposed on both sides of the peroxisomal membrane. The strong interaction of Pex5p with the membrane of the organelle is not affected by mild protease treatment of intact organelles, conditions that result in the partial degradation of Pex13p. Cytosolic Pex5p is a monomeric protein. In contrast, virtually all peroxisomal Pex5p was found to be part of a stable 250-kDa protein assembly. This complex was isolated and shown to comprise just two subunits, Pex5p and Pex14p.

Interaction of peroxisomes with microtubules. In vitro studies using a novel peroxisome-microtubule binding assay.

The association of membrane-bounded cell organelles to microtubules is crucial for determination of their shape, intracellular localization and translocation. We have shown previously the high affinity binding of peroxisomes to microtubules which appears to be of static nature as in vivo studies indicate that only a few peroxisomes move along the microtubular tracks. In order to characterize the interactions of peroxisomes with microtubules, we have developed a semiquantitative in vitro binding assay, which is based on the association of highly purified rat liver peroxisomes to microtubules coated onto microtiterplates. The binding was visualized by differential interference contrast and immunofluorescence using a confocal laser scanning microscope. The binding was concentration dependent and saturable, being affected by time, temperature, and pH. Addition of ATP or the motor proteins kinesin and dynein increased the binding capacity, while ATP-depletion or microtubule associated proteins (MAPs) decreased it. KCl treatment of peroxisomes reduced the binding, which was restored by dialyzed KCl-stripping eluate as well as by rat liver cytosol. The reconstituting effect of cytosol was abolished by its pretreatment with proteases or N-ethylmaleimide. Moreover, the treatment of peroxisomes with proteases or N-ethylmaleimide reduced their binding, which was not reversed by cytosol. These results suggest the involvement of a peroxisomal membrane protein and cytosolic factor(s) in the binding of peroxisomes to microtubules. This notion is supported by the observation that distinct subfractions of dialyzed KCl-stripping eluate obtained by gel chromatography augmented the binding. Those subfractions, as well as purified peroxisome fractions, exhibited strong immunoreactivity with an antibody to cytoplasmic linker protein (CLIP)-115, revealing a 70-kDa polypeptide. Moreover, immunodepletion of KCl-stripping eluate and its subfractions with an antibody to the conserved microtubule binding domain of CLIPs, abolished their promoting effect on the binding, thus suggesting the involvement of a CLIP-related protein in the binding of peroxisomes to microtubules.

Subcellular organization of bile acid amidation in human liver: a key issue in regulating the biosynthesis of bile salts

To extend our knowledge of how the synthesis of free bile acids and bile salts is regulated within the hepatocyte, bile acid-CoA:amino acid N-acyltransferase and bile acid-CoA thioesterase activities were measured in subcellular fractions of human liver homogenates. Some bile acids, both conjugated and unconjugated, have been reported to be natural ligands for the farnesoid X receptor (FXR), an orphan nuclear receptor. The conversion of [14C]choloyl-CoA and [14C]chenodeoxycholoyl-CoA into the corresponding tauro- and glyco-bile acids or the free bile acids was measured after high-pressure liquid radiochromatography. There was an enrichment of the N-acyltransferase in the cytosolic and the peroxisomal fraction. Bile acid-CoA thioesterase activities were enriched in the cytosolic, peroxisomal, and mitochondrial fractions. The highest amidation activities of both choloyl-CoA and chenodeoxycholoyl-CoA were found in the peroxisomal fraction (15–58 nmol/mg protein/min). The Km was higher for glycine than taurine both in cytosol and the peroxisomal fraction.These results show that the peroxisomal de novo synthesis of bile acids is rate limiting for peroxisomal amidation, and the microsomal bile acid-CoA synthetase is rate limiting for the cytosolic amidation. The peroxisomal location may explain the predominance of glyco-bile acids in human bile. Both a cytosolic and a peroxisomal bile acid-CoA thioesterase may influence the intracellular levels of free and conjugated bile acids.

Insulin-degrading enzyme exists inside of rat liver peroxisomes and degrades oxidized proteins.

Insulin-degrading enzyme (IDE) was detected by immunoblot analysis in highly purified rat liver peroxisomes. IDE in the peroxisomal fraction was resistant to proteolysis by trypsin and chymotrypsin under conditions where the peroxisomal membranes remained intact. After sonication of the peroxisomal fraction, IDE was recovered in the supernatant fraction. Further, the localization of IDE in the peroxisomes was shown by immunoelectron microscopy. In addition, IDE isolated from peroxisomes degraded insulin as well as oxidized lysozyme as a model substrate for oxidized proteins. These results suggest that IDE exists in an active form in the matrix of rat liver peroxisomes and is involved in elimination of oxidized proteins in peroxisomes.

Presence of choloyl- and chenodeoxycholoyl-coenzyme A thioesterase activity in human liver

In human liver homogenate the formation of bile acid-CoA thioesters is localized both to the microsomal fraction catalysed by an ATP-dependent synthetase and to the peroxisomal fraction catalysed by the thiolase in the last step of the beta-oxidative cleavage of the 5beta-cholestanoyl side chain. The cytosolic bile acid-CoA:amino acid N-acyltransferase catalyse the conjugation of the CoA-activated bile acids with taurine or glycine prior to secretion into bile. The formation of bile acid-CoA esters is considered the rate-limiting step in bile acid amidation. So far, a bile acid-CoA cleaving activity has not been assessed in the research of bile acid amidation in human liver. In this work, a bile acid-CoA cleaving activity has been demonstrated at a rate that may influence the concentration of bile acid-CoA thioesters, free bile acids and amidated bile acids within the hepatocyte. Recently, it was shown that free chenodeoxycholic acid, formed by the thioesterase, is the physiological ligand of the farnesoid X receptor. A multiorganelle distribution of the bile acid-CoA hydrolytic activity was found. In the postnuclear fraction of human liver homogenate, apparent Km and Vmax for the cleavage of choloyl-CoA were 7.7 x 10-5 mol/L and 3.6 nmol x mg-1 x min-1 respectively. The corresponding values for chenodeoxycholoyl-CoA cleavage were 7.1 x 10-5 mol/L and 4.8 nmol x mg-1 x min-1. Hydrolytic activities were detected in the microsomal and the peroxisomal fractions where the bile acid-CoA esters are formed as well as in cytosol housing the N-acyltransferase activity. Compared to the bile acid-CoA synthetase activities, the hydrolytic activities were considerably higher, both in the postnuclear fraction and in the microsomal fraction. The thioesterase activities were in the same range as detected for the N-acyltransferase activities both in the postnuclear fraction and in the cytosolic fraction. The mere presence of thioesterase in microsomes, peroxisomes and cytosol seems counterproductive to bile acid amidation. The thioesterases may have an indirect regulatory function on the bile acid synthesis and are important for the regulation of bile acid synthesis by providing free chenodeoxycholic acid, the most potent activator of the farnesoid X receptor.

Peroxisomal and mitochondrial targeting of serine:pyruvate/alanine:glyoxylate aminotransferase in rat liver.

Serine:pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) of rat liver is a unique enzyme of dual subcellular localization, and exists in both mitochondria and peroxisomes. To characterize a peroxisomal targeting signal of rat liver SPT, a number of C-terminal mutants were constructed and their subcellular localization in transfected COS-1 cells was examined. Deletion of C-terminal NKL, and point mutation of K2 (the second Lys from the C-terminus), K4 and E15 caused accumulation of translated products in the cytoplasm. This suggests that the PTS of SPT is not identical to PTS1 (the C-terminal SKL motif) in that it is not restricted to the C-terminal tripeptide. In vitro synthesized precursor for mitochondrial SPT was highly sensitive to the proteinase K digestion, whereas peroxisomal SPT (SPTp) was fairly resistant to the protease. In in vitro import experiment with purified peroxisomes, however, SPTp recovered in the peroxisomal fraction was very sensitive to the protease. These results suggest that the mitochondrial precursor is synthesized as an unfolded form and is translocated into the mitochondrial matrix, whereas SPTp is synthesized as a folded form and its conformation changes to an unfolded form just before translocation into peroxisomes.

Very-long-chain fatty acid metabolism in adrenoleukodystrophy protein-deficient mice.

X-linked adrenoleukodystrophy (X-ALD) is characterized by progressive mental and motor deterioration, with demyelination of the central and peripheral nervous system. Its principal biochemical abnormality is the accumulation of very-long-chain fatty acids (VLCFAs) in tissues and body fluids, caused by the impairment of peroxisomal beta-oxidation. The authors have generated a line of mice deficient in ALD protein (ALDP) by gene targeting. ALDP-deficient mice appeared normal clinically, at least up to 12 mo. Western blot analysis showed absence of ALDP in the brain, spinal cord, lung, and kidney. The amounts of C26:0 increased by 240% in the spinal cord. VLCFA beta-oxidation in cultured hepatocytes was reduced to 50% of normal. The authors investigated the roles of ALDP in VLCFA beta-oxidation using the ALDP-deficient mice. Very-long-chain acyl-CoA synthetase (VLACS) is functionally deficient in ALD cells. The impairment of VLCFA beta-oxidation in the ALDP-deficient fibroblasts was not corrected by over-expression of VLACS only, but was done by co-expression of VLACS and ALDP, suggesting that VLACS requires ALDP to function. VLACS was detected in the peroxisomal and microsomal fractions of the liver from both types of mice. Peroxisomal VLACS was clearly decreased in the ALDP-deficient mouse. Thus, ALDP is involved in the peroxisomal localization of VLACS.

Peroxisomal NADP-Dependent Isocitrate Dehydrogenase. Characterization and Activity Regulation during Natural Senescence.

The peroxisomal localization and characterization of NADP-dependent isocitrate dehydrogenase (perICDH) in young and senescent pea (Pisum sativum) leaves was studied by subcellular fractionation, kinetic analysis, immunoblotting, and immunoelectron microscopy. The subunit molecular mass for perICDH determined by immunoblotting was 46 kD. By isoelectric focusing (IEF) of the peroxisomal matrix fraction, the NADP-ICDH activity was resolved into four isoforms, perICDH-1 to perICDH-4, with isoelectric points (pIs) of 6.0, 5.6, 5.4, and 5.2, respectively. The kinetic properties of the NADP-ICDH in peroxisomes from young and senescent pea leaves were analyzed. The maximum initial velocity was the same in peroxisomes from young and senescent leaves, while the Michaelis constant value in senescent leaf peroxisomes was 11-fold lower than in young leaf peroxisomes. The protein levels of NADP-ICDH in peroxisomes were not altered during senescence. The kinetic behavior of this enzyme suggests a possible fine control of enzymatic activity by modulation of its Michaelis constant during the natural senescence of pea leaves. After embedding, electron microscopy immunogold labeling of NADP-ICDH confirmed that this enzyme was localized in the peroxisomal matrix. Peroxisomal NADP-ICDH represents an alternative dehydrogenase in these cell organelles and may be the main system for the reduction of NADP to NADPH for its re-utilization in the peroxisomal metabolism.