Reactive oxygen species (ROS) can surely be considered as multifunctional biofactors within the cell. They are known to participate in regular cell functions, for example, as signal mediators, but overproduction under oxidative stress conditions leads to deleterious cellular effects, cell death and diverse pathological conditions. Peroxisomal function has long been linked to oxygen metabolism due to the high concentration of H(2)O(2)-generating oxidases in peroxisomes and their set of antioxidant enzymes, especially catalase. Still, mitochondria have been very much placed in the centre of ROS metabolism and oxidative stress. This review discusses novel findings concerning the relationship between ROS and peroxisomes, as they revealed to be a key player in the dynamic spin of ROS metabolism and oxidative injury. An overview of ROS generating enzymes as well as their antioxidant counterparts will be given, exemplifying the precise fine-tuning between the opposing systems. Various conditions in which the balance between generation and scavenging of ROS in peroxisomes is perturbed, for example, exogenous manipulation, ageing and peroxisomal disorders, are addressed. Furthermore, peroxisome-derived oxidative stress and its effect on mitochondria (and vice versa) are discussed, highlighting the close interrelationship of both organelles.

The classic method of Leighton et al. [(1968) J. Cell Biol. 37, 482-5131 for the isolation of peroxisomes from rat liver involves the use of Triton WR-1339 which alters the biochemical properties of this organelle and requires the specialized type Beaufay-rotor which is not easily available. We have employed Metrizamide as the gradient medium and a commercial type vertical rotor to obtain highly purified and structurally well-preserved peroxisomes from normal untreated animals.The livers were homogenized in buffered 0.25 M sucrose and a slightly modified 'light mitochondria1 fraction' was prepared by differential centrifugation. This was loaded on top of a linear Metrizamide gradient (1.12 -1.26 g/cm3) and subjected to an integrated force of 1.252 x lo6 x (g x min) using a Beckman VTi 50 vertical rotor. Peroxisomes banded at the density of 1.245 g/cm3. In the isolated fraction 95% of the protein was contributed by peroxisomes, which exhibited a strong activity for cyanide-insensitive lipid p-oxidation. The purity of fractions was also confirmed by morphometry, which revealed that 98% of isolated particles consisted of peroxisomes. The latency for catalase was about 90% indicating a high degree of peroxisomal integrity. This corresponded to the low level of extraction of catalase in 3,3'-diaminobenzidine-~tained filter preparations. The entire procedure took about five hours. Highly purified and structurally well preserved peroxisomes should be useful in further elucidation of the function of this organelle and especially in studies of peroxisomal enzymes with multiple intracellular localizations.A simple and reproducible procedure for the isolation of an organelle in highly purified and morphologically well preserved form facilitates its detailed investigation. As far as the peroxisomes are concerned there are three main obstacles : (a) their relative paucity (in liver they make up 2% of the total protein); (b) their density in sucrose, which is very close to that of mitochondria and lysosomes; and (c) their extreme fragility. In the past fifteen years, several methods for the isolation of this organelle have been described [l -

Most newly synthesized peroxisomal proteins are imported in a receptor-mediated fashion, depending on the interaction of a peroxisomal targeting signal (PTS) with its cognate targeting receptor Pex5 or Pex7 located in the cytoplasm. Apart from this classic mechanism, heterologous protein complexes that have been proposed more than a decade ago are also to be imported into peroxisomes. However, it remains still unclear if this so-called piggyback import is of physiological relevance in mammals. Here, we show that Cu/Zn superoxide dismutase 1 (SOD1), an enzyme without an endogenous PTS, is targeted to peroxisomes using its physiological interaction partner 'copper chaperone of SOD1' (CCS) as a shuttle. Both proteins have been identified as peroxisomal constituents by 2D-liquid chromatography mass spectrometry of isolated rat liver peroxisomes. Yet, while a major fraction of CCS was imported into peroxisomes in a PTS1-dependent fashion in CHO cells, overexpressed SOD1 remained in the cytoplasm. However, increasing the concentrations of both CCS and SOD1 led to an enrichment of SOD1 in peroxisomes. In contrast, CCS-mediated SOD1 import into peroxisomes was abolished by deletion of the SOD domain of CCS, which is required for heterodimer formation. SOD1/CCS co-import is the first demonstration of a physiologically relevant piggyback import into mammalian peroxisomes.

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.

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We have studied the effects of TNF-a on the mRNAs coding for the peroxisome proliferator activated receptor a (PPAR-a), and for catalase (Cat), acyl-CoA oxidase (AOX), multifunctional enzyme (PH), and P-actin in rat liver. Total RNA was isolated from livers of male SD-rats 16 h after administration of a single dose of 25 u.g TNF-a and mRNAs were analyzed by a novel dot blot RNase protection assay. The mRNAs for PPAR-a and for Cat, AOX and PH were significantly reduced by TNF-treatment. In addition, the level of PPAR-a protein was also decreased after TNF. In contrast, the mRNA for P-actin was markedly increased implying that the effect of TNF on PPAR-a and the peroxisomal mRNAs is highly selective. This effect may have important implications in perturbation of the lipid metabolism induced by TNF-a.

The aim of the present study was to compare the plasma and erythrocyte fatty acid (FA) status in a cohort of 200 women over 75 yr of age living at home with that of a control group of 50 young female volunteers aged 20-48 yr. The data were related to the dietary habits and food intakes which were evaluated by two different methods of dietary investigation. The FA composition of total plasma lipids, plasma triglycerides, cholesterol esters (CE), and erythrocytes were determined by capillary column gas-liquid chromatography using H 2 as carrier gas.The n-6 series precursor linoleic acid (18:2n-6) was significantly lower in elderly women (EW) than in the control group in plasma triglycerides and CE (P = 0.029 and P = 0.014, respectively), suggesting that dietary intake of this essential fatty acid was lower in this group than in the control group. This is supported by the high correlation we observed between linoleic acid in CE and vegetables, vegetal fat, .polyunsaturated fatty acid and vitamin E intakes (P < 0.001 each). In CE, the n-3 series precursor o~-linolenic acid (18:n-3) was also lower in EW (P < 0.04) and was highly correlated to carbohydrates, bread, fibers, and vegetal protein intakes (P < 0.0001 each), suggesting again a lower dietary supply. In agreement with this observation, palmitoleic acid (16: I n-7) in CE was higher in the EW group (P < 0.0001), an observation supported by logistic regression analysis showing that higher 16:1n-7 values were associated with the EW group in all plasma lipid fractions. These data show a tendency to EFA deficiency in the EW group.The 20:4n-6/20:3n-6 ratio was lower in CE and phospholipids (P < 0.02 and P < 0.005, respectively) for EW, suggesting some impairment in the A5 desaturation, whereas 18:3n-6/18:2n-6 ratio was not reduced in EW, supporting an unaltered A6-desaturase activity. Contrasting with the suspected lower linoleic acid intake and A5-desaturase activity, no difference was observed in arachidonic acid levels. Dietary investigations suggested that high dietary protein intakes largely contributed in EW to 20:4n-6 supply by diet meat.In PL, 22:6n-3/20:5n-3 was lower in EW (P < 0.002), suggesting impairment in the apparent "A4-desaturase" pathway. Since A6-desaturase capacity seemed unchanged, alteration of the peroxisomat retroconversion could be implicated. Such a peroxisomal defect has been observed in aging rodents (1,2), whereas increased A4-activity was observed in fibratetreated rats (3). A fragile equilibrium largely dependent on exogenous supply of long chain FA characterizes the essential fatty acid status of the group of EW studied. It is suggested that (i) the high protein intake contributing to 20:4n-6 homeostasis in free living EW should be maintained and (ii) caution is required before dietary supplementation with large amounts of very long chain polyunsaturated fatty acids in these populations until potential peroxisomal [3-oxidation deficiency can be ruled out.

Abstract. According to Poole et al. (1970. J. Cell Biol. 45:408-415), newly synthesized peroxisomal proteins are incorporated uniformly into peroxisomes (PO) of different size classes, suggesting that rat hepatic PO form a homogeneous population. There is however increasing cytochemical and biochemical evidence that PO in rat liver are heterogenous, undergoing significant modulations in shape and size in process of PO morphogenesis (Yamamoto and Fahimi, 1987. J. Cell Biol. 105:713-722). In the present study, the kinetics of incorporation of newly synthesized proteins into distinct PO-subpopulations have been studied using short-term in vivo labeling (5-90 min). Two distinct "heavy" and "light" crude PO fractions were prepared by differential pelleting from normal and regenerating liver, and highly purified PO were subsequently isolated by density-dependent metrizamide gradient centrifugation according to V61kl and Fahimi (1985. Eur. J. Biochem. 149:25%265). The peroxisomal fractions banded at 1.20 and 1.24 g x cm -3. They differed in their mean diameters and form-factors and particularly in respect to the activity of/3-oxidation enzymes which was higher in the "light" PO. Whereas the "light" PO exhibited a single immunoreactive band with the antibody to the 70-kD peroxisomal membrane protein the "heavy" PO contained an additional (68 kD) band. In pulse-labeling experiments "light" PO showed clearly a higher initial rate of incorporation than the "heavy" PO. The relative specific activity in the "heavy" PO fraction, however increased progressively reaching that of "light" PO by 90 min. These observations provide evidence for the existence of different PO populations in rat liver which differ in their morphological and biochemical properties as well as in their rates of incorporation of new proteins.