Intact Peroxisomes Research Articles

The presence of a short redox chain in the membrane of intact potato tuber peroxisomes and the association of malate dehydrogenase with the peroxisomal membrane

The presence of a short redox chain in the membrane of intact potato tuber peroxisomes and the association of malate dehydrogenase with the peroxisomal membrane

Synthesis and subcellular location of peroxisomal membrane proteins in a peroxisome-deficient mutant of the yeast Hansenula polymorpha.

We have studied the synthesis and subcellular location of peroxisomal membrane proteins (PMPs) in cells of a peroxisome-deficient (per) mutant of the methylotrophic yeast Hansenula polymorpha. Western blot analysis of methanol-induced cells of the per mutant, which had been growing in a continuous culture on a glucose/methanol mixture, indicated that various PMPs were normally synthesized. As in wild type (WT) cells, the levels of PMP synthesis appeared to be dependent on specific cultivation conditions, e.g. the carbon source used for growth. In contrast to WT controls, PMPs in methanol-induced per mutants were not subject to proteolytic degradation. Biochemical and immuno(cyto)chemical studies suggested that the PMPs in methanol-induced per cells were located in small proteinaceous aggregates, separated from peroxisomal matrix proteins that were also present in the cytosol. Vesicular membranous structures, resembling the morphology of intact peroxisomes, were never detected irrespective of the growth conditions employed.

Histochemistry of peroxisomal enzyme activities: a tool in the diagnosis of Zellweger syndrome.

The localization of the activity of the peroxisomal enzymes D-amino acid oxidase and hydroxyacid oxidase was studied at the light-microscopical level in livers and kidneys of control subjects and patients with Zellweger syndrome, an inherited disease characterized by a lack of intact peroxisomes. D-Amino acid oxidase and hydroxyacid oxidase activities were demonstrated in unfixed cryostat sections with the cerium-diaminobenzidine-cobalt-hydrogen peroxide procedure, in which cerium ions capture hydrogen peroxide, the product of both enzymes. In a second step reaction decomposition of cerium perhydroxide gives rise to a diaminobenzidine polymer complexed with cobalt ions. D-Amino acid oxidase and hydroxyacid oxidase activities were found in peroxisomes of liver parenchymal cells, and only D-amino acid oxidase in peroxisomes of proximal tubular cells of kidneys of control humans. The activities of these enzymes were not detectable in livers and kidneys of Zellweger patients. It is concluded that the cerium-diaminobenzidine-cobalt-hydrogen peroxide procedure enables the demonstration of peroxisomal enzyme activities in human tissues at the light-microscopical level and is an important tool in detecting patients with Zellweger syndrome.

Exclusive localization in peroxisomes of dihydroxyacetone phosphate acyltransferase and alkyl-dihydroxyacetone phosphate synthase in rat liver.

Dihydroxyacetone phosphate acyl transferase (DHAP-AT), alkyl dihydroxyacetone phosphate synthase (alkyl-DHAP-synthase), and glycerol-3-phosphate acyltransferase (GPAT) activities were investigated under optimal assay conditions using highly purified organelle preparations. The data presented clearly indicate that GPAT activity was mainly localized in mitochondria and microsomes, whereas DHAP-AT and alkyl-DHAP-synthase activities were exclusively localized in peroxisomes. A small fraction of the total DHAP-AT and alkyl-DHAP-synthase activities observed in purified mitochondrial preparations was due to the presence of intact peroxisomes. DHAP-AT and alkyl-DHAP-synthase activities were very low in purified microsomes (< 1% compared to peroxisomes) and these activities are thought to be due to sedimentation of peroxisomal fragments (generated during homogenization of liver and processing of liver homogenate) with microsomes. The results indicate that the dihydroxyacetone phosphate pathway does not contribute to the synthesis of glycerolipids other than ether lipids in rat liver. The ether bond formation occurs exclusively in peroxisomes, and all the biosynthetic reactions for plasmalogen synthesis may also be operating within peroxisomes in rat liver.

Expression and targeting of a 47 kDa integral peroxisomal membrane protein of Candida boidinii in wild type and a peroxisome-deficient mutant of Hansenula polymorpha

A 47 kDa integral peroxisomal membrane protein (PMP47) of Candida boidinii was expressed in wild type (WT) and a temperature-sensitive (Ts6) peroxisome-deficient ( per) mutant of Hansenula polymorpha. The subcellular location of PMP47 appeared to be dependent on the level of expression. At low expression levels PMP47 was sorted to the peroxisomal membrane; however, in Ts6 cells grown at restrictive temperatures (which lack intact peroxisomes) PMP47 was solely located in small cytosolic aggregates, together with homologous H. polymorpha PMP's. At enhanced expression levels, however, part of the protein also became incorporated into mitochondria, both in transformed WT and Ts6 cells.

Membrane topology of the 22 kDa integral peroxisomal membrane protein

In order to study the membrane topology and the possible function of the rat liver 22 kDa integral peroxisomal membrane protein (PMP 22) at a molecular level, we have cloned PMP 22 from a λgt11 expression library and sequenced its cDNA. Hydropathy analysis of the deduced primary structure indicates 4 putative transmembrane segments. The accessibility to exogenous aminopeptidase of PMP 22 in intact peroxisomes suggests that the N-terminus faces the cytosol. A model of the topology of PMP 22 in the peroxisomal membrane is discussed. Homology studies revealed a striking similarity with the Mpv 17 gene product. Lack of this membrane protein causes nephrotic syndrome in mice.

Mitochondrial and peroxisomal fractions derived from human white adipocytes

1.1. A procedure is described for the separation of intact peroxisomes from human white adipocytes using a linear metrizamide gradient (20–50% w/v).2.2. Peroxisomes were found in the high density region of the gradient in an intact form.3.3. Mitochondria were distributed in the high density and low density regions of the gradient.4.4. Lysosomes separated well from the peroxisomes, occurring only in the low density region of the gradient.5.5. Low levels of glyoxylate cycle enzyme activities (isocitrate lyase and malate synthase) were detected within the light and heavy mitochondrial pellet fractions.

Isolation and characterization of peroxisomal protein import (Pim−) mutants of Hansenula polymorpha

AbstractIn the course of our studies on the molecular mechanisms involved in peroxisome biogenesis, we have isolated several mutants of the methylotrophic yeast Hansenula polymorpha impaired in the import of peroximal matrix proteins. These mutants are characterized by the presence of small intact peroxisomes, while the bulk of the peroxisomal matrix protein is not imported and resides in the cytosol (Pim− phenotype). Genetic analysis of back‐crossed mutants revealed five different complementation groups, which were designated PERI–PER5. Mapping studies to determine the linkage relationships indicated that the observed Pim− phenotypes were determined by single recessive nuclear mutations.The different mutants had comparable phenotypes: (i) they were impaired to utilize methanol as the sole source of carbon and energy but grew well on various other compounds, including nitrogen sources, the metabolism of which is known to be mediated by peroxisome‐borne enzymes in wild‐type cells; (ii) all peroxisomal enzymes tested were induced, assembled and activated as in wild‐type cells although their activities varied between the different representative mutants; (iii) all peroxisomal proteins, whether constitutive or inducible, were found both in the cytosol and in the small peroxisomes. These results suggest that a general, major import mechanism is affected in all mutants.

Malonyl-CoA inhibition of peroxisomal carnitine octanoyltransferase.

Although the malonyl-CoA sensitivity of peroxisomal carnitine octanoyltransferase (COT) is reportedly lost on solubilization, we show that malonyl-CoA does inhibit the purified enzyme. Assay conditions such as buffer composition, pH, acyl-CoA substrate and the presence or absence of BSA can affect the observed inhibition. When assayed in the absence of BSA, COT shows simple competitive inhibition by malonyl-CoA. The Ki value for inhibition of purified COT is high (106 microM) compared with physiological concentrations (1-6 microM) and other short-chain acyl-CoA esters inhibit COT to the same degree. However, when COT is assayed in intact peroxisomes, the Ki for malonyl-CoA is almost 20-fold lower than found with the purified enzyme, whereas inhibition by other short-chain acyl-CoA esters does not change significantly. Several features of the inhibition of peroxisomal COT, including the specificity of malonyl-CoA over other short-chain acyl-CoA esters, resemble those of carnitine palmitoyltransferase (CPT)-I, suggesting that the regulation of COT and CPT-I in parallel may be necessary for the control of cellular fatty acid metabolism.

Induction and characterization of two types of ATPase on rat liver peroxisomes.

Clofibrate increased oligomycin-resistant ATPase activity in peroxisomes more than 17-fold (5.15 +/- 0.71 milliunits/mg protein) in rat liver. The activity was dependent on divalent cations (Mg2+ > Ca2+) with an optimum pH of 7.5. This activity was partially inhibited by N-ethylmaleimide (NEM), 4,4'-dithiocyanatostilbene-2,2'-disulfonic acid (DIDS), silicotungstic acid (STA), and high concentrations of N,N'-dicyclohexylcarbodiimide (DCCD). Proteinase K digestion of intact peroxisomes severely reduced the NEM-sensitive activity, but little affected the NEM-resistant activity. NEM-sensitive and -resistant ATPases showed Km values for ATP of 780 and 73 microM, respectively. The NEM-sensitive activity was inhibited completely by DIDS, 7-chloro-4- nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), tributyltin chloride (TBT), and quercetin, and partially by DCCD and STA, whereas the NEM-resistant activity was totally insensitive to these chemicals except for STA. These activities had unique requirements for divalent cations, anions, and substrates, respectively. They were partially separated by gel filtration chromatography and had molecular masses of 520 kDa (NEM-sensitive enzyme) and 450 kDa (NEM-resistant enzyme), respectively.

Partial phenotypic suppression of a peroxisome-deficient animal cell mutant treated with aminoglycoside G418.

Certain enzymes normally associated with peroxisomes, such as the dihydroxyacetone phosphate (DHAP) acyltransferase involved in plasmalogen biosynthesis, are present at low levels in peroxisome-deficient mutants of Chinese hamster ovary (CHO) cells. We now show that the aminoglycoside G418 increases the residual DHAP acyltransferase in mutant ZR-82 by 60-fold. This is accompanied by a dose- and time-dependent restoration of the plasmalogen content. G418 treatment of ZR-82 also increases residual peroxisomal beta-oxidation activity by 3.8-fold. G418 does not affect wild-type CHO cells (CHO-K1) or a different peroxisome-deficient mutant, ZR-78.1. The effects of G418 on ZR-82 are transient, since plasmalogens and DHAP-acyltransferase decline to basal levels 5 days after G418 withdrawal. Other aminoglycosides and lysosomotropic agents do not alter plasmalogen levels in ZR-82. The subcellular distribution of catalase (an enzyme of the peroxisomal matrix which is present in normal amounts in peroxisome-deficient mutants but is mislocalized in the cytosol) is unaffected by G418 treatment of ZR-82, demonstrating that G418 does not restore peroxisomes. Localization of catalase by immunofluorescence microscopy confirms a total absence of intact peroxisomes in ZR-82, either before or after exposure to G418. This study is the first to demonstrate that some peroxisome-deficient mutants can be induced to accumulate functional DHAP acyltransferase and other peroxisomal enzymes, usually missing in the absence of peroxisomes. G418 may have some therapeutic value in selected patients with inborn errors of peroxisome assembly, such as Zellweger syndrome.

Increased cholesterol synthesis in Chinese hamster ovary cells deficient in peroxisomes

In a previous study we have shown that Chinese hamster ovary (CHO) cells deficient in intact peroxisomes, lack the nonspecific lipid transfer protein (nsL-TP; sterol carrier protein 2) (van Heusden, G.P.H., Bos, K., Raetz, C.R.H. and Wirtz, K.W.A. (1990) J. Biol. Chem. 265, 4105-4110). The consequences of the absence of peroxisomes and of nsL-TP on intracellular cholesterol metabolism have been investigated in two peroxisome-deficient CHO cell lines (CHO-82 and CHO-78). Compared with wild-type cells (CHO-K1), the incorporation of [3H]acetate into cholesterol was 3-fold higher in the CHO-82 cells and 2-fold higher in the CHO-78 cells. In agreement with an increased synthesis of cholesterol, a 2-3-fold higher 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was measured in both mutant cell lines. On the other hand, addition of low density lipoprotein (LDL), mevalonate (30 mM) or 25-hydroxycholesterol (2 micrograms/ml) to cells grown in lipoprotein-deficient serum, demonstrated that in both mutant cell lines the down-regulation of HMG-CoA reductase and of cholesterol synthesis were comparable to that in wild-type cells. These results strongly suggest that, in addition to down-regulation by LDL-derived cholesterol, mevalonate and 25-hydroxycholesterol, HMG-CoA reductase activity is under control of peroxisomes and/or nsL-TP.

Ischemia-reperfusion injury: Biochemical alterations in peroxisomes of rat kidney

Exogenously supplied catalase, a peroxisomal enzyme, has been found to be of therapeutic value in ischemic injury. Therefore, we examined the effect of ischemic-reperfusion injury on the structure and function of kidney peroxisomes. Ischemic injury changed the density of peroxisomes from 1.21 g/cm 3 (peak I) to a lighter density of 1.14 g/cm 3 (peak II). The number of peroxisomes moving from the normal density population (peak I) to a lower density population (peak II) increased with an increase in ischemic injury. Latency experiments indicated both populations of peroxisomes to be of intact peroxisomes. Immunoblot analysis with antibodies against peroxisomal matrix and membrane proteins demonstrated that after 90 min of ischemia a significant number of matrix proteins were lost in the peak II population, suggesting that functions of these peroxisomes may be severally affected. Reperfusion following ischemic injury resulted in loss of peroxisomal matrix proteins in both peaks I and II, suggesting that peroxisomal functions may be drastically compromised. This change in peroxisomal functions is reflected by a significant decrease in peroxisomal catalase activity (35%) and β-oxidation of lignoceric acid (43%) observed following 90 min of ischemia. The decrease in catalase activity was more pronounced in reperfused kidneys even after a shorter term of ischemic injury. Reperfusion restored the normal peroxisomal β-oxidation in kidneys exposed up to 60 min of ischemia. However, 90 min of ischemia was irreversible as there was a further decrease in β-oxidation upon reperfusion. The decrease in catalase activity during ischemia alone was due to the formation of an inactive complex, whereas during reperfusion, following 90 min of ischemia, inactivation and proteolysis or decreased synthesis of catalase contributed equally toward the injury. The observed changes in the structure and function of peroxisomes as a result of ischemic-reperfusion injury and the ubiquitous distribution of peroxisomes underlines the importance of this organelle in the pathophysiology of vascular injury in general.

Metabolism of prostaglandin F2 alpha in Zellweger syndrome. Peroxisomal beta-oxidation is a major importance for in vivo degradation of prostaglandins in humans.

We have recently shown in vitro that the peroxisomal fraction of a rat liver homogenate has the highest capacity to beta-oxidize prostaglandins. In order to evaluate the relative importance of peroxisomes for this conversion also in vivo, we administered [3H]prostaglandin F2 alpha to an infant suffering from Zellweger syndrome, a congenital disorder characterized by the absence of intact peroxisomes. As a control, labeled compound was administered to two healthy volunteers. Urine was collected, fractionated on a SEP-PAK C18 cartridge, and subjected to reversed-phase high-performance liquid chromatography. The Zellweger patient was found to excrete prostaglandin metabolites considerably less polar than those of the control subjects. The major urinary metabolite in the control subjects was practically absent in the urine from the Zellweger patient. The major urinary prostaglandin F2 alpha metabolite from the Zellweger patient was identified as an omega-oxidized C20-prostaglandin, 9,11-dihydroxy-15-oxoprost-5-ene-1,20-dioic acid. The major urinary prostaglandin F2 alpha metabolite from the control subjects had chromatographic properties of a tetranor (C16) prostaglandin, in accordance with earlier published data. The present results, in combination with our previous in vitro data, indicate that peroxisomal beta-oxidation is of major importance for in vivo chain shortening of prostaglandins.

Hydroxyeicosatetraenoic acid oxidation in chinese hamster ovary cells: a peroxisomal metabolic pathway

To evaluate the peroxisomal requirement for beta-oxidation of hydroxyeicosatetraenoic acids (HETES), we tested 5-, 12- and 15-HETE oxidation in wild-type and mutant Chinese hamster ovary (CHO) cells. Mutant CHO cells contain peroxisomal ghosts, have random cytosolic localization of catalase and lack two of the enzymes necessary for peroxisomal beta-oxidation. Reverse-phase HPLC indicated that 33% of 12-HETE radioactivity was converted by wild-type CHO cells during a 2 h incubation to one major and several minor polar metabolites. Wild-type CHO cells also converted 15-HETE to one major and several minor polar metabolites. Neither 12- nor 15-HETE were converted to any metabolites by the mutant CHO cell lines, despite appreciable cellular uptake of these hydroxyeicosanoids. 5-HETE was not converted to any metabolic products by either the wild-type or the mutant CHO cells. Docosahexaenoic acid beta-oxidation was substantially reduced in the mutants as compared to the wild-type cells, palmitic acid beta-oxidation was reduced to an intermediate extent in the mutants, but octanoate beta-oxidation and citrate synthase activity were not impaired. Protein immunoblotting for mitochondrial manganese superoxide dismutase indicated a single band of identity at 20 kDa in both wild-type and mutant CHO cells. Since mutant CHO cells fail to convert 12- and 15-HETE to oxidative metabolites but contain normal mitochondrial enzymatic activities, intact peroxisomes appear to be the organelle responsible for HETE oxidation.

Methanol metabolism in a peroxisome-deficient mutant of Hansenula polymorpha: a physiological study

We have studied methanol-utilization in a peroxisome-deficient (PER) mutant of Hansenula polymorpha. In spite of the fact that in carbon-limited chemostat cultures under induced conditions the enzymes involved in methanol metabolism were present at wild-type (WT) levels, this mutant is unable to grow on methanol as a sole carbon and energy source. Addition of methanol to glucose-limited (SR = 12.5 mM) chemostat cultures of the PER mutant only resulted in an increase in yield when small amounts were used (up to 22.5 mM). At increasing amounts however, a gradual decrease in cell density was observed which, at 80 mM methanol in the feed, had dropped below the original value of the glucose-limited culture. This reduction in yield was not observed when increasing amounts of formate instead of methanol were used as supplements for the glucose-limited mutant culture and also not in WT cells, used as control in these experiments. The effect of addition of methanol to a glucose-limited PER culture was also studied in the transient state during adaptation of the cells to methanol. The enzyme patterns obtained suggested that the ultimate decrease in yield observed at enhanced methanol concentrations was due to an inefficient methanol-metabolism as a consequence of the absence of peroxisomes. The absence of intact peroxisomes results in two major problems namely i) in H2O2-metabolism, which most probably is no longer mediated by catalase and ii) the inability of the cell to control the fluxes of formaldehyde, generated from methanol. The energetic consequences of this metabolism, compared to the WT situation with intact peroxisomes, are discussed.

Topography of very-long-chain-fatty-acid-activating activity in peroxisomes from rat liver

We have investigated the localization of palmitoyl-CoA (hexadecanoyl-CoA) synthetase (EC 6.2.1.3) and cerotoyl-CoA (hexacosanoyl-CoA) synthetase in peroxisomes isolated from rat liver. Palmitoyl-CoA and cerotoyl-CoA synthetases, like acyl-CoA: dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42), are present in the peroxisomal membrane. Trypsin treatment of intact peroxisomes led to the disappearance of both palmitoyl-CoA and cerotoyl-CoA synthetase activities but had little, if any, effect on L-alpha-hydroxy-acid oxidase (EC 1.1.3.15), D-amino acid oxidase (EC 1.4.3.3) or acyl-CoA:dihydroxyacetone phosphate acyltransferase. The latter three enzymes were inactivated if the trypsin treatment was preceeded by disruption of the peroxisomes by sonication. These results show that the active site, or at least domains essential for the activity of cerotoyl-CoA synthetase, like that of palmitoyl-CoA synthetase, is located on the cytosolic face of the peroxisomal membrane.

Peroxisome proliferation and oxidative stress mediated by activated oxygen species in plant peroxisomes

The existence of a relationship between clofibrate-induced peroxisome proliferation and oxidative stress mediated by activated oxygen species was studied in intact peroxisomes purified from Pisum sativum L. plants. Incubation of leaves with 1 m m clofibrate produced a remarkable increase in the peroxisomal activity of acyl-CoA oxidase and, to a lesser extent, of xanthine oxidase, whereas there was a nearly complete loss of catalase activity and a decrease in Mn-superoxide dismutase. Ultrastructural studies of intact leaves showed that clofibrate induced a five- and twofold proliferation of the peroxisomal and mitochondrial populations, respectively, in comparison with those in control leaves. Prolonged incubation with clofibrate produced considerable alterations in the ultrastructure of cells. In peroxisomal membranes, the NADH-induced generation of O 2 − radicals, as well as the lipid peroxidation of membranes, increased as a result of treatment of plants with clofibrate. In intact peroxisomes treated with this hypolipidemic drug, the H 2O 2 concentration was higher than in peroxisomes from control plants. These results demonstrate that clofibrate stimulates the production of activated oxygen species (O 2 − and H 2O 2) inside peroxisomes, as well as the lipid peroxidation of peroxisomal membranes. This effect is concomitant with a decrease of catalase and Mn-SOD activities, the main peroxisomal enzymatic defenses against H 2O 2 and O 2 −, and indicates that in the toxicity of clofibrate, at the level of peroxisomes, an oxidative stress mechanism mediated by activated oxygen species is involved.

72 Topographical localization of Peroxisomal Acyl-CoA Ligases: Differential localization of Palmitoyl-CoA and Lignoceroyl-CoA Ligases

We found that peroxisomal licjnoceroyl-CoA ligase, like palmitoyl-CoA ligase, is present in the peroxisomal membrane. To further define the role of peroxisomal acyl-CoA ligases we examined the transverse topographical localization of enzymatic sites of palmitoyl-CoA and lignoceroyl-CoA ligases in the peroxisomal membranes. Proteolytic enzyme treatment irihibited palmitoyl-CoA ligase activity in intact peroxisomes and ligrraceroyl-CoA ligase activity was inhibited only if peroxisomes were disrupted prior to trypsin treatment. Antibodies to palmitoyl-CoA ligase and to peroxisomal membrane proteins (PMP) inhibited palmtoyl-CoA ligase in intact peroxisomes, and no pool of “latent” activity appeared when antibody-treated peroxisomes were disrupted. On the other hand, disruption of PMP antibody-treated peroxisomes with detergent resulted in the appearance of a “latent” pool of lignoceroyl-CoA ligase activity. These results demonstrate that the enzymatic site of palmitoyl-CoA ligase is on the cytoplasmic surface whereas that for ligncceroyl-CoA ligase is on the luminal surface of peroxisomal membranes. The implication of these findings to X-adrenoleukodystiuphy will be discussed.

Topographical localization of peroxisomal acyl-CoA ligases: differential localization of palmitoyl-CoA and lignoceroyl-CoA ligases

We found that peroxisomal lignoceroyl-CoA ligase, like palmitoyl-CoA ligase, is present in the peroxisomal membrane whereas the peroxisomal beta-oxidation enzyme system is localized in the matrix. To further define the role of peroxisomal acyl-CoA ligases (membrane component) in providing acyl-CoA for peroxisomal beta-oxidation, we examined the transverse topographical localization of enzymatic sites of palmitoyl-CoA and lignoceroyl-CoA ligases in the peroxisomal membranes. The disruption of peroxisomes by various techniques resulted in the release of a "latent" pool of lignoceroyl-CoA ligase activity while palmitoyl-CoA ligase activity remained the same. Proteolytic enzyme treatment inhibited palmitoyl-CoA ligase activity in intact peroxisomes but had no effect on lignoceroyl-CoA ligase activity. Lignoceroyl-CoA ligase activity was inhibited only if peroxisomes were disrupted with detergent before trypsin treatment. Antibodies to palmitoyl-CoA ligase and to peroxisomal membrane proteins (PMP) inhibited palmitoyl-CoA ligase in intact peroxisomes, and no pool of "latent" activity appeared when antibody-treated peroxisomes were disrupted with detergent. On the other hand, disruption of PMP antibody-treated peroxisomes with detergent resulted in the appearance of a "latent" pool of lignoceroyl-CoA ligase activity. These results demonstrate that the enzymatic site of palmitoyl-CoA ligase is on the cytoplasmic surface whereas that for lignoceroyl-CoA ligase is on the luminal surface of peroxisomal membranes. This implies that palmitoyl-CoA is synthesized on the cytoplasmic surface and is then transferred to the matrix through the peroxisomal membrane for beta-oxidation in the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)