Hydrogen peroxide permeation across liposomal membranes: a novel method to assess structural flaws in liposomes

Hydrogen peroxide permeation across large multilamellar vesicles of defined and complex lipid composition was shown to obey precise kinetic relationships for the activity of the occluded catalase. Careful assay conditions precluded simultaneous peroxidative damage to the lipids. The kinetic data was consistent with a barrier role for the bilayer for hydrogen peroxide permeation. More interestingly, hydrogen peroxide permeation across liposomes of complex lipid mixtures exhibited osmotic inhibition of permeation of hydrogen peroxide. On the other hand, purified egg lecithin vesicles did not exhibit any effect of external osmolality on hydrogen peroxide permeation in an experimentally defined non-lytic zone of external osmolarity. These results argue in favour of a heterogeneous, heteroporous structure of bilayers with complex lipid composition.     

Effect of ciprofibrate on the activation and oxidation of very long chain fatty acids

The effect of ciprofibrate, a hypolipidemic drug, was examined in the metabolism of palmitic (C_16:0) and lignoceric (C_24:0) acids in rat liver. Ciprofibrate is a peroxisomal proliferating drug which increases the number of peroxisomes. The palmitoyl-CoA ligase activity in peroxisomes, mitochondria and microsomes from ciprofibrate treated liver was 3.2, 1.9 and 1.5-fold higher respectively and the activity for oxidation of palmitic acid in peroxisomes and mitochondria was 8.5 and 2.3-fold higher respectively. Similarly, ciprofibrate had a higher effect on the metabolism of lignoceric acid. Treatment with ciprofibrate increased lignoceroyl-CoA ligase activity in peroxisomes, mitochondria and microsomes by 5.3, 3.3 and 2.3-fold respectively and that of oxidation of lignoceric acid was increased in peroxisomes and mitochondria by 13.4 and 2.3-fold respectively. The peroxisomal rates of oxidation of palmitic acid (8.5-fold) and lignoceric acid (13.4-fold) were increased to a different degree by ciprofibrate treatment. This differential effect of ciprofibrate suggests that different enzymes may be responsible for the oxidation of fatty acids of different chain length, at least at one or more step(s) of the peroxisomal fatty acid -oxidation pathway.     

Localization of Glyoxylate Cycle Enzymes in Glyoxysomes in Euglena*

SYNOPSIS. We demonstrated previously microbodies in Euglena gracilis grown in the dark on 2-carbon substrates. We have now established in Euglena the particulate nature of enzymes known in other organisms to be localized in microbodies (glyoxysomes and leaf peroxisomes). On a linear sucrose gradient the glyoxylate cycle enzymes band together at a nigner equilibrium density (1.20 g/cm3) than mitochondrial marker enzymes (1.17 g/cm3), establishing the existence in Euglena of glyoxysomes similar to those of higher plants. Glyoxylate (hydroxypyruvate) reductase and, under certain conditions, also glycolate dehydrogenase co-band with the glyoxylate cycle enzymes, suggesting that Euglena glyoxysomes, like those of higher plants, may contain peroxisomal-type enzymes. Catalase, an enzyme characteristic of microbodies from a variety of sources, was not detected in Euglena.     

Molecular chaperones and the biogenesis of mitochondria and peroxisomes

Summary— A review of the proteinaceous machinery involved in protein sorting pathways and protein folding and assembly in mitochondria and peroxisomes is presented. After considering the various sorting pathways and targeting signals of mitochondrial and peroxisomal proteins, we make a comparative dissection of the protein factors involved in: i) the stabilization of cytosolic precursor proteins in a translocation competent conformation; ii) the membrane import apparatus of mitochondria and peroxisomes; iii) the processing of mitochondrial precursor proteins, and the eventual processing of certain peroxisomal precursor, in the interior of the organelles; and iv) the requirement of molecular chaperones for appropriate folding and assembly of imported proteins in the matrix of both organelles. Those aspects of mitochondrial biogenesis that have developed rapidly during the last few years, such as the requirement of molecular chaperones, are stressed in order to stimulate further parallel investigations aimed to understand the origin, biochemistry, molecular biology and pathology of peroxisomes. In this regard, a brief review of findings from our group and others is presented in which the role of the F₁-ATPase α-subunit is pointed out as a molecular chaperone of mitochondria and chloroplasts. In addition, data are presented that could question our previous indication that the immunoreactive protein found in the rat liver peroxisomes is due to the presence of the F₁-ATPase α-subunit.     

Cadmium causes the oxidative modification of proteins in pea plants

In pea (Pisum sativum L.) leaves from plants grown in the presence of 50 µm CdCl₂ the oxidative production of carbonyl groups in proteins, the rate of protein degradation and the proteolytic activity were investigated. In leaf extracts the content of carbonyl groups measured by derivatization with 2,4‐dinitrophenylhydrazine (DNPH), was two‐fold higher in plants treated with Cd than in control plants. The identification of oxidized proteins was carried out by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of proteins derivatized with DNPH and immunochemical detection with an antibody against DNPH. The intensity of the reactive bands was higher in plants exposed to Cd than in controls. By using different antibodies some of the oxidized proteins were identified as Rubisco, glutathione reductase, manganese superoxide dismutase, and catalase. The incubation of leaf crude extracts with increasing H₂O₂ concentrations showed a progressive enhancement in carbonyl content and the pattern of oxidized proteins was similar to that found in Cd‐treated plants. Oxidized proteins were more efficiently degraded, and the proteolytic activity increased 20% due to the metal treatment. In peroxisomes purified from pea leaves a rise in the carbonyl content similar to that obtained in crude extracts from Cd‐treated plants was observed, but the functionality of the peroxisomal membrane was not apparently affected by Cd. Results obtained demonstrate the participation of both oxidative stress, probably mediated by H₂O₂, and proteolytic degradation in the mechanism of Cd toxicity in leaves of pea plants, and they appear to be involved in the Cd‐induced senescence previously reported in these plants.     

The role of peroxisomes in xylose alcoholic fermentation in the engineered Saccharomyces cerevisiae

Xylose is a second‐most abounded sugar after glucose in lignocellulosic hydrolysates and should be efficiently fermented for economically viable second‐generation ethanol production. Despite significant progress in metabolic and evolutionary engineering, xylose fermentation rate of recombinant Saccharomyces cerevisiae remains lower than that for glucose. Our recent study demonstrated that peroxisome‐deficient cells of yeast Ogataea polymorpha showed a decrease in ethanol production from xylose. In this work, we have studied the role of peroxisomes in xylose alcoholic fermentation in the engineered xylose‐utilizing strain of S. cerevisiae. It was shown that peroxisome‐less pex3Δ mutant possessed 1.5‐fold decrease of ethanol production from xylose. We hypothesized that peroxisomal catalase Cta1 may have importance for hydrogen peroxide, the important component of reactive oxygen species, detoxification during xylose alcoholic fermentation. It was clearly shown that CTA1 deletion impaired ethanol production from xylose. It was found that enhancing the peroxisome population by modulation the peroxisomal biogenesis by overexpression of PEX34 activates xylose alcoholic fermentation.     

Silencing of OsCV (chloroplast vesiculation) maintained photorespiration and N assimilation in rice plants grown under elevated CO2

High CO₂ concentrations stimulate net photosynthesis by increasing CO₂ substrate availability for Rubisco, simultaneously suppressing photorespiration. Previously, we reported that silencing the chloroplast vesiculation (cv) gene in rice increased source fitness, through the maintenance of chloroplast stability and the expression of photorespiration-associated genes. Because high atmospheric CO₂ conditions diminished photorespiration, we tested whether CV silencing might be a viable strategy to improve the effects of high CO₂ on grain yield and N assimilation in rice. Under elevated CO₂, OsCV expression was induced, and OsCV was targeted to peroxisomes where it facilitated the removal of OsPEX11-1 from the peroxisome and delivered it to the vacuole for degradation. This process correlated well with the reduction in the number of peroxisomes, the decreased catalase activity and the increased H₂O₂ content in wild-type plants under elevated CO₂. At elevated CO₂, CV-silenced rice plants maintained peroxisome proliferation and photorespiration and displayed higher N assimilation than wild-type plants. This was supported by higher activity of enzymes involved in NO₃⁻ and NH₄⁺ assimilation and higher total and seed protein contents. Co-immunoprecipitation of OsCV-interacting proteins suggested that, similar to its role in chloroplast protein turnover, OsCV acted as a scaffold, binding peroxisomal proteins.     

Determinants of the assembly,integrity and maintenance of the endoplasmic reticulum‐peroxisome tether

Organelle tethering and intercommunication are crucial for proper cell function. We previously described a tether between peroxisomes and the endoplasmic reticulum (ER) that acts in peroxisome population control in the yeast, Saccharomyces cerevisiae. Components of this tether are Pex3p, an integral membrane protein of both peroxisomes and the ER and Inp1p, a connector that links peroxisomes to the ER. Here, we report the analysis of random Inp1p mutants that enabled identification of regions in Inp1p required for the assembly and maintenance of the ER‐peroxisome tether. Interaction analysis between Inp1p mutants and known Inp1p‐binding proteins demonstrated that Pex3p and Inp1p do not constitute the sole components of the ER‐peroxisome tether. Deletion of these Inp1p interactors whose steady‐state localization is outside of ER‐peroxisome tethers affected peroxisome dynamics. Our findings are consistent with the presence of regulatory cues that act on ER‐peroxisome tethers and point to the existence of membrane contact sites between peroxisomes and organelles other than the ER.      

Effects of dietary polyamines and clofibrate on metabolism of polyamines in the rat

The activities of catalase, polyamine oxidase, diamine oxidase, ornithine decarboxylase, and peroxisomal β-oxidation were assayed in homogenates from liver and small intestinal mucosa of rats which had been fed either a diet very low in polyamines or a diet containing five times the levels of dietary polyamines (putrescine, spermine, and spermidine) found in a standard rat diet. In rats fed the high polyamine diet, hepatic activities of catalase and polyamine oxidase were significantly decreased. Levels of the other activities were unchanged, except that intestinal ornithine decarboxylase was decreased. In rats treated simultaneously with clofibrate, the high polyamine diet restored activities of catalase, ornithine decarboxylase, and polyamine oxidase back to levels found in rats fed the low polyamine diet. The expected increase in activity of peroxisomal β-oxidation was observed, although this was somewhat diminished in rats fed the high polyamine diet. Intestinal diamine oxidase activity was stimulated by clofibrate, particularly in rats fed the high polyamine diet. For the duration of the experiment (20 days), levels of putrescine, spermine, and spermidine in blood remained remarkably constant irrespective of treatment, suggesting that polyamine homeostasis is essentially independent of dietary supply of polyamines. It is suggested that intestinal absorption/metabolism of polyamines is of significance in this respect. Treatment with clofibrate appeared to alter polyamine homeostasis.     

Peroxisomal β-Oxidation Enzymes

The enzymes involved in -oxidation spiral are schematically classified into two groups. The first group consists of palmitoyl-CoA oxidase, the L-bifunctional protein, which has been called as the bifunctional protein, and 3-ketoacyl-CoA thiolase. The second group consists of the newly confirmed enzymes, branched chain oxidase, the D-bifunctional protein, and sterol carrier protein x. The enzymes of the first group are inducible and act on the straight chain acyl-CoA substrates. But the enzymes of the second group are non-inducible and act on branched chain acyl-CoAs. Accordingly, bile acid formation and oxidation of pristanic acid derived from phytol are catalyzed by the enzymes of the second group but not by those of the first group. The functions of the peroxisomal system and methods of analysis of the enzymes are briefly summarized.