Browsing by Author "LEIGHTON, F"

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    ACYL-COA SYNTHETASE AND THE PEROXISOMAL ENZYMES OF BETA-OXIDATION IN HUMAN-LIVER - QUANTITATIVE-ANALYSIS OF THEIR SUBCELLULAR-LOCALIZATION
    (1984) BRONFMAN, M; INESTROSA, NC; NERVI, FO; LEIGHTON, F
    The presence of acyl CoA synthetase (EC 6.2.1.3) in peroxisomes and the subcellular distribution of .beta.-oxidation enzymes in human liver were investigated by using a single-step fractionation method of whole liver homogenates in metrizamide continuous density gradients and a novel procedure of computer analysis of results. Peroxisomes contain 16% of the liver palmitoyl CoA synthetase activity, and 21% and 60% of the enzyme activity was localized in mitochondria and microsomal fractions, respectively. Fatty acyl CoA oxidase was localized exclusively in peroxisomes, confirming previous results. Human liver peroxisomes contribute 13%, 17% and 11% of the liver activities of crotonase, .beta.-hydroxyacyl CoA dehdyrogenase and thiolase, respectively. The absolute activities found in peroxisomes for the enzymes investigated suggest that in human liver fatty acyl CoA oxidase is the rate-limiting enzyme of the peroxisomal .beta.-oxidation pathway, when palmitic acid is the substrate.
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    CARNITINE ACYLTRANSFERASE AND ACYL-COENZYME-A HYDROLASE ACTIVITIES IN HUMAN-LIVER - QUANTITATIVE-ANALYSIS OF THEIR SUBCELLULAR-LOCALIZATION
    (1984) BRONFMAN, M; LEIGHTON, F
    The subcellular localizations of carnitine acyltransferase and acyl CoA hydrolase activities with different chain-length substrates were quantitatively evaluated in human liver by fractionation of total homogenates in metrizamide density gradients and by differential centrifugation. Peroxisomes contain 8-37% of the liver acyltransferase activity, the relative amount depending on the chain length of the substrate. The remaining activity was ascribed to mitochondria, except for carnitine octanoyltransferase, for which 25% of the activity was present in microsomal fractions. In contrast with rat liver, where the activity in peroxisomes is very low or absent, human liver peroxisomes contain about 20% of the carnitine palmitoyltransferase. Short-chain acyl CoA hydrolase activity was localized mainly in the mitochondrial and soluble compartments; the long-chain activity was present in both microsomal fractions and the soluble compartment. Particle-bound acyl CoA hydrolase activity for medium-chain substrates exhibited an intermediate distribution, in mictochondria and microsomal fractions, with 30-40% of the activity in the soluble fraction. No acyl CoA hydrolase activity appears to be present in human liver peroxisomes.
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    CHARACTERIZATION OF PEROXISOMES IN CHINESE-HAMSTER OVARY CELLS IN CULTURE
    (1985) SANTOS, MJ; GARRIDO, J; OLIVER, C; ROBBINS, AR; LEIGHTON, F
    In order to explore the potential value of Chinese hamster overay (CHO) cells for the isolation of peroxisomal mutants defective in the peroxisomal fatty acid oxidation system, some characteristics of their peroxisomes were studied. Catalase was detected biochemically and histochemically in peroxisome-like particles in cells or in subcellular fractions prepared by differential centrifugation or isopyknic equilibrium in Percoll or Metrizamide with catalase in the high density fraction of the isopyknic equilibrium gradients. By oxidation system, exhibited an unusually high specific activity, 2.46 .+-. 1.09 mU/mg protein, in CHO cell homogenates, a value comparable to that of rat liver. This enzyme copurifies with catalase in the high density fractions of the isopycnic equilibrium gradients. By analogy with other cell types and from the ultrastructural analysis, it is concluded that these enzymes are contained in peroxisomes. These findings support the value of CHO cells for studies of peroxisomal function and organization.
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    DETECTION OF AN ATPASE ACTIVITY IN RAT-LIVER PEROXISOMES
    (1988) DELVALLE, R; SOTO, U; NECOCHEA, C; LEIGHTON, F
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    FATTY-ACID OXIDATION BY HUMAN-LIVER PEROXISOMES
    (1979) BRONFMAN, M; INESTROSA, NC; LEIGHTON, F
    A cyanide insensitive fatty acid oxidation system was detected in human liver by subcellular fractionation in Metrizamide continuous density gradients and was localized in peroxisomes. Fatty acyl-CoA oxidase, its characteristic enzyme, acts maximally on C12-C18 saturated fatty acids and oleoyl-CoA and requires FAD. These results, and the already established properties of the system in rat liver support its potential contribution to lipid metabolism and to the hypolipidemic effect of Clofibrate and related drugs in humans.
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    FLAVONOIDS AS STABILIZERS OF FISH-OIL - AN ALTERNATIVE TO SYNTHETIC ANTIOXIDANTS
    (1993) NIETO, S; GARRIDO, A; SANHUEZA, J; LOYOLA, LA; MORALES, G; LEIGHTON, F; VALENZUELA, A
    The antioxidant activities against fish oil oxidation of six commercially available flavonoids and of five flavonoids purified from two Chilean native plants were compared to those of dl-alpha-tocopherol and of two synthetic antioxidants, butylated hydroxytoluene and butylated hydroxyanisole. Among the commercial flavonoids, catechin, morin and quercetin showed a higher activity when fish oil oxidation (either spontaneous or Fe2+-induced) was assessed from the formation of peroxides or thiobarbituric acid-reactive substances. Among the native flavonoids, the 5,3',4'-trihydroxy-7-methoxy flavanone (designated as Pt-2) showed the highest antioxidant activity. Mixtures of quercetin or of Pt-2 with dl-alpha-tocopherol produced better inhibitory effects when compared to that of each substance assayed by itself. Also, when Pt-2 and quercetin were assayed in combination (0.3 g/kg oil and 0.7 g/kg oil, respectively), a synergistic antioxidant effect was observed. Results indicate that several flavonoids could be used as natural antioxidants as a means to replace those synthetic antioxidants, the use of which has been questioned.
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    FUNCTION OF PEROXISOMES IN ANIMAL-CELLS
    (1982) LEIGHTON, F; LAZO, O
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    LARGE CATION-SELECTIVE PORES FROM RAT-LIVER PEROXISOMAL MEMBRANES INCORPORATED TO PLANAR LIPID BILAYERS
    (1986) LABARCA, P; WOLFF, D; SOTO, U; NECOCHEA, C; LEIGHTON, F
    Fusion of a highly purified fraction of rat liver peroxisomal membranes to planar lipid bilayers incorporates large, cation-selective voltage-dependent pores. The PK/PCl ratio of these pores estimated in KCl gradients, is close to 4. The pores display several conductance states and spend most of the time open at voltages near 0 mV, closing at more positive and negative voltages. At voltages near 0 mV the most frequent open state has a conductance of 2.4 nS in 0.3 M KCl. At voltages more positive and more negative than 10 mV the most frequent open state displays a conductance of 1.2 nS in 0.3 M KCl. With these results pore diameters of 3 and 1.5 nm, respectively, can be estimated. We suggest that these pores might account for the unusually high permeability of peroxisomes to low molecular weight solutes. Fusion also incorporates a perfectly anion-selective, two-open states channel with conductances of 50 and 100 pS in 0.1 m KCl.
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    PEROXISOMAL FATTY-ACID OXIDATION AND INHIBITORS OF THE MITOCHONDRIAL CARNITINE PALMITOYLTRANSFERASE-I IN ISOLATED RAT HEPATOCYTES
    (1992) SKORIN, C; NECOCHEA, C; JOHOW, V; SOTO, U; GRAU, AM; BREMER, J; LEIGHTON, F
    Fatty acid oxidation was studied in the presence of inhibitors of carnitinic palmitoyltransferase I (CPT I), in normal and in peroxisome-proliferated rat hepatocytes. The oxidation decreased in mitochondria, as expected, but in peroxisomes it increased. These two effects were seen, in variable proportions, with (+)-decanoylcarnitine, 2-tetradecylglycidic acid (TDGA) and etomoxir. The decrease in mitochondrial oxidation (ketogenesis) affected saturated fatty acids with 12 or more carbon atoms, whereas the increase in peroxisomal oxidation (H2O2 production) affected saturated fatty acids with 8 or more carbon atoms. The peroxisomal increase was sensitive to chlorpromazine, a peroxisomal inhibitor. To study possible mechanisms, palmitoyl-, octanoyl- and acetyl-carnitine acyltransferase activities were measured, in homogenates and in subcellular fractions from control and TDGA-treated cells. The palmitoylcarnitine acyltransferase was inhibited, as expected, but the octanoyltransferase activity also decreased. The CoA derivative of TDGA was synthesized and tentatively identified as being responsible for inhibition of the octanoylcarnitine acyltransferase. These results show that inhibitors of the mitochondrial CPT 1 may also inhibit the peroxisomal octanoyl transferase; they also support the hypothesis that the octanoyltransferase has the capacity to control or regulate peroxisomal fatty acid oxidation.
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    PEROXISOMAL ORGANIZATION IN NORMAL AND CEREBROHEPATORENAL (ZELLWEGER) SYNDROME FIBROBLASTS
    (1985) SANTOS, MJ; OJEDA, JM; GARRIDO, J; LEIGHTON, F
    The reported absence of morphologically detectable peroxisomes in liver and kidney tissue cells from patients affected by the autosomic recessive, inherited metabolic disease known as cerebrohepatorenal, or Zellweger, syndrome was studied in fibroblasts, assuming it to be a generalized defect. Normal cultured fibroblasts were shown to contain peroxisomes according to morphological, biochemical, and subcellular fractionation criteria: particle-bound catalase and fatty acyl-CoA oxidase copurify in subcellular fractionation by differential centrifugation or isopycnic equilibrium in continuous density gradients and peroxidase-positive organelles of .apprxeq. 0.1 .mu.m in diameter are detected in the cytoplasm. In Zellweger cultured fibroblasts, these peroxisomal enzymes are present; however, they behave as cytosolic enzymes in the different subcellular fractionation procedures employed and peroxisomes are not detected cytochemically. These findings support the hypothesis that the lack of peroxisomes in this genetic disease is the consequence of a defect in the assembly of the peroxisomal constituents. Furthermore, the value of fibroblasts for subcellular analysis of peroxisomal defects is illustrated.
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    PURIFICATION OF THE PEROXISOMAL FATTY ACYL-COA OXIDASE FROM RAT-LIVER
    (1980) INESTROSA, NC; BRONFMAN, M; LEIGHTON, F
    Fatty acyl-CoA oxidase, the rate limiting enzyme of the peroxisomal fatty acid oxidizing system, was purified from rat liver to near homogeneity by a procedure involving affinity chromatography of its apoenzyme on FAD-Sepharose. The oxidase presents an absolute requirement for the dinucleotide which is weakly bound to the apoenzyme (KD, 0.6 .mu.M). The highest specific activity obtained was 27 units/mg protein. The purified enzyme was 2 major polypeptides with apparent MW of 45,000 and 22,000. The enzyme is a flavoprotein with non-covalently bound flavin adenin dinucleotide composed of 4 subunits, 2 of 45,000 MW and 2 of 22,000 MW.
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