ABSTRACT
We studied the effect of fenoprofen on the activation of palmitic acid (C16:0), lignoceric acid (C24:0) and cerotic acid (C26:0) in microsomal and peroxisomal fractions from rat liver. Fenoprofen was found to inhibit the formation of palmitoyl-CoA in both microsomal and peroxisomal fractions whereas the formation of lignoceroyl-CoA and cerotoyl-CoA was not inhibited at all. In freshly isolated rat hepatocytes palmitic acid beta-oxidation was progressively inhibited at increasing concentrations of fenoprofen, most probably due to its inhibitory effect on palmitoyl-CoA synthetase activity. On the other hand, fenoprofen was also found to inhibit the beta-oxidation of lignoceric acid and cerotic acid in rat hepatocytes. It is shown that the acyl-CoA oxidase activity with lignoceroyl-CoA as substrate was inhibited by fenoprofen whereas the palmitoyl-CoA and pristanoyl-CoA oxidase activities were not inhibited by fenoprofen. This finding provides an explanation for the inhibitory effect of fenoprofen on lignocerate and cerotate beta-oxidation in hepatocytes.
Subject(s)
Fatty Acids/metabolism , Fenoprofen/pharmacology , Liver/metabolism , Animals , Dose-Response Relationship, Drug , Male , Microbodies/metabolism , Oxidation-Reduction , Oxidoreductases/metabolism , Palmitic Acid , Palmitic Acids/metabolism , Rats , Rats, Wistar , Subcellular Fractions/metabolismABSTRACT
The adrenoleukodystrophies refer to three genetically distinct disorders all characterized by the accumulation of very long-chain fatty acids. In this paper we will review the biochemical aspects of these leukodystrophies with particular emphasis on the methods used to measure very long-chain fatty acid levels in plasma and their reliability. Furthermore, we will concentrate on the primary defect in the X-linked form of adrenoleukodystrophy.
Subject(s)
Adrenoleukodystrophy/genetics , X Chromosome , Adrenoleukodystrophy/diagnosis , Coenzyme A Ligases/deficiency , Fatty Acids/analysis , Fatty Acids/metabolism , Humans , Microbodies/metabolism , Mitochondria/metabolism , Oxidation-ReductionABSTRACT
We studied the effect of sodium 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA), a potent inhibitor of mitochondrial carnitine palmitoyltransferase I, on fatty acid oxidation by rat brain cells. In cultured glial cells as well as in dissociated brain cells from adult rats palmitic acid (16:0) oxidation was inhibited by about 85% of control values when 25 microM POCA was added to the medium, whereas no inhibition of cerotic acid (26:0) oxidation was observed. Furthermore, omission of carnitine from the culture medium resulted in a 57.7% decrease in palmitic acid oxidation in cultured glial cells, whereas cerotic acid oxidation was not influenced. These results indicate that rat brain peroxisomes contribute only little (about 15%) to palmitic acid oxidation and provide conclusive evidence that cerotic acid is oxidized exclusively in rat brain peroxisomes.
Subject(s)
Brain/metabolism , Fatty Acids/metabolism , Microbodies/metabolism , Animals , Brain/drug effects , Carnitine/pharmacology , Carnitine O-Palmitoyltransferase/antagonists & inhibitors , Cells, Cultured , Epoxy Compounds/pharmacology , Microbodies/drug effects , Mitochondria/metabolism , Neuroglia/drug effects , Neuroglia/metabolism , Oxidation-Reduction , RatsABSTRACT
In this paper we describe a fluorimetric assay for the measurement of long-chain acyl-CoA synthetase activity in rat liver postnuclear supernatants. The method is based upon the use of acyl-CoA oxidase which catalyzes the dehydrogenation of acyl-CoA esters to yield enoyl-CoA esters and H2O2. H2O2 subsequently reacts with homovanillic acid in a horseradish peroxidase-catalyzed reaction to form a highly fluorescent dimer (see G. G. Guilbault, P. J. Brignac, and M. Zimmer (1968) Anal. Chem. 40, 190-196). The increase in fluorescence can be followed either continuously or discontinuously. The method described is able to detect acyl-CoA synthetase activities as low as 20 microU/ml which is almost as sensitive as the standard isotopic assay used in most laboratories. The method is applicable to measure the activation of a variety of fatty acids. Finally, the method provides a simple means of carrying out kinetic studies.
Subject(s)
Coenzyme A Ligases/analysis , Fluorometry/methods , Acyl-CoA Oxidase , Adenosine Triphosphate/chemistry , Animals , Flavin-Adenine Dinucleotide/chemistry , Homovanillic Acid/chemistry , Hydrogen Peroxide/chemistry , Hydrogen-Ion Concentration , Kinetics , Liver/enzymology , Oxidoreductases , Rats , Sensitivity and Specificity , Time FactorsABSTRACT
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.
Subject(s)
Coenzyme A Ligases/metabolism , Liver/enzymology , Microbodies/enzymology , Repressor Proteins , Saccharomyces cerevisiae Proteins , Animals , Coenzyme A Ligases/analysis , Intracellular Membranes/enzymology , Intracellular Membranes/ultrastructure , Kinetics , Microbodies/ultrastructure , Rats , Trypsin/pharmacologyABSTRACT
We have investigated the palmitic acid (C16:0) and cerotic acid (C26:0) activating activities in rat-liver microsomes and peroxisomes. The activation of the two fatty acids showed similar dependencies on ATP and coenzyme A, reflected in about equal apparent Km values both in microsomes and peroxisomes. In microsomes and peroxisomes similar apparent Km values for palmitic acid were found (15 microM and 22.8 microM, respectively), whereas apparent Km values for cerotic acid were 8.4 microM and 1.0 microM in microsomes and peroxisomes, respectively. The activation of cerotic acid was found to be inhibited to a progressively greater extent by increasing concentrations of 1-pyrenedecanoic acid (P10) as compared to the activation of palmitic acid, both in microsomes and peroxisomes. The inhibition by P10 of palmitic acid activation and cerotic acid activation was non-competitive in both organelles. From the observation that P10 activation is not affected by palmitic acid and cerotic acid, we conclude that P10 is activated by a distinct enzyme. Furthermore, our results are in accordance with earlier suggestions that activation of cerotic acid is brought about by an enzyme distinct from the palmitoyl-CoA synthetase.
