Showing papers on "Acyl-CoA published in 1996"

Acyl-CoA binding proteins: Multiplicity and function

Abstract: The physiological role of long-chain fatty acyl-CoA is thought to be primarily in intermediary metabolism of fatty acids. However, recent data show that nM to μM levels of these lipophilic molecules are potent regulators of cell functionsin vitro. Although long-chain fatty acyl-CoA are present at several hundred μM concentration in the cell, very little long-chain fatty acyl-CoA actually exists as free or unbound molecules, but rather is bound with high affinity to membrane lipids and/or proteins. Recently, there is growing awareness that cytosol contains nonenzymatic proteins also capable of binding long-chain fatty acyl-CoA with high affinity. Although the identity of the cytosolic long-chain fatty acyl-CoA binding protein(s) has been the subject of some controversy, there is growing evidence that several diverse nonenzymatic cytosolic proteins will bind long-chain fatty acyl-CoA. Not only does acyl-CoA binding protein specifically bind medium and long-chain fatty acyl-CoA (LCFA-CoA), but ubiquitous proteins with multiple ligand specificities such as the fatty acid binding proteins and sterol carrier protein-2 also bind LCFA-CoA with high affinity. The potential of these acyl-CoA binding proteins to influence the level of free LCFA-CoA and thereby the amount of LCFA-CoA bound to regulatory sites in proteins and enzymes is only now being examined in detail. The purpose of this article is to explore the identity, nature, function, and pathobiology of these fascinating newly discovered long-chain fatty acyl-CoA binding proteins. The relative contributions of these three different protein families to LCFA-CoA utilization and/or regulation of cellular activities are the focus of new directions in this field.

Higher-plant medium- and short-chain acyl-CoA oxidases: identification, purification and characterization of two novel enzymes of eukaryotic peroxisomal beta-oxidation.

Abstract: Medium- and short-chain acyl-CoA oxidases were identified in and subsequently purified from dark-grown maize plantlets. The oxidase showing preference for medium-chain fatty acyl-CoAs (C10-C14) was purified to homogeneity. The oxidase showing preference for short-chain fatty acyl-CoAs (C4-C8) was purified over 150-fold. Various catalytic properties confirmed these enzymes to be true acyl-CoA oxidases. They produced trans-2-enoyl-CoA and H2O2 from the saturated acyl-CoA, as verified by various independent assay techniques. They also exhibited FAD-dependent activity; i.e. removal of loosely bound FAD by gel filtration markedly reduced activity, which could be restored upon re-addition of FAD. They showed apparent Km values between 2 and 10 microM for the acyl-CoA substrate giving maximal activity, no activity with the corresponding free fatty acid, high pH optima (8.3-8.6) and a peroxisomal subcellular location. The medium-chain acyl-CoA oxidase was determined to be a monomeric protein with a molecular mass of 62 kDa. The short-chain acyl-CoA oxidase was shown to have a native molecular mass of 60 kDa, but exhibited a labile multimeric structure, as indicated by the elution of multiple peaks of activity during several chromatographic steps, and ultimately by the purification of a subunit of molecular mass 15 kDa. The medium- and short-chain acyl-CoA oxidases were demonstrated to be distinct from the maize equivalent of the cucumber glyoxysomal long-chain acyl-CoA oxidase previously purified and characterized [Kirsch, Loffler and Kindl (1986) J. Biol. Chem. 261, 8570-8575]. The maize long-chain acyl-CoA oxidase was partially purified to permit determination of its substrate specificity; it showed activity with a broad range of acyl-CoAs of chain length greater than C8, and maximal activity with C16. The implications of the existence of multiple acyl-CoA oxidases in the regulation of plant peroxisomal beta-oxidation are discussed.

beta-Oxidation in human alcoholic and non-alcoholic hepatic steatosis.

Abstract: 1. The CoA and carnitine ester intermediates of mitochondrial beta-oxidation have not previously been quantified in liver disease, although there is some evidence that beta-oxidation is inhibited in alcoholic fatty liver. Mitochondria were isolated from needle liver biopsies from normal subjects, from patients with alcoholic fatty liver and patients with fatty liver of other aetiologies, incubated with 60 mumol/l [U-14C]hexadecanoate and the resultant CoA and carnitine esters were measured. 2. Although there was no significant difference in beta-oxidation flux between the patient groups, there was a significant rise in the proportion of 3-hydroxyacyl-CoA and 2-enoyl-CoA esters in patients with alcoholic fatty liver compared with normal subjects, and in patients with non-alcoholic fatty liver, suggesting an inhibition at the level of 3-hydroxyacyl-CoA dehydrogenase activity. 3. In alcoholic patients this difference could not be accounted for on the basis of the measured activity of short and long-chain 3-hydroxyacyl-CoA dehydrogenases, and it is suggested that either an inhibition of complex I activity or diminished amounts of ubiquinone are likely to be responsible for the observed accumulation of CoA and carnitine esters, which may contribute to the accumulation of triacylglycerols in alcoholic steatosis. In fatty liver of other aetiologies, short- and long-chain 3-hydroxyacyl-CoA dehydrogenase activities were decreased.

A New Inhibitor of Mitochondrial Fatty Acid Oxidation

Abstract: The mitochondrial enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein (trifunctional protein) plays a major role in mitochondrial fatty acid oxidation. The enzyme complex consists of four molecules of alpha-subunit containing both hydratase and dehydrogenase domains and four molecules of beta-subunit containing the thiolase domain. The primary structure of a gastrin-binding protein (GBP) was highly homologous to that of the alpha-subunit of the trifunctional protein. Here, we report that gastrin inhibits the hydratase, dehydrogenase, and thiolase activities of the trifunctional protein. The gastrin/cholecystokinin receptor antagonist benzotript, which inhibited binding of gastrin to the GBP, also inhibited all three activities of the trifunctional protein. In addition, benzotript inhibits the activities of multifunctional enzymes having similar structures, such as the peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein and the Pseudomonas fragi fatty acid oxidation enzyme complex. This reagent, however, hardly inhibited various monofunctional enzymes involved in fatty acid oxidation.

Acyl Coenzyme a Synthetase and the Transport of Long-Chain Fatty Acids

Abstract: Long-chain fatty acids and their derivatives represent essential components of membranes, importance sources of metabolic energy and important effector molecules that regulate metabolism. Prior to any type of metabolic transformation, exogenous long-chain fatty acids (C12 - C18) must pass through the cell membrane. A saturable long-chain fatty acid uptake process that is effective at nanomolar concentrations has been described in a number of different cells type suggesting these compounds enter the cell by a facilitated mechanism. This suggestion is supported by work showing that this process is also rapid, blocked by fatty acid analogues and protease sensitive1-6. Once long-chain fatty acids are taken up by the cell, they become bound to intracellular fatty acid binding proteins (FABPs) or are activated into CoA thioesters. Fatty acyl CoA thioesters serve as substrates for both β-oxidation and phospholipid synthesis.

Regulation of the pyruvate dehydrogenase complex during the aerobic/anaerobic transition in the development of the parasitic nematode, Ascaris suum

Abstract: The pyruvate dehydrogenase complex (PDC) occupies a pivotal position in the novel, anaerobic, mitochondrial metabolism of the parasitic nematode, Ascaris suum (Kita, 1992; Komuniecki and Komuniecki, 1995). Adult ascarid muscle mitochondria use unsaturated organic acids, instead of oxygen, as terminal electron-acceptors and acetate, propionate, succinate, and the 2-methyl branched-chain fatty acids, 2-methylbutyrate and 2-methylvalerate, accumulate as end products of carbohydrate metabolism. The tricarboxylic acid cycle is not operative and the NADH-dependent reductions of fumarate and 2-methyl branched-chain enoyl CoAs are coupled to site 1, electron-transport associated energy-generation (Kita, 1992; Ma et al., 1993). Most importantly, from the perspective of pyruvate metabolism, intramitochondrial NADH/NAD+ and acyl CoA/CoA ratios appear to be dramatically elevated, when compared to the corresponding aerobic organelles, and serve as the driving force for the reversal of β–oxidation and the synthesis of branched-chain fatty acids (Kita, 1992; Komuniecki and Komuniecki, 1995). Therefore, it was initially surprising to find a functional PDC and PDHa kinase in these organelles, given the potential for these elevated ratios to reduce PDC activity, either through end product inhibition or stimulation of PDHa kinase and its subsequent phosphorylation and inactivation of the complex. In fact, the PDC is significantly overexpressed in adult ascarid muscle mitochondria and is present in amounts substantially greater than those reported from other eukaryotic sources (Song and Komuniecki, 1994; Thissen et al., 1986). Not surprisingly, both its subunit composition and regulatory properties differ significantly from complexes isolated from aerobic tissues.