The subcellular distribution of acyl CoA: Dihydroxyacetone phosphate acyl transferase in guinea pig liver

Upon differential centrifugation, the enzyme acyl CoA: dihydroxyacetone phosphate acyl transferase (EC 2.3.1.42) in guinea pig liver is shown to sediment in a lysosomal-peroxisomal fraction. Comparison of the distribution of the marker enzymes and of DHAP acyl transferase indicates that the acyl transferase is localized in peroxisomes (microbodies).     

Acyl CoA:Cholesterol acyl transferase activity in human placental microsomes: Inhibition by progesterone

The characteristics of acyl CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) in microsomes prepared from human term placenta were studied and the rate of incorporation of [1-¹⁴C] oleoyl CoA into cholesteryl esters was measured. The apparent K_m of the enzyme for [1-¹⁴C] oleoyl CoA was 38 ± 9 μm and the V for the reaction was 15 ± 6 pmol × mg^? protein × min^?1. The Hill coefficient for the reaction was 1.2, indicative of some degree of positive cooperativity. Cholesterol, added to the incubation mixture, did not influence ACAT activity, indicating that endogenous microsomal cholesterol served as an effective substrate for the placental ACAT enzyme. However, [1,2-³H]cholesterol in the presence of oleoyl CoA was incorporated into cholesteryl esters by placental microsomes. When progesterone was present in the incubation mixture at a concentration of 20 μm, ACAT activity was inhibited 50%. Pregnenolone, 5α-dihydroprogesterone, 17α-hydroxyprogesterone, deoxycorticosterone, dehydroisoandrosterone, androstenedione, testosterone, and estradiol-17β also inhibited ACAT activity, whereas corticosterone, cortisol, and estriol had little effect. These results are supportive of the view that ACAT activity in human placenta may be regulated by endogenously synthesized steroid hormones.     

Short term regulation of acyl CoA: Cholesterol acyl transferase (ACAT) activity in the regenerating and perinatal liver

Acyl coenzyme A:cholesterol acyltransferase (ACAT), the enzyme catalyzing the hepatic cholesterol esterification could be involved in the modified availability of cholesterol detectable in proliferating systems. While no significant variations are detectable in the regenerating liver, the modified ACAT activity during liver development and its differential sensitivity to the in vitro stimulation of modulatory systems suggest an involvement of the enzyme in this proliferating process.     

Purification and properties of an acyl CoA transferase from Ascaris suum muscle mitochondria

An acyl CoA transferase has been purified to electrophoretic homogeneity from the soluble compartment of Ascaris suum muscle mitochondria. From SDS-PAGE, isoelectric focusing and molecular exclusion chromatography, homogeneity was confirmed and the enzyme appears to be composed of two similar or identical subunits of apparent mol. wts of 50,000 resulting in an apparent mol. wt of 100,000 for the holoenzyme. The apparent isoelectric point was 5.6 +/- 0.1 by both chromatofocusing columns and slab gel isoelectric focusing. The transferase was relatively specific for the short, straight-chain acyl CoA donors as well as the CoA acceptors, being active on acetyl CoA, propionyl CoA, butyryl CoA, valeryl CoA and hexanoyl CoA as donors to acetate and propionate. Neither succinyl CoA nor succinate were appreciably active as CoA donor or acceptor, respectively. This enzyme cannot serve physiologically to activate succinate for decarboxylation to propionate, but may serve to ensure a supply of propionyl CoA which appears to be required in catalytic amounts for the decarboxylation of succinate.     

Regulation of acyl CoA: Cholesterol acyl transferase (ACAT) activity by mevalonate and cholesterol in isolated rat hepatocytes during perinatal development

Acyl CoA: cholesterol acyl transferase (ACAT) activity presents marked oscillations and differential sensitivity to the in vitro stimulation of the kinase-phosphatase modulatory system in the perinatal rat liver.The regulation of this enzyme activity by some modulators generally active in adulthood, such as cholesterol, lipoproteins and mevalonate, has been studied in hepatocytes isolated at different developmental stages. A lack of effect of mevalonate and a positive effort of lipoprotein cholesterol have been observed at the fetal and neonatal stages.A differential prevalence is suggested of one of the two modulatory mechanisms (phosphorylation-dephosphorylation system, or substrate effect) at each developmental stage.     

Erythrocyte membrane acyl:CoA synthetase activity

The presence of long-chain acyl:CoA synthetases in mammalian microsomes and mitochondria has been established previously [(1971) Biochim. Biophys. Acta 231, 32-47]. The presence of a plasma membrane-associated enzyme was investigated in human erythrocyte ghost plasma membranes, where an enzyme exhibiting high activity, and with a preferred substrate of 18 carbon chain length, was discovered. The results are consistent with the presence of a single enzyme. The effect of the degree of unsaturation of the fatty acid substrates was not as pronounced as that arising from the length of the carbon chain. The pattern of substrate preference of the enzyme was omega 3 polyenoics greater than omega 6 polyenoics greater than omega 9 monoenoics greater than saturated fatty acids. This may relate to the similar substrate preference pattern exhibited by the fatty acyl desaturase enzymes. However, the role played by long-chain acyl:CoA synthetase in erythrocyte metabolism is uncertain, but may relate to the transportation of polyenoic fatty acids in the circulation.     

Oligosaccharyl transferase: gatekeeper to the secretory pathway

Oligosaccharyl transferase is part of the macromolecular machinery that processes nascent proteins in the endoplasmic reticulum. The enzyme is highly conserved, catalyzes the initial step in the biosynthesis of N-linked glycoproteins and acts as a 'gatekeeper' for the secretory pathway. As more proteins associated with oligosaccharyl transferase are identified, the intricacies of the enzyme and the relationship with other proteins in the lumen of the endoplasmic reticulum are starting to be unraveled.     

Palmitoyl transferase activity of lecithin retinol acyl transferase

Lecithin retinol acyl transferase (LRAT) has the essential role of catalyzing the transfer of an acyl group from the sn-1 position of lecithin to vitamin A to generate all-trans-retinyl esters (tREs). In vitro studies had shown previously that LRAT also can exchange palmitoyl groups between RPE65, a tRE binding protein essential for vision, and tREs. This exchange is likely to be of regulatory significance in the operation of the visual cycle. In the current study, the substrate specificity of LRAT is explored with palmitoylated amino acids and dipeptides as RPE65 surrogates. Both O- and S-substituted palmitoylated analogues are excellent substrates for tLRAT, a readily expressed and readily purified form of LRAT. Using vitamin A as the palmitoyl acceptor, tREs are readily formed. The cognate of these reactions occurs in crude retinal pigment epithelial (RPE) membranes as well. RPE membranes containing LRAT transfer palmitoyl groups from radiolabeled [1-(14)C]-l-alpha-dipalmitoyl diphosphatidylcholine (DPPC) to RPE65. Palmitoyl transfer is abolished by preincubation with a specific LRAT antagonist both in membranes and with purified tLRAT. These experiments are consistent with an expanded role for LRAT function as a protein palmitoyl transferase.     

The heterotrimeric G protein Go2 regulates catecholamine uptake by secretory vesicles.

Secretory vesicles store neurotransmitters that are released by exocytosis. Their membrane contains transporters responsible for transmitter loading that are driven by an electrochemical proton gradient across the vesicle membrane. We have now examined whether uptake of noradrenaline is regulated by heterotrimeric G proteins. In streptolysin O-permeabilized PC 12 cells, GTP-analogues and AlF4- inhibited noradrenaline uptake, an effect that was sensitive to treatment with pertussis toxin. Inhibition of uptake was prevented by Galphao-specific antibodies and mimicked by purified activated Galphao2. No effect was seen when Galphao2 in its inactive GDP-bound form or purified activated Galphao1, Galphai1 and Galphai2 were tested. Down-regulation of uptake remained unchanged when exocytosis was inhibited by the light chain of tetanus toxin. Vesicular acidification was not affected whereas binding of [3H]reserpine was reduced by GTPgammaS and Galphao2. These data suggest that the monoamine transporter rather than the vacuolar ATPase is affected. We conclude that catecholamine uptake is controlled by Galphao2, suggesting a novel function for heterotrimeric G proteins in the control of neurotransmitter storage.     

Catalytic role of the conformational change in succinyl-CoA:3-oxoacid CoA transferase on binding CoA

Catalysis by succinyl-CoA:3-oxoacid CoA transferase proceeds through a thioester intermediate in which CoA is covalently linked to the enzyme. To determine the conformation of the thioester intermediate, crystals of the pig enzyme were grown in the presence of the substrate acetoacetyl-CoA. X-ray diffraction data show the enzyme in both the free form and covalently bound to CoA via Glu305. In the complex, the protein adopts a conformation in which residues 267-275, 280-287, 357-373, and 398-477 have shifted toward Glu305, closing the enzyme around the thioester. Enzymes provide catalysis by stabilizing the transition state relative to complexes with substrates or products. In this case, the conformational change allows the enzyme to interact with parts of CoA distant from the reactive thiol while the thiol is covalently linked to the enzyme. The enzyme forms stabilizing interactions with both the nucleotide and pantoic acid portions of CoA, while the interactions with the amide groups of the pantetheine portion are poor. The results shed light on how the enzyme uses the binding energy for groups remote from the active center of CoA to destabilize atoms closer to the active center, leading to acceleration of the reaction by the enzyme.     

And then there were acyl coenzyme A:cholesterol acyl transferase inhibitors

PURPOSE OF REVIEW: The reputation of acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitors has changed profoundly from promising new drugs for cardiovascular prevention to drugs without clinical benefits or possibly even with adverse effects. RECENT FINDINGS: ACAT inhibitors decrease the intracellular conversion of free cholesterol into cholesteryl ester in a number of tissues, including intestine, liver and macrophages. In contrast to promising results in experimental animal models, all subsequent clinical studies in humans with ACAT inhibitors failed to show lipid profile changes as well as reductions in surrogate markers for coronary artery disease. In fact, there was even a tendency towards an increase in atheroma burden in the most recent and well executed clinical trials. In addition, the inhibition of this pivotal enzyme in cholesterol esterification may interfere with reverse cholesterol transport. SUMMARY: In our opinion, the consistent negative findings in recent clinical trials have virtually eliminated the chances for this class of drugs to be introduced for cardiovascular prevention. Possible strategies focused on selective ACAT 2 inhibition or the combination of ACAT inhibitors with compounds that stimulate reverse cholesterol transport may prove to have clinical benefit. This will have to await further clinical research in humans, however, as, obviously, rodent models cannot provide reliable data as to the efficacy of this class of drugs in humans.     

A fluorescent assay to quantitatively measure in vitro acyl CoA:diacylglycerol acyltransferase activity

Triacylglycerols (TG) are the major storage form of energy in eukaryotic organisms and are synthesized primarily by acyl CoA:1,2-diacylglycerol acyltransferase (DGAT) enzymes. In vitro DGAT activity has previously been quantified by measuring the incorporation of either radiolabeled fatty acyl CoA or diacylglycerol (DG) into TG. We developed a modified acyltransferase assay using a fluorescent fatty acyl CoA substrate to accurately quantify in vitro DGAT activity. In the modified assay, radioactive fatty acyl CoA is replaced with fluorescent NBD-palmitoyl CoA, which is used as a substrate by DGAT with DG to produce NBD-TG. After extraction with organic solvents and separation by thin layer chromatography, NBD-TG formation can be detected and accurately quantified using a fluorescent imaging system. We demonstrate that this method can be adapted to detect other acyltransferase activities. Because NBD-palmitoyl CoA is commercially available at a much lower cost compared with radioactive acyl CoA substrates, it is a more economical alternative to radioactive tracers. In addition, the exposure of laboratory personnel to radioactivity is greatly reduced.     

Purification of acyl CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase

Acyl coenzyme A:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase (EC 2.3.1.23) is capable of forming lipid bilayer vesicles from its soluble substrates lysophosphatidylcholine (LPC) and oleoyl CoA. This suggested a purification method in which rat liver microsomes are first washed with deoxycholate to increase specific activity of the endogenous acyltransferase approximately fivefold, then solubilized by the detergent effect of excess LPC and oleoyl CoA in 1:1 stoichiometric ratios. As the LPC is converted to phosphatidylcholine by acyl group transfer, the detergent effect is lost and lipid vesicles containing the enzyme activity are produced. Other microsomal proteins are excluded from the vesicles. The vesicles may be separated by density gradient flotation and are found to contain acyltransferase with a specific activity of 9–10 µmol/mg/min. This reflects a purification of approximately 140-fold, about ten times greater than achieved in previous studies.     

Identification and characterization of a succinyl-coenzyme A (CoA):benzoate CoA transferase in Geobacter metallireducens

Geobacter metallireducens is a Fe(III)-respiring deltaproteobacterium and serves as a model organism for aromatic compound-degrading, obligately anaerobic bacteria. In this study, a genetic system was established for G. metallireducens using nitrate as an alternative electron acceptor. Surprisingly, disruption of the benzoate-induced bamY gene, encoding a benzoate coenzyme A (CoA) ligase, reproducibly showed an increased biomass yield in comparison to the wild type during growth with benzoate but not during growth with acetate. Complementation of bamY in trans converted the biomass yield back to the wild-type level. Growth of the bamY mutant with benzoate can be rationalized by the identification of a previously unknown succinyl-CoA:benzoate CoA transferase activity; it represents an additional, energetically less demanding mode of benzoate activation. The activity was highly enriched from extracts of cells grown on benzoate, yielding a 50-kDa protein band; mass spectrometric analysis identified the corresponding benzoate-induced gene annotated as a CoA transferase. It was heterologously expressed in Escherichia coli and characterized as a specific succinyl-CoA:benzoate CoA transferase. The newly identified enzyme in conjunction with a benzoate-induced succinyl-CoA synthetase links the tricarboxylic acid cycle to the upper benzoyl-CoA degradation pathway during growth on aromatic growth substrates.     

Propionate mitochondrial toxicity in liver and skeletal muscle: acyl CoA levels

Propionic acidemia occasionally produces a toxic encephalopathy resembling Reye syndrome, indicating disruption of mitochondrial metabolism. Understanding the mitochondrial effect of propionate might clarify the pathophysiology. Liver mitochondria are inhibited by propionate (5 mM) while muscle mitochondria are not. Preincubation is required to inhibit liver mitochondria, suggesting that propionate is metabolized to propionyl CoA. Liver and skeletal muscle mitochondria incubated with [1-14C]propionate contain similar quantities of matrix isotope and release comparable [14C]CO2. However, only liver mitochondria accumulated significant propionyl CoA, which was largely (68%) synthesized from propionate. Carnitine reduced the level of liver matrix propionyl CoA. Inhibition of respiratory control ratios by propionate correlated with propionyl CoA levels. These results support the hypothesis that acyl CoA esters are toxic and that carnitine exerts its protective effect by converting acyl CoA esters to acylcarnitine esters.