Purification and properties of carnitine acetyltransferase from human liver

Carnitine acetyltransferase was purified from the supernatant obtained after centrifugation of human liver homogenate to a final specific activity of 78.75 unit.mg-1 with acetyl-CoA as a substrate. Human carnitine acetyltransferase is a monomer of 60.5 kDa with maximum activity in the presence of propionyl-CoA and a pH optimum of 8.7. Apparent Km values for acetyl-CoA are three times lower than for decanoyl-CoA. Km values for L-carnitine in the presence of acetyl-CoA are six times lower than in the presence of decanoyl-CoA. Km values for acetylcarnitine are three times lower than for octanoylcarnitine. The polyclonal antibodies against human carnitine acetyltransferase recognize a 60.5-kDa peptide in the purified preparation of human liver and brain homogenates and in immunoblots of mitochondrial and peroxisomal fractions from human liver. Immunoprecipitation and SDS/PAGE analysis of 35S-labelled proteins produced by human fibroblasts indicate that mitochondrial carnitine acetyltransferase is synthesized as a precursor of 65 kDa. We also purified carnitine acetyltransferase from the pellet obtained after centrifugation of liver homogenate. The pellet was extracted by sonication in the presence of 0.5% Tween 20. The chromatographic procedures for the purification and the kinetic, physical and immunological properties of pellet-extracted carnitine acetyltransferase are similar to those of carnitine acetyltransferase purified from the supernatant of human liver homogenate.     

The sidedness of carnitine acetyltransferase and carnitine octanoyltransferase of rat liver endoplasmic reticulum

The location of carnitine acetyltransferase and carnitine octanoyltransferase on the inner and outer surfaces of rat liver microsomes was investigated. Latency of mannose-6-phosphate phosphatase showed that the microsomes were 90–94% sealed. All of the octanoyltransferase is associated with the cytosolic face, while the acetyltransferase is distributed between the cytosolic face (68–73%) and the lumen face (27–32%) of the endoplasmic reticulum membrane. Small amounts of trypsin inhibit the carnitine octanoyltransferase equally in either sealed or permeable microsomes but the acetyltransferase of sealed microsomes is stimulated. Large amounts of trypsin inhibit all transferase activities by about 60%, except for acetyltransferase of sealed microsomes. Other studies show that 0.1% Triton X-100 partially inhibits carnitine octanoyltransferase of microsomes but does not inhibit the acetyltransferase or any of the mitochondrial carnitine acyltransferase.     

Biosynthesis and turnover of carnitine acetyltransferase in rat liver

Male Wistar rats were fed on a diet with and without di(2-ethylhexyl)phthalate (DEHP) for 2 weeks. Carnitine acetyltransferase in the liver was increased about 100-fold by administration of DEHP. The results of in vivo experiments showed that the incorporation of L-[4,5-3H]leucine into the enzyme was 12-fold higher and the half-life of the labeled enzyme was elongated by a factor 4.6. The results of in vitro translation experiments with total hepatic RNA in a rabbit reticulocytelysate system and the results concerning the synthesis of the enzyme in isolated hepatocytes indicate that the translatable mRNA for the enzyme was increased upon administration of DEHP and that the enzyme is synthesized as a precursor (Mw = 69,000) larger than the mature enzyme (Mw = 67,500). RNA in the free polysomes directed the synthesis of the enzyme precursor five times more actively than RNA in membrane-bound polysomes.     

Identification and purification of the carnitine carrier from rat liver mitochondria

The carnitine carrier from rat liver mitochondria, solubilized in Triton X-100 and partially purified on hydroxyapatite, was identified and completely purified by specific elution from celite in the presence of cardiolipin. On SDS-gel electrophoresis, the purified celite fraction consisted of a single band with an apparent Mr of 32,500. When reconstituted into liposomes the carnitine transport protein catalyzed an N-ethylmaleimide-sensitive carnitine/carnitine exchange. It was purified 970-fold with a recovery of 43% and a protein yield of 0.04% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e., requirement for a countersubstrate, substrate specificity and inhibitor sensitivity, were similar to those of the carnitine transport system as characterized in intact mitochondria.     

Presence of carnitine acetyltransferase in peroxisomes and in mitochondria of oleic acid-grown Saccharomyces cerevisiae

Abstract Paracoccidiodes brasiliensis , the agent of paracoccidioidomycosis, when grown in a synthetic medium, expresses at the cell surface of both yeast and mycelial forms acidic glycoconjugates containing N -acetlyneuraminic acid units. Sialic acids were extracted using mild hydrolytic conditions, and were identified by thin-layer and gas chromatography, standard colorimetry, reaction with periodate-resorcinol and mass spectrometry. Their surface location was inferred from fluorescent-lectin ( Limulus polyphemus agglutinin) binding to whole cells abrogated by previous treatment with neuraminidase. Expression of sialic acids on virulent yeast forms of P. brasiliensis (3.7 × 10⁶ residues per cell) may inhibit fungal phagocytosis during early infection, when the immunological response is still being built up.     

Induction of carnitine acetyltransferase by clofibrate in rat liver.

Administration of the anti-hypercholesterolaemic drug clofibrate to the rat increases the activity of carnitine acetyltransferase (acetyl-CoA-carnitine O-acetyltransferase, EC 2.3.1.7) in liver and kidney. The drug-mediated increase in enzyme activity in hepatic mitochondria shows a time lag during which the activity increases in the microsomal and peroxisomal fractions. The enzyme induced in the particulate fractions is identical with one normally present in mitochondria. The increase in enzyme activity is prevented by inhibitors of RNA and general protein synthesis. Mitochondrial protein-synthetic machinery does not appear to be involved in the process. Immunoprecipitation shows increased concentration of the enzyme protein in hepatic mitochondria isolated from drug-treated animals. In these animals, the rate of synthesis of the enzyme is increased 7-fold.     

The substrate specificity of carnitine acetyltransferase

1. A study of the acyl group specificity of the carnitine acetyltransferase reaction [acyl-(-)carnitine+CoASH right harpoon over left harpoon (-)-carnitine+acyl-CoA] has been made with the enzyme from pigeon breast muscle. Acyl groups containing up to 10 carbon atoms are transferred and detailed kinetic investigations with a range of acyl-CoA and acylcarnitine substrates are reported. 2. Acyl-CoA derivatives with 12 or more carbon atoms in the acyl group are potent reversible inhibitors of carnitine acetyltransferase, competing with acetyl-CoA. Lauroyl- and myristoyl-CoA show a mixed inhibition with respect to (-)-carnitine, but palmitoyl-CoA competes strictly with this substrate also. Palmitoyl-dl-carnitine shows none of these effects. 3. Ammonium palmitate inhibits the enzyme competitively with respect to (-)-carnitine and non-competitively with respect to acetyl-CoA. 4. It is suggested that a hydrophobic site exists on the carnitine acetyltransferase molecule. The hydrocarbon chain of an acyl-CoA derivative containing eight or more carbon atoms in the acyl group may interact with this, which results in enhanced acyl-CoA binding. Competition occurs between ligands bound to this hydrophobic site and the carnitine binding site. 5. The possible physiological significance of long-chain acyl-CoA inhibition of this enzyme is discussed.     

Kinetic characterization of the reconstituted carnitine carrier from rat liver mitochondria

The carnitine carrier was purified from rat liver mitochondria and reconstituted into liposomes by removing the detergent from mixed micelles by Amberlite. Optimal transport activity was obtained with 1 microgram/ml and 12.5 mg/ml of protein and phospholipid concentration, respectively, with a Triton X-100/phospholipid ratio of 1.8 and with 16 passages through the same Amberlite column. The activity of the carrier was influenced by the phospholipid composition of the liposomes, being increased in the presence of cardiolipin and decreased in the presence of phosphatidylinositol. In the reconstituted system the incorporated carnitine carrier catalyzed a carnitine/carnitine exchange which followed a first-order reaction. The maximum transport rate of external [3H]carnitine was 1.7 mmol/min per g protein at 25 degrees C and was independent of the type of countersubstrate. The half-saturation constant (Km) for carnitine was 0.51 mM. The affinity of the carrier for acylcarnitines was in the microM range and depended on the carbon chain length. The activation energy of the carnitine/carnitine exchange was 133 kJ/mol. The carrier function was independent of the pH in the range between 6 and 8 and was inhibited at pH below 6.     

The intracellular localization and properties of carnitine acetyltransferase from ram spermatozoa

Although spermatozoa possess a very active carnitine acetyltransferase, there is no satisfactory explanation for such a high activity. In order to help elucidate possible roles for carnitine acetyltransferase in spermatozoa, we examined the intracellular location and properties of carnitine acetyltransferase from ejaculated ram spermatozoa. The spermatozoa were disrupted by hypotonic treatment with 10 mm phosphate buffer (pH 7.4), followed by mild sonication. The resulting homogenate was separated by sucrose step-gradient centrifugation into soluble, plasma membrane, acrosomal membrane, and mitochondrial fractions. These fractions were characterized by electron microscopy and marker enzyme assays. The particulate fractions were made soluble by treatment with 0.1% deoxycholate and then were assayed for carnitine acetyltransferase activity. Carnitine acetyltransferase activity was found exclusively in the mitochondrial fraction with a specific activity of 0.151 μmol CoASH · min^?1 · mg^?1. The apparent K_m values for acetyl-CoA and l-carnitine were 1.1 × 10^?5 and 1.3 × 10^?4m respectively.     

Localization and solubilization of a rat liver microsomal carnitine acetyltransferase.

Carnitine acetyltransferase activity had been previously shown to occur in peroxisomes, mitochondria, and a membranous fraction of rat and pig hepatocytes. When components of this third subcellular fraction (plasma membranes, components of the Golgi apparatus, and microsomes) were further separated, carnitine acetyltransferase fractionated with the microsomes. Microsomes isolated by three different methods (isopycnic sucrose density zonal centrifugation, high-speed differential centrifugation, and aggregation with Ca²⁺ followed by low-speed differential centrifugation) all contained carnitine acetyltransferase activity. The lability of carnitine acetyltransferase in microsomes isolated by different methods and in different isolation media is reported.When total microsomes were subfractionated into rough and smooth components, carnitine acetyltransferase activity was found to the same extent in both and was tightly associated with the microsomal membrane. The microsomal enzyme was rapidly inactivated in 0.25 m sucrose or 0.1 m phosphate, but was stable for at least 2 weeks in 0.4 m KCl. Extensive treatment with high ionic strength salt solutions, 1% Triton X-100, or a combination of the two was used to solubilize microsomal carnitine acetyltransferase activity.Carnitine octanoyltransferase activity was also found in the microsomal fractions isolated by three different methods, but no carnitine palmitoyltransferase was detected in the microsomal fractions. It is proposed that microsomal carnitine acetyl- and octanoyltransferases could be involved in the transfer of acyl groups across the microsomal membrane, thereby providing a source of acetyl and other acyl CoA's at sites of acetylation reactions and synthesis.     

Purification and properties of the soluble carnitine palmitoyltransferase from bovine liver mitochondria.

A new carnitine palmitoyltransferase (CPT) was purified to homogeneity from bovine liver mitochondria which were 96% free of peroxisomal contamination, as judged by catalase and glutamate dehydrogenase activities. The enzyme is easily removed from mitochondria, without the use of detergent. It is monomeric (Mr 63,500), unlike other preparations of CPT from mitochondria, and is most active with myristoyl-CoA and palmitoyl-CoA. The Km values are between 0.8 and 4 microM for a range of substrates from hexanoyl-CoA to stearoyl-CoA; these are much lower than values reported for other purified CPT preparations. The Km for L-carnitine is 185 microM measured with palmitoyl-CoA, and does not vary greatly with the chain length. This is also lower than the values reported for other CPT preparations, but higher than those cited for the medium-chain transferases. Kinetic and inhibitor studies were consistent with a rapid-equilibrium random-order mechanism. 2-Bromopalmitoyl-CoA, which is an inhibitor of the outer CPT, inhibited the enzyme competitively with palmitoyl-CoA as the variable substrate, when added without preincubation. If the enzyme was preincubated with 2-bromopalmitoyl-CoA and carnitine, the activity did not reappear after gel filtration of the protein. The inhibitor was bound in a 1:1 stoichiometry per subunit of enzyme.     

Characterization of the unidirectional transport of carnitine catalyzed by the reconstituted carnitine carrier from rat liver mitochondria

The carnitine carrier from rat liver mitochondria was purified by chromatography on hydroxyapatite and celite and reconstituted in egg yolk phospholipid vesicles by adsorbing the detergent on polystyrene beads. In the reconstituted system, in addition to the carnitine/carnitine exchange, the purified protein catalyzed a uni-directional transport (uniport) of carnitine measured as uptake into unloaded proteoliposomes as well as efflux from prelabelled proteoliposomes. In both cases the reaction followed a first-order kinetics with a rate constant of 0.023-0.026 min-1. Besides carnitine, also acylcarnitines were transported in the uniport mode. N-Ethylmaleimide inhibited the uni-directional transport of carnitine completely. The uniport of carnitine is not influenced by the delta pH and the electric gradient across the membrane. The activation energy for uniport was 115 kJ/mol and the half-saturation constant on the external side of the proteoliposomes was 0.53 mM. The maximal rate of the uniport at 25 degrees C was 0.2 mumol/min per mg protein, i.e. about 10 times lower than that of the reconstituted carnitine transport in exchange mode.     

Phosphoenolpyruvate synthesis by fetal guinea-pig liver mitochondria

Liver mitochondria isolated from fetal and newborn guinea pigs synthesized phosphoenolpyruvate at 4–6 nmol/min per mg protein with 2 mM malate, succinate, and α-ketoglutarate as substrates. These rates were 90–110% of that by adult liver mitochondria and were not substantially altered in the second half of gestation or within 24 h after birth. Both palmitoyl- and octanoylcarnitine were inhibitory to phosphoenolpyruvate synthesis in adult and fetal preparations, but free octanoate was inhibitory only in adult liver mitochondria.     

Active-site-directed inhibition of carnitine acetyltransferase

Methoxycarbonyl-CoA disulfide has been used as an active-site-directed inhibitor of carnitine acetyltransferase. Stoichiometric addition of methoxycarbonyl-CoA disulfide to carnitine acetyltransferase showed the modification of one sulfhydryl group with concomitant loss of about 80% enzyme activity. The rate of modification of this sulfhydryl group is an order of magnitude faster than that of the remaining sulfhydryl groups in the enzyme. Methoxycarbonyl-CoA disulfide inactivation is biphasic: k₁ = 1.09 × 10²m^?1s^?1, k₂ = 1.1 × 10¹m^?1s^?1. This modification, Enz-SS-CoA is covalent; it can be reversed with either dithioerythritol or thiocholine. Acetyl-carnitine and acetyl-CoA protected the enzyme against methoxycarbonyl-CoA disulfide inactivation; however, carnitine did not. These results indicate the presence of a sulfhydryl group in carnitine acetyltransferase at the site of acetyl group transfer. Titration of carnitine acetyltransferase with nonspecific sulfhydryl reagents, DTNB, and ?-nitrophenoxycarbonyl methyl disulfide, revealed that four sulfhydryl groups were preferentially modified by these reagents. The results also show that seven other sulfhydryl groups are available for modification.