Carnitine Acyltransferase Activities Research Articles

Carnitine-acylcarnitine translocase. Inhibition by alpha-cyano-4-hydroxycinnamate and evidence for separate identity from the pyruvate transporting system of mitochondria.

Some of the known inhibitors of pyruvate transport inhibited the activity of carnitine-acylcarnitine translocase. Their order of effectiveness with millimolar concentration required for 50% inhibition given in parentheses, was: Compound UK-5099 (alpha-cyano-beta-(1-phenylindol-3-yl)acrylate) (0.1); alpha-cyano-4-hydroxycinnamate (0.17); alpha-cyano-3-hydroxycinnamate (1); alpha-cyanocinnamate (1); alpha-fluorocinnamate (7); transcinnamate (10); p-hydroxycinnamate (10); phenylpyruvate (22); p-hydroxyphenylpyruvate (25). Kinetically, the alpha-cyano-4-hydroxycinnamate inhibition was mixed and the p-hydroxyphenylpyruvate inhibition was noncompetitive with respect to external (-)-carnitine. The alpha-cyano-4-hydroxycinnamate inhibition was reversible and resulted from its ability to act as a thiol reagent. In general, alpha-cyanocinnamate and its derivatives inhibit carnitine transport at concentrations 100 to 5000 times as high as those known to pyruvate transport. At millimolar concentrations, alpha-cyano-4-hydroxycinnamate inhibited the mitochondrial transport of molecules other than carnitine as well as the activity of carnitine acyltransferases. Pyruvate and carnitine did not complete for transport into and out of mitochondria. These results establish that transmitochondrial transport mechanisms for carnitine and pyruvate involve different carriers.

Experimental lipid storage myopathy: A quantitative ultrastructural and biochemical study

A lipid storage myopathy was induced in rats treated with daily doses of 2.5 g/kg brominated vegetable oil. As in human lipid storage myopathies, type I fibers were selectively severely affected, the lipid deposits were surrounded by mitochondria and the mitochondrial fraction of the affected fibers was increased. The oxidation of [U- 14C]palmitate, [1- 14C]octanoate and β-[3- 14C]hydroxybutyrate was significantly depressed but [1- 14C]palmitate, as well as labeled pyruvate and succinate were oxidized at normal rates. The activities of long-chain, medium-chain and short-chain carnitine acyltransferases and the muscle carnitine levels were normal. The lipid storage is attributed to impaired beta oxidation of medium-chain and short-chain fatty acyl residues. An approach to the investigation of those human lipid storage myopathies not due to defects in the carnitine system is suggested.

Comparison of the carnitine acyltransferase activities from rat liver peroxisomes and microsomes

Carnitine acyltransferase activities for acetyl- and octanoyl-CoA (coenzyme A) occur in isolated peroxisomal, mitochondrial, and microsomal fractions from rat and pig liver. Solubility studies indicated that both peroxisomal carnitine acyltransferases were in the soluble matrix. In contrast, the microsomal carnitine acyltransferases were tightly associated with their membrane. The microsomal short-chain transferase, carnitine acetyltransferase, was solubilized and stabilized by extensive treatment of the membrane with 0.4 m KCl or 0.3 m sucrose in 0.1 m pyrophosphate at pH 7.5. The same treatment only partially solubilized the microsomal medium-chain transferase, carnitine octanoyltransferase. Although half of the total carnitine acetyltransferase activity in rat liver resides in peroxisomes and microsomes, previous reports have only investigated the mitochondrial activity. Transferase activity for acetyl- and octanoyl-CoA were about equal in peroxisomal and in microsomal fractions. A 200-fold purification of peroxisomal and microsomal carnitine acetyltransferases was achieved using O-(diethylaminoethyl)-cellulose and cellulose phosphate chromatography. This short-chain transferase preparation contained less than 5% as much carnitine octanoyltransferase and acyl-CoA deacylase activities. This fact, plus differences in solubility and stability of the microsomal transferase system for acetyl- and octanoyl-CoA indicate the existence of two separate enzymes: a carnitine acetyltransferase and a carnitine octanoyltransferase in peroxisomes and in microsomes. Peroxisomal and microsomal carnitine acetyltransferases had similar properties and could be the same protein. They showed identical chromatographic behavior and had the same pH activity profiles and major isoelectric points. They also had the same apparent molecular weight by gel filtration (59,000) and the same relative velocities and K m values for several short-chain acyl-CoA substrates. Both were active with propionyl-, acetyl-, malonyl-, and acetyacetyl-CoA, but not with succinyl- and β-hydroxy-β-methylglutaryl-CoA as substrates.

Hormonal control of ketogenesis. Rapid activation of hepatic ketogenic capacity in fed rats by anti-insulin serum and glucagon.

The enhanced capacity for long-chain fatty acid oxidation and ketogenesis that develops in the rat liver between 6 and 9 h after the onset of starvation was shown to be inducible much more rapidly by administration of anti-insulin serum or glucagon to fed rats. After only 1 h of treatment with either agent, the liver had clearly switched from a "nonketogenic" to a "ketogenic" profile, as determined by rates of acetoacetate and b-hydroxybutyrate production on perfusion with oleic acid. As was the case after starvation, the administration of insulin antibodies or glucagon resulted in depletion of hepatic glycogen stores and a proportional increase in the ability of the liver to oxidize long-chain fatty acids and (-)-octanoylcarnitine, suggesting that all three treatment schedules activated the carnitine acyltransferase system of enzymes. In contrast to anti-insulin serum, which produced marked elevations in plasma glucose, free fatty acid, and ketone body concentrations, glucagon treatment had little effect on any of these parameters, presumably due to enhanced insulin secretion after the initial stimulation of glycogenolysis. Thus, after treatment with glucagon alone, it was possible to obtain a "ketogenic" liver from a nonketotic animal. The results are consistent with the possibility that the activity of carnitine acyltransferase, and thus ketogenic capacity, is subject to bihormonal control through the relative blood concentrations of insulin and glucagon, as also appears to be the case with hepatic carbohydrate metabolism.

The Subcellular Distribution of Carnitine Acyltransferases in Mammalian Liver and Kidney: A NEW PEROXISOMAL ENZYME

Abstract Liver and kidney organelles from rat and pig were separated by isopycnic sucrose density gradient centrifugation and located by marker enzymes. Carnitine palmitoyltransferase was shown to be exclusively a mitochondrial enzyme. In liver, approximately 52% of carnitine acetyltransferase activity was mitochondrial, 14% peroxisomal, and 34% located in a lipid-rich membranous fraction. Microsomes were a component of this last fraction and, when isolated by differential centrifugation, contained carnitine acetyltransferase activity. This enzyme has not previously been reported to be in peroxisomes. The specific activity of carnitine acetyltransferase in liver peroxisomes was two to three times greater than in the mitochondria or microsomes. Partial fractionation of broken rat liver peroxisomes into core, membranes, and the soluble matrix indicated that carnitine acetyltransferase had a similar distribution to the matrix enzyme, catalase. In gradients of rat and pig kidney, carnitine acetyltransferase was found primarily in the mitochondrial fractions. This enzyme was also not detected in microbodies, mitochondria, or microsomes from plants. Carnitine acetyltransferase activity in liver fractions was confirmed by three separate assays—an 1(-)-carnitine-dependent release of coenzyme A (CoA) from acetyl-CoA, identification of the 14C-labeled reaction product acetylcarnitine, and the 1(-)-acetylcarnitine-dependent formation of acetyl-CoA from CoA. Carnitine acyltransferase activity for octanoyl-CoA in hepatic peroxisomes and microsomes was about equal to activity for acetyl-CoA. In the mitochondria, activity for octanoyl-CoA was six times greater than for acetyl-CoA.

In vitro studies on the metabolism of hexadecanedioic acid and its mono- l-carnitine- ester

1. 1. A method for the synthesis of the mono-L-carnitine ester of hexadecanedioic acid (hexadecanedioylcarnitine) is described. A thin-layer chromatography system for the separation of long-chain monocarboxylic and long-chain dicarboxylic acylcarnitines was developed. 2. 2. Hexadecanedioic acid can be activated by rat liver mitochondria in the presence of CoA, ATP, and Mg 2+. Since Triton X-Ioo does not increase the activation capacity, the enzyme(s) activating long-chain dicarboxylic acids seems to be localized outside the inner mitochondrial compartment. 3. 3. Hexadecanedioylcarnitine is substrate for a solubilized preparation of hexadecanoyl- CoA: carnitine O- hexadecanoyltransferase (EC 2.3.1.-), but the reaction rate is only about 1/10 of that with palmitylcarnitine. Accordingly, mitochondrial CoA can be acylated by hexadecanoylcarnitine, but much slower than by palmitylcarnitine. 4. 4. Hexadecanedioylcarnitine competes with palmitylcarnitine in the reaction catalyzed by the enzyme. 5. 5. In fasted, ketotic streptozotocin-diabetic, and clofibrate-fed rats the carnitine acyltransferase activity was significantly increased. The relative increases were about the same whether hexadecanedioylcarnitine or palmitylcarnitine were used as substrates. 6. 6. The observations under points 4 and 5 indicate that the same enzyme ( hexadecanoyl- CoA: carnitine O- hexadecanoyltransferase, EC 2.3.1.-) may transport activated hexadecanedioic as well as palmitic acid across the inner mitochondrial membrane. 7. 7. The O 2 uptake of rat heart and liver mitochondria with hexadecanedioylcarnitine as substrate was very low and transitory. Hexadecanedioylcarnitine did not inhibit the oxidation of palmitylcarnitine. 8. 8. The present in vitro studies demonstrate a possible mechanism for the degradation of long-chain dicarboxylic acids, viz. activation by CoA and transportation into the inner mitochondrial compartment as carnitine esters followed by β-oxidation.

Erucic acid—an inhibitor of fatty acid oxidation in the heart

In the present work the carnitine ester of erucic acid has been studied. The oxygen uptake of heart and liver mitochondria from young rats was much lower with erucylcarnitine as the substrate than with palmitylcarnitine. The presence of erucylcarnitine caused a significant inhibition of the mitochondrial oxidation of palmitylcarnitine. The inhibition was more pronounced with heart mitochondria than with liver mitochondria. With liver mitochondria the inhibitory effect of erucylcarnitine on the oxygen uptake was stronger in the presence of malonate than with malate. Erucylcarnitine did not inhibit the formation of intramitochondrial long-chain acyl-CoA from added palmitylcarnitine. Erucylcarnitine did not inhibit the activity of long-chain carnitine acyltransferase with palmitylcarnitine as the substrate. The presence of erucylcarnitine did not inhibit the acylation of glycerophosphate by palmityl groups in heart and liver homogenate. The findings suggest that a mitochondrial metabolite of erucic acid inhibits the mitochondrial oxidation of other fatty acids, especially in the heart, and that this causes the accumulation of triglycerides in the hearts of rats fed rapeseed oil.

The activity of the different carnitine acyltransferases in the liver of clofibrate-fed rats

1. 1. Rats were fed 0.3% clofibrate in the stock diet for one and two weeks. 2. 2. Clofibrate caused a variable increase in carnitine acyltransferase activities in the liver dependent on the chain length of the substrates. The following factors for the increase in specific activity were observed: 5.3 (short-chain acyltransferase), 2.8 (medium-chain transferase), and 1·7 (long-chain transferase). 3. 3. These results support our earlier conclusion (H.E. Solberg, FEBS Lett., 12 (1971) 134) that a carnitine medium-chain acyltransferase exists. 4. 4. Clofibrate also caused a small increase in short-chain and medium-chain acyl-CoA hydrolase activity.

Different carnitine acyltransferases in calf liver

1. 1. Carnitine acyltransferases were partially purified from calf liver. 2. 2. Studies of acyl group specificity showed chain length optima at C 3, C 6, C 12 and C 16. 3. 3. Three different levels of apparent K m values for acyl- l-carnitines were obtained: 470–638 μM (short-chain acylcarnitines), 62–265 μM (medium-chain acyl-carnitines) and 24–36 μM (long-chain acylcarnitines). 4. 4. Carnitine acyltransferase activities assayed with short-chain, medium-chain or long-chain substrates showed different distributions in fractions obtained by precipitation with ammonium sulphate and by gel filtration on Sephadex G-200. 5. 5. Complete separation of different carnitine acyltransferases was not achieved because solubilized, lipid-free transferases had similar molecular weights and isoelectric points (pH 6.0). 6. 6. The present results support our earlier conclusion (H. E. Solberg, FEBS Lett. 12 (1971) 134) that a separate carnitine medium-chain acyltransferase exists.

Properties of a purified carnitine palmitoyltransferase, and evidence for the existence of other carnitine acyltransferases.

Carnitine palmitoyltransferase (CPT) has been purified, and some of its properties are described. The purified preparations had specific activities of 10–38 μmoles/min/mg protein at 35°. The apparent molecular weight of CPT was approximately 75 000, or a multiple thereof. The K′m value for palmitoyl-CoA was 1.8 × 10−5 M, while the K′s values for (–)-carnitine, CoA–SH, and (–)-palmitoylcarnitine were estimated to be 4.5 × 10−4 M, 5.5 × 10−6 M, and 1.4 × 10−4 M, respectively. The enzyme was subject to inhibition by all substrates and products. CPT had a relatively broad fatty acyl chain-length specificity, with highest Vmax values obtained for the transfer of myristoyl and palmitoyl groups. K′m values were lowest with long-chain fatty acylcarnitinè derivatives (1.9 × 10−4 M for (–)-stearoylcarnitine versus 1.2 × 10−3 M for (–)-decanoylcarnitine).Evidence is presented demonstrating the existence of two additional protein fractions possessing carnitine acyltransferase activities with different fatty acyl chain-length specificities. One, which displayed highest relative activity towards the transfer of octanoyl groups, is tentatively designated carnitine octanoyltransferase. The other, which displayed a high specificity toward the transfer of palmitoyl and stearoyl groups from acylcarnitine when preincubated with CoA–SH, is tentatively designated carnitine palmitoyltransferase II.

Some aspects of phospholipid metabolism in the red cell

1. (1) Long-chain carnitine acyl transferase activity in red cell membranes was demonstrated by their capacity to incorporate [1- 14C]oleic acid and [1- 14C]linoleic acid into the respective acylcarnitines. The addition of (−)-carnitine to red cell membranes enhanced [1- 14C]palmitate incorporation into palmitylcarnitine, whereas (+)-carnitine was without effect. (+)-Palmitylcarnitine resulted in a depression of [1- 14C]palmitate incorporation into palmitylcarnitine, and this inhibition was reversed by the addition of (−)-carnitine. 2. (2) Fatty acylcarnitine derivatives were shown not to be obligatory intermediates in the incorporation of fatty acyi CoA into lecithin, and the addition of (−)-carnitine increased incorporation of [1- 14C]palmitate into palmitylcarnitine while decreasing incorporation of [1- 14C]palmitate into lecithin. 3. (3) ‘Methyltransferase’ activity was demonstrated in human and sheep erythrocytes. Significant incorporation into lecithin, sphingomyelin, methylated phosphatidylethanolamine derivatives and lysolecithin was found with S-[ Me- 14C]adenosylmethionine. Methionine-activating enzyme activity was absent.

The Role of Carnitine and Carnitine Acyltransferase in Biological Acetylations and Fatty Acid Synthesis

The effect of (-)-carnitine on the conversion of citrate-1-14C and pyruvate-2-14C to 14CO2 and either 14C-fatty acids or 14C-acetylsulfanilamide was studied in pigeon liver homogenates. Carnitine stimulated the conversion of pyruvate-2-14C to fatty acids and acetylsulfanilamide, but had no effect on citrate. In the fasted state both fatty acid synthesis and citrate cleavage enzyme activity were decreased, whereas the conversion of pyruvate-2-14C to 14C-acetylsulfanilamide and carnitine acyltransferase activity were increased. (+)-Carnitine, a competitive inhibitor of carnitine acyltransferase, decreased the conversion of pyruvate-2-14C to 14C-acetylsulfanilamide. It was concluded from calculations of 14CO2 and 14C-fatty acids derived from citrate-1-14C and pyruvate-2-14C that the major pathway of supply of acetyl groups to the extramitochondrial portion of the cell is an indirect one via citrate and the citrate cleavage enzyme, but that a direct pathway via carnitine acyltransferase does exist. A role of carnitine acyltransferase in biological acetylations is discussed.