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.     

Glycerolipid synthetic capacity of rat liver peroxisomes

Investigations on rat liver peroxisomal glycerolipid synthetic capability were performed. Highly purified peroxisomal preparations contained dihydroxyacetone-phosphate acyltransferase, acyldihydroxyacetone-phosphate reductase, and fatty acid-CoA ligase activities. Glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, diacylglycerol cholinephosphotransferase, diacylglycerol ethanolaminephosphotransferase and ethanol acyltransferase activities were low in activity or not detected. These results suggest that the peroxisomes are specialized to contribute to the synthesis of ether-linked glycerolipids. If peroxisomes contribute towards the synthesis of non-ether-linked glycerolipids (i.e., ester-linked) then translocation of acyl glycerophosphatide (acyl dihydroxyacetone phosphatide) from peroxisomes to endoplasmic reticulum would be expected to occur.     

The medium-chain carnitine acyltransferase activity associated with rat liver microsomes is malonyl-CoA sensitive

The data presented herein show that both rough and smooth endoplasmic reticulum contain a medium-chain/long-chain carnitine acyltransferase, designated as COT, that is strongly inhibited by malonyl-CoA. The average percentage inhibition by 17 microM malonyl-CoA for 25 preparations is 87.4 +/- 11.7, with nine preparations showing 100% inhibition; the concentrations of decanoyl-CoA and L-carnitine were 17 microM and 1.7 mM, respectively. The concentration of malonyl-CoA required for 50% inhibition is 5.3 microM. The microsomal medium-chain/long-chain carnitine acyltransferase is also strongly inhibited by etomoxiryl-CoA, with 0.6 microM etomoxiryl-CoA producing 50% inhibition. Although palmitoyl-CoA is a substrate at low concentrations, the enzyme is strongly inhibited by high concentrations of palmitoyl-CoA; 50% inhibition is produced by 11 microM palmitoyl-CoA. The microsomal medium-chain/long-chain carnitine acyltransferase is stable to freezing at -70 degrees C, but it is labile in Triton X-100 and octylglucoside. The inhibition by palmitoyl-CoA and the approximate 200-fold higher I50 for etomoxiryl-CoA clearly distinguish this enzyme from the outer form of mitochondrial carnitine palmitoyltransferase. The microsomal medium-chain/long-chain carnitine acyltransferase is not inhibited by antibody prepared against mitochondrial carnitine palmitoyltransferase, and it is only slightly inhibited by antibody prepared against peroxisomal carnitine octanoyltransferase. When purified peroxisomal enzyme is mixed with equal amounts of microsomal activity and the mixture is incubated with the antibody prepared against the peroxisomal enzyme, the amount of carnitine octanoyltransferase precipitated is equal to all of the peroxisomal carnitine octanoyltransferase plus a small amount of the microsomal activity. This demonstrates that the microsomal enzyme is antigenically different than either of the other liver carnitine acyltransferases that show medium-chain/long-chain transferase activity. These results indicate that medium-chain and long-chain acyl-CoA conversion to acylcarnitines by microsomes in the cytosolic compartment is also modulated by malonyl-CoA.     

Carnitine acyltransferase and acyl-coenzyme A hydrolase activities in human liver. Quantitative analysis of their subcellular localization.

The subcellular localizations of carnitine acyltransferase and acyl-CoA hydrolase activities with different chain-length substrates were quantitatively evaluated in human liver by fractionation of total homogenates in metrizamide density gradients and by differential centrifugation. Peroxisomes were found to contain 8-37% of the liver acyltransferase activity, the relative amount depending on the chain length of the substrate. The remaining activity was ascribed to mitochondria, except for carnitine octanoyltransferase, for which 25% of the activity was present in microsomal fractions. In contrast with rat liver, where the activity in peroxisomes is very low or absent, human liver peroxisomes contain about 20% of the carnitine palmitoyltransferase. Short-chain acyl-CoA hydrolase activity was found to be localized mainly in the mitochondrial and soluble compartments, whereas the long-chain activity was present in both microsomal fractions and the soluble compartment. Particle-bound acyl-CoA hydrolase activity for medium-chain substrates exhibited an intermediate distribution, in mitochondria and microsomal fractions, with 30-40% of the activity in the soluble fraction. No acyl-CoA hydrolase activity appears to be present in human liver peroxisomes.     

Developmental changes in the activities of peroxisomal and mitochondrial beta-oxidation in chicken liver

The activities of peroxisomal and mitochondrial beta-oxidation and carnitine acyltransferases changed during the process of development from embryo to adult chicken, and the highest activities of peroxisomal beta-oxidation, palmitoyl-CoA oxidase, and carnitine acetyltransferase were found at the hatching stage of the embryo. The profiles of these alterations were in agreement with those of the contents of triglycerides and free fatty acids in the liver. The highest activities of mitochondrial beta-oxidation and palmitoyl-CoA dehydrogenase were observed at the earlier stages of the embryo; then the activities decreased gradually from embryo to adult chicken. The ratio of activities of carnitine acetyltransferase in peroxisomes and mitochondria (peroxisomes/mitochondria) increased from 0.54 to 0.82 during the development from embryo to adult chicken. The ratio of activities of carnitine palmitoyltransferase decreased from 0.82 to 0.25 during the development. The affinity of fatty acyl-CoA dehydrogenase toward the medium-chain acyl-CoAs (C6 and C8) was high in the embryo and decreased with development, whereas the substrate specificity of fatty acyl-CoA oxidase did not change. The substrate specificity of mitochondrial carnitine acyltransferases did not change with development. The affinity of peroxisomal carnitine acyltransferases toward the long-chain acyl-CoAs (C10 to C16) was high in the embryo, but low in adult chicken.     

Rat liver dihydroxyacetone-phosphate acyltransferases and their contribution to glycerolipid synthesis

Differential and isopycnic centrifugation of rat liver homogenates showed that, besides its established localization in peroxisomes and endoplasmic reticulum, dihydroxyacetone-phosphate acyltransferase is also present in mitochondria. The three activities differed in a number of properties (pH optimum, palmitoyl-CoA and dihydroxyacetone-phosphate dependence, and sensitivity toward N-ethylmaleimide) and are therefore likely associated with three distinct proteins. Glycerol 3-phosphate (5 mM) did not inhibit peroxisomal dihydroxyacetone-phosphate acyltransferase but inhibited the extraperoxisomal activities virtually completely. Peroxisomal dihydroxyacetone-phosphate acyltransferase was located at the inner aspect of the peroxisomal membrane, but the enzyme was not latent. Purified microsomes, from which intact peroxisomes had been removed, were still contaminated with peroxisomal membranes as deduced from the presence of two dihydroxyacetone-phosphate acyltransferase activities: a glycerol 3-phosphate-resistant activity with properties similar to those of peroxisomal dihydroxyacetone-phosphate acyltransferase and a glycerol 3-phosphate-sensitive "true" microsomal dihydroxyacetone-phosphate acyltransferase. We propose that, assayed in the presence of 5mM glycerol 3-phosphate, dihydroxyacetone-phosphate acyltransferase can be used as a marker enzyme for peroxisomal membranes. Such a marker enzyme has not hitherto been available. The differential effect of 5 mM glycerol 3-phosphate on peroxisomal and extraperoxisomal dihydroxyacetone-phosphate acyltransferases enabled us to determine the relative contribution of these activities to overall dihydroxyacetone-phosphate acylation in whole liver homogenates. At near-physiological pH and at near-physiological concentrations of unbound palmitoyl-CoA and of dihydroxyacetone-phosphate plus glycerol 3-phosphate, peroxisomes contributed 50-75%. The remaining percentage was mostly accounted for by the microsomal enzyme. At near-physiological concentrations of glycerol 3-phosphate plus dihydroxyacetone-phosphate, glycerolphosphate acyltransferase contributed 93% and dihydroxyacetone-phosphate acyltransferase 7% to overall glycerolipid synthesis in homogenates. This suggests that the dihydroxyacetone-phosphate pathway is of minor quantitative importance in overall hepatic glycerolipid synthesis but that its main function lies in the synthesis of ether lipids, which have acyldihydroxyacetone-phosphate as obligatory precursor.(ABSTRACT TRUNCATED AT 400 WORDS)     

The presence of peroxisomal carnitine palmitoyltransferase in chick embryo liver

Hepatic peroxisomes and mitochondria from 20-day-old chick embryo were separated by sucrose density gradient centrifugation and the characteristics of carnitine acyltransferases in these organelles were studied. The carnitine acyltransferase activities in peroxisomes were increased markedly by the treatment of chick embryo with clofibrate, while those in mitochondria did not change. In the liver of clofibrate-treated chick embryo, approximately 50% of total liver carnitine palmitoyltransferase (CPT) activity was present in the peroxisomal fraction. Peroxisomal CPT activity was easily solubilized, in contrast with mitochondrial CPT. The solubilized protein solutions from isolated peroxisomes and mitochondria were separately chromatographed on a column of Blue Sepharose CL-6B after the gel filtration on Sephadex G-25. Peroxisomal CPT was completely bound to a Blue Sepharose CL-6B column and was eluted below 0.25 M KCl, whereas mitochondrial CPT was not retained on the column. The substrate specificity profile of peroxisomal CPT with long-chain acyl-CoAs (C8 to C18) was similar to that of mitochondrial CPT, and the apparent Km value of peroxisomal CPT for palmitoyl-CoA was 5.2 microM, being similar to that of mitochondrial CPT. It is concluded that carnitine long-chain acyltransferase, which is different from mitochondrial CPT and is induced by clofibrate treatment, is present in peroxisomes of chick embryo liver.     

Metabolic interactions between peroxisomes and mitochondria with a special focus on acylcarnitine metabolism

Carnitine plays an essential role in mitochondrial fatty acid β-oxidation as a part of a cycle that transfers long-chain fatty acids across the mitochondrial membrane and involves two carnitine palmitoyltransferases (CPT1 and CPT2). Two distinct carnitine acyltransferases, carnitine octanoyltransferase (COT) and carnitine acetyltransferase (CAT), are peroxisomal enzymes, which indicates that carnitine is not only important for mitochondrial, but also for peroxisomal metabolism. It has been demonstrated that after peroxisomal metabolism, specific intermediates can be exported as acylcarnitines for subsequent and final mitochondrial metabolism. There is also evidence that peroxisomes are able to degrade fatty acids that are typically handled by mitochondria possibly after transport as acylcarnitines. Here we review the biochemistry and physiological functions of metabolite exchange between peroxisomes and mitochondria with a special focus on acylcarnitines.     

Changes in the activities of dihydroxyacetone phosphate and glycerol-3-phosphate acyltransferases in rat liver under various conditions

Activities of enzymes relating to the acyl dihydroxyacetone phosphate (acyl DHAP) pathway were determined in rat liver under conditions known to elevate the peroxisomal β-oxidation activity. In fasted and streptozotocin-induced diabetic rats, DHAP acyltransferase activity showed a small but significant increase, though the activities of glycerol-3-phosphate (GP) acyltransferase and alkyl DHAP synthase were not changed. After 2 weeks, feeding of 20% partially hydrogenated marine oil, the activity of DHAP acyltransferase also increased to 140% of the control. The feeding of 0.25% clofibrate and 2% di(2-ethylhexyl)phthalate (DEHP) increased the activities of both DHAP and GP acyltransferases by 2- to 3-fold, whereas alkyl DHAP synthase activity decreased under the same conditions. A fractionation study showed that the increases in the activities of DHAP acyltransferase and acyl /alkyl DHAP reductase in the liver of rats treated with DEHP occurred mainly in peroxisomes and microsomes, respectively. The phospholipid contents per mg protein of the isolated hepatic peroxisomes from rats were as follows (percent of the control): fasting, 62%; diabetic, 69%; high fat-diet, 89%; clofibrate-treated, 126%; DEHP-treated, 119%. These results suggest that glycerophospholipid metabolism might also be controlled by peroxisomal enzymes under physiological and pathological conditions.     

Structural and biochemical studies of the substrate selectivity of carnitine acetyltransferase

Carnitine acyltransferases catalyze the exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids, and are attractive targets for drug discovery against diabetes and obesity. These enzymes are classified based on their substrate selectivity for short-chain, medium-chain, or long-chain fatty acids. Structural information on carnitine acetyltransferase suggests that residues Met-564 and Phe-565 may be important determinants of substrate selectivity with the side chain of Met-564 located in the putative binding pocket for acyl groups. Both residues are replaced by glycine in carnitine palmitoyltransferases. To assess the functional relevance of this structural observation, we have replaced these two residues with small amino acids by mutagenesis, characterized the substrate preference of the mutants, and determined the crystal structures of two of these mutants. Kinetic studies confirm that the M564G or M564A mutation is sufficient to increase the activity of the enzyme toward medium-chain substrates with hexanoyl-CoA being the preferred substrate for the M564G mutant. The crystal structures of the M564G mutant, both alone and in complex with carnitine, reveal a deep binding pocket that can accommodate the larger acyl group. We have determined the crystal structure of the F565A mutant in a ternary complex with both the carnitine and CoA substrates at a 1.8-A resolution. The F565A mutation has minor effects on the structure or the substrate preference of the enzyme.     

Hepatic subcellular distribution of short-chain beta-ketoacyl coenzyme A reductase and trans-2-enoyl coenzyme A hydratase: 25- to 50-fold stimulation of microsomal activities by the peroxisome proliferator, di-(2-ethylhexyl)phthalate

The present study demonstrates unequivocally the existence of short-chain trans-2-enoyl coenzyme A (CoA) hydratase and beta-ketoacyl CoA reductase activities in the endoplasmic reticulum of rat liver. Subcellular fractionation indicated that all four fractions, namely, mitochondrial, peroxisomal, microsomal, and cytosolic contained significant hydratase activity when crotonyl CoA was employed as the substrate. In the untreated rat, based on marker enzymes and heat treatment, the hydratase activity, expressed as mumol/min/g liver, wet weight, in each fraction was: mitochondria, 684; peroxisomes, 108; microsomes, 36; and cytosol, 60. Following di-(2-ethylhexyl)phthalate (DEHP) treatment (2% (v/w) for 8 days), there was only a 20% increase in mitochondrial activity; in contrast, peroxisomal hydratase activity was stimulated 33-fold, while microsomal and cytosolic activities were enhanced 58- and 14-fold respectively. A portion of the cytosolic hydratase activity can be attributed to the component of the fatty acid synthase complex. Although more than 70% of the total hydratase activity was associated with the mitochondrial fraction in the untreated rat, DEHP treatment markedly altered this pattern; only 11% of the total hydratase activity was present in the mitochondrial fraction, while 49 and 29% resided in the peroxisomal and microsomal fractions, respectively. In addition, all four subcellular fractions contained the short-chain NADH-specific beta-ketoacyl CoA (acetoacetyl CoA) reductase activity. Again, in the untreated animal, reductase activity was predominant in the mitochondrial fraction; following DEHP treatment, there was marked stimulation in the peroxisomal, microsomal, and cytosolic fractions, while the activity in the mitochondrial fraction increased by only 39%. Hence, it can be concluded that both reductase and hydratase activities exist in the endoplasmic reticulum in addition to mitochondria, peroxisomes, and soluble cytoplasm.     

Crystal structure of rat carnitine palmitoyltransferase II (CPT-II)

Carnitine palmitoyltransferase II (CPT-II) has a crucial role in the beta-oxidation of long-chain fatty acids in mitochondria. We report here the crystal structure of rat CPT-II at 1.9A resolution. The overall structure shares strong similarity to those of short- and medium-chain carnitine acyltransferases, although detailed structural differences in the active site region have a significant impact on the substrate selectivity of CPT-II. Three aliphatic chains, possibly from a detergent that is used for the crystallization, were found in the structure. Two of them are located in the carnitine and CoA binding sites, respectively. The third aliphatic chain may mimic the long-chain acyl group in the substrate of CPT-II. The binding site for this aliphatic chain does not exist in the short- and medium-chain carnitine acyltransferases, due to conformational differences among the enzymes. A unique insert in CPT-II is positioned on the surface of the enzyme, with a highly hydrophobic surface. It is likely that this surface patch mediates the association of CPT-II with the inner membrane of the mitochondria.     

Enzymes of carnitine acylation. Is overt carnitine palmitoyltransferase of liver peroxisomal carnitine octanoyltransferase?

Liver mitochondria prepared by differential centrifugation are contaminated by significant quantities of peroxisomes and microsomal fractions. 'Easily solubilized carnitine palmitoyltransferase' prepared from liver mitochondria is thought to originate from the outer surface of the mitochondrial inner membrane. We have characterized the carnitine palmitoyltransferase activities of freeze-thaw extracts of rat liver mitochondrial preparations. Chromatography on Sephadex G-100 yields two broad peaks of carnitine decanoyltransferase activity: one eluted at the end of the void volume, which can be removed (precipitated) by ultracentrifugation; the second peak represents the soluble activity and is eluted at an Mr near 70,000. The activity in the soluble peak is precipitated by an antibody raised against carnitine octanoyltransferase purified from mouse liver peroxisomes. In contrast, antibody raised against carnitine palmitoyltransferase purified from liver mitochondrial membranes had no effect (P. Brady & L. Brady, personal communication). The carnitine acyltransferase activities of the Mr-70,000 peak in the presence or absence of Tween 20 showed maximum activity with decanoyl-CoA and about one-third of this activity with palmitoyl-CoA, similar to peroxisomal carnitine octanoyltransferase. These data show that 7500 g preparations of liver mitochondria isolated by differential centrifugation are enriched by peroxisomal carnitine octanoyltransferase (approx. 20% of the protein of the fraction is peroxisomal) and indicate that this enzyme may be the one reported as 'overt' or 'easily solubilized' mitochondrial carnitine palmitoyltransferase.     

The hypolipidemic peroxisome-proliferating drug, bis(carboxymethylthio)-1.10 decane, a dicarboxylic metabolite of tiadenol, is activated to an acylcoenzyme A thioester

Bis(carboxymethylthio)-1.10 decane (BCMTD), a thiodicarboxylic acid, was shown to be a hypolipidemic peroxisome-proliferating drug as it: (a) decreased the total serum triacylglycerols and cholesterol; (b) induced hepatomegaly; (c) increased the peroxisomal beta-oxidation and catalase activity and the activities of the multiorganelle localized enzymes: palmitoyl-CoA synthetase, palmitoyl-CoA hydrolase, glycerophosphate acyltransferase; (d) decreased the carnitine palmitoyltransferase and urate oxidase activities; and (e) induced the bifunctional eonyl-CoA hydratase in peroxisomes. The present study has confirmed the effect of tiadenol administration on the activities of key enzymes involved in hepatic fatty acid metabolism in male rats. However, the hepatic pleiotropic response was more marked with the dicarboxylic acid than with its alcohol. In a separate dose-response study BCMTD was found to be a more potent inducer of peroxisomal beta-oxidation compared to tiadenol. BCMTD can be activated in vitro to its coenzyme A thioester by a dicarboxyl-CoA synthetase. In control and BCMTD-treated animals, the synthetase activity was found in all cellular fractions except the cytosolic. Whether the acyl-CoA thioesters of peroxisome-proliferating drugs may be mediators of peroxisomal proliferation should be considered.     

Structure of human carnitine acetyltransferase. Molecular basis for fatty acyl transfer

Carnitine acyltransferases are a family of ubiquitous enzymes that play a pivotal role in cellular energy metabolism. We report here the x-ray structure of human carnitine acetyltransferase to a 1.6-A resolution. This structure reveals a monomeric protein of two equally sized alpha/beta domains. Each domain is shown to have a partially similar fold to other known but oligomeric enzymes that are also involved in group-transfer reactions. The unique monomeric arrangement of the two domains constitutes a central narrow active site tunnel, indicating a likely universal feature for all members of the carnitine acyltransferase family. Superimposition of the substrate complex of a related protein, dihydrolipoyl trans-acetylase, reveals that both substrates localize to the active site tunnel of human carnitine acetyltransferase, suggesting the location of the ligand binding sites for carnitine and coenzyme A. Most significantly, this structure provides critical insights into the molecular basis for fatty acyl chain transfer and a possible common mechanism among a wide range of acyltransferases utilizing a catalytic dyad.     

Carnitine octanoyltransferase of mouse liver peroxisomes: properties and effect of hypolipidemic drugs

Carnitine octanoyltransferase (COT) in 500g supernatant fluids from mouse liver has a specific activity at least twice that of carnitine acetyltransferase (CAT) or carnitine palmitoyltransferase (CPT). When mice are fed diets containing the lipid-lowering drugs, clofibrate or nafenopin, the specific activity of COT increases 4- and 11-fold, respectively. Liver homogenates from mice fed a control diet, and diets containing clofibrate, nafenopin, or Wy-14,643 were fractionated by sucrose gradient centrifugation, and the subcellular distribution of carnitine acyltransferases was determined. In the controls, peroxisomes contained about 70% of the total COT. The specific activity of COT in the peroxisomal peak was 12-fold greater than either CAT or CPT, and 20-fold greater than the COT activity in the mitochondrial fraction. Treatment with hypolipidemic drugs increased the specific activity of peroxisomal COT 2- to 3-fold and CAT 6- to 12-fold, while mitochondrial COT increased 5- to 11-fold and CAT 19- to 54-fold. COT was purified to homogeneity from livers of mice treated with Wy-14,643. It had an apparent Mr of 60,000 by Sephadex G-100 and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and a maximum activity for octanoyl-CoA with acetyl-CoA and palmitoyl-CoA having activities of 2 and 10%, respectively, when 100 microM acyl-CoA substrates were used. The Km's for 1-carnitine, octanoyl-CoA, palmitoyl-CoA, and acetyl-CoA were 130, 15, 69, and 155 microM, respectively, in the forward direction; and in the reverse direction were 110, 100, 104, and 783 microM for CoASH, octanoylcarnitine, palmitoylcarnitine, and acetylcarnitine, respectively. With Vmax conditions, acetyl-CoA and palmitoyl-CoA had activities of 8 and 26% of the activity for octanoyl-CoA, and acetylcarnitine and palmitoylcarnitine had activities of 7 and 22%, respectively, of the activity for octanoylcarnitine. It is concluded that COT is a separate enzyme present in large amounts in the matrix of mouse liver peroxisomes, with kinetic properties that greatly favor medium-chain acylcarnitine formation.     

Properties of purified carnitine acyltransferases of mouse liver peroxisomes

The purpose of this study was to characterize the physical, kinetic, and immunological properties of carnitine acyltransferases purified from mouse liver peroxisomes. Peroxisomal carnitine octanoyltransferase and carnitine acetyltransferase were purified to apparent homogeneity from livers of mice fed a diet containing the hypolipidemic drug Wy-14,643 [( 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]-acetic acid). Both enzymes have a molecular weight of 60,000 and a similar pH optimum. Carnitine octanoyltransferase had a maximum activity for C6 moieties while the maximum for carnitine acetyltransferase was with C3 and C4 moieties. The apparent Km values were between 2 and 20 microM for the preferred acyl-CoA substrates, and the Km values for L-carnitine varied depending on the acyl-CoA cosubstrates used. The Hill coefficient, n, was approximately 1 for all acyl-CoAs tested, indicating Michaelis-Menten kinetics. Carnitine octanoyltransferase retained its maximum activity when preincubated with 5,5'-dithiobis-(2-nitrobenzoate) at pH 7.0 or 8.5. Neither carnitine octanoyltransferase nor carnitine acetyltransferase were inhibited by malonyl-CoA. The immunology of carnitine octanoyltransferase is discussed. These data indicate that peroxisomal carnitine octanoyltransferase and carnitine acetyltransferase function in vivo in the direction of acylcarnitine formation, and suggest that the concentration of L-carnitine could influence the specificity for different acyl-CoA substrates.     

OCTN3 is a mammalian peroxisomal membrane carnitine transporter

Carnitine is a zwitterion essential for the beta-oxidation of fatty acids. The role of the carnitine system is to maintain homeostasis in the acyl-CoA pools of the cell, keeping the acyl-CoA/CoA pool constant even under conditions of very high acyl-CoA turnover, thereby providing cells with a critical source of free CoA. Carnitine derivatives can be moved across intracellular barriers providing a shuttle mechanism between mitochondria, peroxisomes, and microsomes. We now demonstrate expression and colocalization of mOctn3, the intermediate-affinity carnitine transporter (Km 20 microM), and catalase in murine liver peroxisomes by TEM using immunogold labelled anti-mOctn3 and anti-catalase antibodies. We further demonstrate expression of hOCTN3 in control human cultured skin fibroblasts both by Western blotting and immunostaining analysis using our specific anti-mOctn3 antibody. In contrast with two peroxisomal biogenesis disorders, we show reduced expression of hOCTN3 in human PEX 1 deficient Zellweger fibroblasts in which the uptake of peroxisomal matrix enzymes is impaired but the biosynthesis of peroxisomal membrane proteins is normal, versus a complete absence of hOCTN3 in human PEX 19 deficient Zellweger fibroblasts in which both the uptake of peroxisomal matrix enzymes as well as peroxisomal membranes are deficient. This supports the localization of hOCTN3 to the peroxisomal membrane. Given the impermeability of the peroxisomal membrane and the key role of carnitine in the transport of different chain-shortened products out of peroxisomes, there appears to be a critical need for the intermediate-affinity carnitine/organic cation transporter, OCTN3, on peroxisomal membranes now shown to be expressed in both human and murine peroxisomes. This Octn3 localization is in keeping with the essential role of carnitine in peroxisomal lipid metabolism.     

Mutagenesis of specific amino acids converts carnitine acetyltransferase into carnitine palmitoyltransferase

Carnitine acyltransferases catalyze the exchange of acyl groups between carnitine and CoA. The members of the family can be classified on the basis of their acyl-CoA selectivity. Carnitine acetyltransferases (CrATs) are very active toward short-chain acyl-CoAs but not toward medium- or long-chain acyl-CoAs. Previously, we identified an amino acid residue (Met(564) in rat CrAT) that was critical to fatty acyl-chain-length specificity. M564G-mutated CrAT behaved as if its natural substrates were medium-chain acyl-CoAs, similar to that of carnitine octanoyltransferase (COT). To extend the specificity of rat CrAT to other substrates, we have performed new mutations. Using in silico molecular modeling procedures, we have now identified a second putative amino acid involved in acyl-CoA specificity (Asp(356) in rat CrAT). The double CrAT mutant D356A/M564G showed 6-fold higher activity toward palmitoyl-CoA than that of the single CrAT mutant M564G and a new activity toward stearoyl-CoA. We show that by performing two amino acid replacements a CrAT can be converted into a pseudo carnitine palmitoyltransferase (CPT) in terms of substrate specificity. To change CrAT specificity from carnitine to choline, we also prepared a mutant CrAT that incorporates four amino acid substitutions (A106M/T465V/T467N/R518N). The quadruple mutant shifted the catalytic discrimination between l-carnitine and choline in favor of the latter substrate and showed a 9-fold increase in catalytic efficiency toward choline compared with that of the wild-type. Molecular in silico docking supports kinetic data for the positioning of substrates in the catalytic site of CrAT mutants.