Butyrate enhances CPT1A activity to promote fatty acid oxidation and iTreg differentiation.

Inducible regulatory T (iTreg) cells play a crucial role in immune suppression and are important for the maintenance of immune homeostasis. Mounting evidence has demonstrated connections between iTreg differentiation and metabolic reprogramming, especially rewiring in fatty acid oxidation (FAO). Previous work showed that butyrate, a specific type of short-chain fatty acid (SCFA) readily produced from fiber-rich diets through microbial fermentation, was critical for the maintenance of intestinal homeostasis and capable of promoting iTreg generation by up-regulating histone acetylation for gene expression as an HDAC inhibitor. Here, we revealed that butyrate could also accelerate FAO to facilitate iTreg differentiation. Moreover, butyrate was converted, by acyl-CoA synthetase short-chain family member 2 (ACSS2), into butyryl-CoA (BCoA), which up-regulated CPT1A activity through antagonizing the association of malonyl-CoA (MCoA), the best known metabolic intermediate inhibiting CPT1A, to promote FAO and thereby iTreg differentiation. Mutation of CPT1A at Arg243, a reported amino acid required for MCoA association, impaired both MCoA and BCoA binding, indicating that Arg243 is probably the responsible site for MCoA and BCoA association. Furthermore, blocking BCoA formation by ACSS2 inhibitor compromised butyrate-mediated iTreg generation and mitigation of mouse colitis. Together, we unveil a previously unappreciated role for butyrate in iTreg differentiation and illustrate butyrate-BCoA-CPT1A axis for the regulation of immune homeostasis.

Acetyl CoA Carboxylase 2 Is Dispensable for CD8+ T Cell Responses.

Differentiation of T cells is closely associated with dynamic changes in nutrient and energy metabolism. However, the extent to which specific metabolic pathways and molecular components are determinative of CD8+ T cell fate remains unclear. It has been previously established in various tissues that acetyl CoA carboxylase 2 (ACC2) regulates fatty acid oxidation (FAO) by inhibiting carnitine palmitoyltransferase 1 (CPT1), a rate-limiting enzyme of FAO in mitochondria. Here, we explore the cell-intrinsic role of ACC2 in T cell immunity in response to infections. We report here that ACC2 deficiency results in a marginal increase of cellular FAO in CD8+ T cells, but does not appear to influence antigen-specific effector and memory CD8+ T cell responses during infection with listeria or lymphocytic choriomeningitis virus. These results suggest that ACC2 is dispensable for CD8+ T cell responses.

Effector T cells require fatty acid metabolism during murine graft-versus-host disease.

Activated T cells require increased energy to proliferate and mediate effector functions, but the metabolic changes that occur in T cells following stimulation in vivo are poorly understood, particularly in the context of inflammation. We have previously shown that T cells activated during graft-versus-host disease (GVHD) primarily rely on oxidative phosphorylation to synthesize adenosine 5'-triphosphate. Here, we demonstrate that alloreactive effector T cells (Teff) use fatty acids (FAs) as a fuel source to support their in vivo activation. Alloreactive T cells increased FA transport, elevated levels of FA oxidation enzymes, up-regulated transcriptional coactivators to drive oxidative metabolism, and increased their rates of FA oxidation. Importantly, increases in FA transport and up-regulation of FA oxidation machinery occurred specifically in T cells during GVHD and were not seen in Teff following acute activation. Pharmacological blockade of FA oxidation decreased the survival of alloreactive T cells but did not influence the survival of T cells during normal immune reconstitution. These studies suggest that pathways controlling FA metabolism might serve as therapeutic targets to treat GVHD and other T-cell-mediated immune diseases.

Partial characterization of a malonyl-CoA-sensitive carnitine O-palmitoyltransferase I from Macrobrachium borellii (Crustacea: Palaemonidae).

The shuttle system that mediates the transport of fatty acids across the mitochondrial membrane in invertebrates has received little attention. Carnitine O-palmitoyltransferase I (EC 2.3.1.21; CPT I) is a key component of this system that in vertebrates controls long-chain fatty acid beta-oxidation. To gain knowledge on the acyltransferases in aquatic arthropods, physical, kinetic, regulatory and immunological properties of CPT of the midgut gland mitochondria of Macrobrachium borellii were assayed. CPT I optimum conditions were 34 degrees C and pH=8.0. Kinetic analysis revealed a Km for carnitine of 2180+/-281 microM and a Km for palmitoyl-CoA of 98.9+/-8.9 microM, while V(max) were 56.5+/-6.6 and 36.7+/-4.8 nmol min(-1) mg protein(-1), respectively. A Hill coefficient, n~1, indicate a Michaelis-Menten behavior. The CPT I activity was sensitive to regulation by malonyl-CoA, with an IC(50) of 25.2 microM. Electrophoretic and immunological analyses showed that a 66 kDa protein with an isoelectric point of 5.1 cross-reacted with both rat liver and muscle-liver anti CPT I polyclonal antibodies, suggesting antigenic similarity with the rat enzymes. Although CPT I displayed kinetic differences with insect and vertebrates, prawn showed a high capacity for energy generation through beta-oxidation of long-chain fatty acids.

A novel brain-expressed protein related to carnitine palmitoyltransferase I.

Malonyl-CoenzymeA acts as a fuel sensor, being both an intermediate of fatty acid synthesis and an inhibitor of the two known isoforms of carnitine palmitoyltransferase I (CPT I), which control mitochondrial fatty acid oxidation. We describe here a novel CPT1 family member whose mRNA is present predominantly in brain and testis. Chromosomal locations and genome organization are reported for the mouse and human genes. The protein sequence contains all the residues known to be important for both carnitine acyltransferase activity and malonyl-CoA binding in other family members. Yeast expressed protein has no detectable catalytic activity with several different acyl-CoA esters that are good substrates for other carnitine acyltransferases, including the liver isoform of CPT I, which is also expressed in brain; however, it displays high-affinity malonyl-CoA binding. Thus this new CPT I related protein may be specialized for the metabolism of a distinct class of fatty acids involved in brain function.

Topology of carnitine palmitoyltransferase I in the mitochondrial outer membrane.

The topology of carnitine palmitoyltransferase I (CPT I) in the outer membrane of rat liver mitochondria was studied using several approaches. 1. The accessibility of the active site and malonyl-CoA-binding site of the enzyme from the cytosolic aspect of the membrane was investigated using preparations of octanoyl-CoA and malonyl-CoA immobilized on to agarose beads to render them impermeant through the outer membrane. Both immobilized ligands were fully able to interact effectively with CPT I. 2. The effects of proteinase K and trypsin on the activity and malonyl-CoA sensitivity of CPT I were studied using preparations of mitochondria that were either intact or had their outer membranes ruptured by hypo-osmotic swelling (OMRM). Proteinase K had a marked but similar effect on CPT I activity irrespective of whether only the cytosolic or both sides of the membrane were exposed to it. However, it affected sensitivity more rapidly in OMRM. By contrast, trypsin only reduced CPT I activity when incubated with OMRM. The sensitivity of the residual CPT I activity was unaffected by trypsin. 3. The proteolytic fragments generated by these treatments were studied by Western blotting using three anti-peptide antibodies raised against linear epitopes of CPT I. These showed that a proteinase K-sensitive site close to the N-terminus was accessible from the cytosolic side of the membrane. No trypsin-sensitive sites were accessible in intact mitochondria. In OMRM, both proteinase K and trypsin acted from the inter-membrane space side of the membrane. 4. The ability of intact mitochondria and OMRM to bind to each of the three anti-peptide antibodies was used to study the accessibility of the respective epitopes on the cytosolic and inter-membrane space sides of the membrane. 5. The results of all these approaches indicate that CPT I adopts a bitopic topology within the mitochondrial outer membrane; it has two transmembrane domains, and both the N- and C-termini are exposed on the cytosolic side of the membrane, whereas the linker region between the transmembrane domains protrudes into the intermembrane space.

Immunodetection of rat Walker 256 tumour mitochondrial carnitine palmitoyltransferase I and II: evidence for the control of CPT II expression by insulin.

The purpose of the study was to separate the mitochondrial proteins of rat Walker 256 tumour tissue and perform immunodetection studies to identify the carnitine palmitoyltransferase I (CPT I) and CPT II proteins previously reported to be present in this tumour. CPT I protein was undetectable using antibody raised against rat liver CPT I and was therefore considered to be immunologically different from that found in normal rat tissues such as heart, liver and skeletal muscle. In contrast, CPT II protein was readily detected in Walker 256 tumour and had an apparent Mr of approximately 70,000, as was found for rat liver. The in vivo treatment of tumour-bearing rats with insulin caused an increase in the expression of CPT II protein in the tumour tissue. The data confirm that CPT II can be regulated by insulin and also demonstrate that tumour CPT I may be a different isoform from that present in rat liver.

Development and characterization of a polyclonal antibody against rat liver mitochondrial overt carnitine palmitoyltransferase (CPT I). Distinction of CPT I from CPT II and of isoforms of CPT I in different tissues.

The [3H]tetradecylglycidyl-CoA (TDG-CoA)-binding protein (Mr approx. 88,000) of purified outer membranes from rat liver mitochondria was identified by SDS/PAGE. The region in which it migrated was shown to contain another protein which stained strongly with periodic acid-Schiff reagent and could be removed from membrane extracts by incubation with Sepharose-concanavalin A. Amounts of TDG-CoA-binding protein were prepared from lectin-treated extracts using preparative SDS/PAGE and used to raise a polyclonal antibody in a sheep. The IgG fraction purified from this anti-serum reacted strongly with a protein of Mr approximately 88,000 on Western blots, and much more weakly with two other proteins of Mr approximately 76,000 and Mr approximately 53,000 in extracts of rat liver mitochondrial outer membranes. The crude IgG fraction and immunopurified IgG both removed carnitine palmitoyltransferase (CPT) I activity from very pure outer membrane extracts, suggesting that the TDG-CoA-binding protein against which the antiserum was raised also expresses CPT I activity. This was confirmed by the demonstration of a strong positive correlation between CPT I activity and the amount of immunoreactive protein of Mr approximately 88,000 in mitochondria prepared from rats in different physiological states. By contrast, the antibody did not react with CPT II either in mitochondria or in purified form. Similarly, an anti-(CPT II) antibody did not cross-react with CPT I on Western blots, proving conclusively that CPT I and CPT II are immunologically distinct proteins, as well as being of different functional molecular sizes [Zammit, Corstophine & Kelliher (1988) Biochem. J. 250, 415-420]. Immunoblots of mitochondrial proteins obtained from different tissues indicated that, of the rat tissues tested, only kidney cortex mitochondria contain the same isoform of CPT I as that in liver. Heart, skeletal muscle and brown adipose tissue mitochondria contain a slightly smaller isoform which was only weakly reactive with anti-(rat liver CPT I) antibody, indicating that these tissues contain a molecularly quite distinct isoenzyme. This would explain the previous observations that CPT I in these tissues has markedly different kinetic characteristics from the isoenzyme present in liver mitochondria.

Infantile form of carnitine palmitoyltransferase II deficiency with hepatomuscular symptoms and sudden death. Physiopathological approach to carnitine palmitoyltransferase II deficiencies.

Reported cases of carnitine palmitoyltransferase II (CPT II) deficiency are characterized only by a muscular symptomatology in young adults although the defect is expressed in extra-muscular tissues as well as in skeletal muscle. We describe here a CPT II deficiency associating hypoketotic hypoglycemia, high plasma creatine kinase level, heart beat disorders, and sudden death in a 3-mo-old boy. CPT II defect (-90%) diagnosed in fibroblasts is qualitatively similar to that (-75%) of two "classical" CPT II-deficient patients previously studied: It resulted from a decreased amount of CPT II probably arising from its reduced biosynthesis. Consequences of CPT II deficiency studied in fibroblasts differed in both sets of patients. An impaired oxidation of long-chain fatty acids was found in the proband but not in patients with the "classical" form of the deficiency. The metabolic and clinical consequences of CPT II deficiency might depend, in part, on the magnitude of residual CPT II activity. With 25% residual activity CPT II would become rate limiting in skeletal muscle but not in liver, heart, and fibroblasts. As observed in the patient described herein, CPT II activity ought to be more reduced to induce an impaired oxidation of long-chain fatty acids in these tissues.

Purification of carnitine palmitoyltransferase from heart mitochondria by hydrophobic affinity chromatography.

Carnitine palmitoyltransferase II of rat heart mitochondria was purified to homogeneity by a rapid method exploiting the hydrophobic nature of the protein. The method involves solubilization of mitochondrial membrane proteins by detergents and subsequent fractionation by hydrophobic affinity chromatography. Sepharose, cross-linked via a primary amino group of 1,omega-diaminoalkane, 4-aminobutyric acid, 6-aminocaproic acid, or 6-aminohexanol, was found to reversibly bind carnitine palmitoyltransferase under nondenaturing conditions. A homologous series of n-alkyl-agarose resins with n = 2 to 8 and phenyl-Sepharose were also found to reversibly bind the enzyme. Alkyl-Superose, phenyl-Superose, and Superose 12 chromatographies were also very useful in fractionating the enzyme. Successive chromatography on three or four hydrophobic columns yielded a highly pure enzyme preparation. The purified preparation appeared as one major protein band on polyacrylamide electrophoresis gels in the presence of sodium dodecyl sulfate (M(r) 68,000). The isolated enzyme had significant activity (sp act = 15.0 mumol/min/mg protein when 80 microM palmitoyl-CoA and 20 mM carnitine were used as substrates). Antibodies against this protein recognized (in immunoblots) one major protein band in crude preparations of rat heart mitochondria (M(r) 68,000), indistinguishable from purified carnitine palmitoyltransferase II. L-Palmitoylcarnitine (0.1 mM) and coenzyme A (0.1 mM), products of the enzyme-catalyzed reaction, inhibited carnitine palmitoyltransferase activity 66 and 71%, respectively. D-Palmitoylcarnitine (0.1 mM), however, did not inhibit the activity. Malonyl-CoA, a specific inhibitor of membrane-bound carnitine palmitoyltransferase I, did not show significant inhibition.

The relationship of rat liver overt carnitine palmitoyltransferase to the mitochondrial malonyl-CoA binding entity and to the latent palmitoyltransferase.

1. Confirming previous work [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382], malonyl-CoA-inhibitable carnitine palmitoyltransferase (CPT1) from rat liver was found to be localized in outer rather than in inner mitochondrial membranes. 2. Antisera were raised against a liver mitochondrial CPT of Mr 68,000, which was presumed to be the latent from of the enzyme (CPT2). These antisera cross-reacted with solubilized CPT extracted from liver inner mitochondrial membranes and with polypeptides of Mr 68,000 and 60,000 in immunoblots of both inner and outer mitochondrial membranes. The antisera also precipitated CPT activity from detergent-treated total membrane and outer-membrane preparations. 3. The antisera did not precipitate [14C]malonyl-CoA binding material obtained either from total membranes or from outer membranes. 4. It was concluded that liver CPT1 and CPT2 have some epitopes in common and may have a similar subunit size. In addition, CPT1 and the entity that binds malonyl-CoA must be separated polypeptides.

Immunoquantitative analysis of human carnitine palmitoyltransferase I and II defects.

Carnitine palmitoyltransferase deficiency realizes two distinct clinical forms. We previously showed and confirmed in the present work that CPTII (identified as the carnitine palmitoyltransferase activity assayable in detergent conditions) is decreased in the muscular form whereas it is unaffected and CPTI is decreased in the hepatic form. The antibody previously prepared against human liver mitochondrial CPTII recognizes the same enzyme in muscle, liver, and fibroblasts. Immunoprecipitation experiments were performed in fibroblasts from patients with the muscular and hepatic forms of the defect. As compared with controls, cell lines from two patients with the hepatic form of the defect did not exhibit any qualitative nor quantitative abnormality of cross-reacting material, whereas cell lines from two patients with the muscular form of the defect exhibited a decreased amount of cross-reacting material. These data suggest that CPTII deficiency could result from a decreased production of protein. The amount of cross-reacting material in the two sets of patients only correlates with CPTII activity, which is decreased in the muscular presentation and unaffected in the hepatic form. These results strengthen the hypothesis of distinct proteins supporting CPTI and CPTII activities.

Regulation of carnitine palmitoyltransferase in vivo by glucagon and insulin.

Carnitine palmitoyltransferase (CPT total) activity and synthesis increase in states where the insulin/glucagon ratio is low, such as starvation and diabetes [Brady & Brady (1987) Biochem. J. 246, 641-646]. However, the effect of glucagon and insulin on CPT synthesis is unknown. The present experiments were designed to determine the effect of glucagon, cAMP [8-(chlorophenylthio) cyclic AMP], and insulin + cAMP on CPT transcription and mRNA amounts over time after injection. The CPT protein that was purified, used to generate antibody, and cloned in these studies was the 68 kDa mitochondrial protein described previously [Brady & Brady (1987) Biochem. J. 246, 641-646; Brady, Feng & Brady (1988) J. Nutr. 118, 1128-1136; Brady & Brady (1989) Diabetes 38, in the press]. Saline-injected control rats exhibited a 2-fold increase in hepatic CPT transcription rate and CPT mRNA over the 5 h experiment from 09:00 to 14:00 h. The effect was most probably due to the fasting state of the rats during the day. Glucagon injection caused an 8-fold increase in transcription rate by 90 min and a 4-fold increase in CPT mRNA by 90-120 min. The cAMP effect had reached a peak by the first time point taken (15 min). Transcription rate was increased 4-fold and CPT mRNA was increased 3-fold at this time. The combination of cAMP + insulin injection did not produce any significant increase in transcription rate or CPT mRNA over the saline-injected controls. CPT mRNA and transcription rate showed a clear dose-response to glucagon injection from 0 to 150 micrograms/100 g body wt. Total CPT activity and immunoreactive CPT were not increased during these experiments. The data indicate that glucagon and insulin interact in control of transcription rate and amount of CPT mRNA, but that increases in CPT immunoreactive protein and activity are temporally delayed. This lag probably relates to the half-life of the CPT protein in vivo, which has been estimated as 2-7 days.

The soluble carnitine palmitoyltransferase from bovine liver. A comparison with the enzymes from peroxisomes and from the mitochondrial inner membrane.

The properties of two carnitine acyltransferases (CPT) purified from bovine liver are compared to confirm that they are different proteins. The soluble CPT and the inner CPT from mitochondria differ in subunit Mr, native Mr, pI and reactivity with thiol reagents. All eight free thiol groups in soluble CPT react with 5,5'-dithiobis-(2-nitrobenzoate) in the absence of any unfolding reagent, and activity is gradually lost. The inner CPT activity is completely stable in the presence of 5,5'-dithiobis-(2-nitrobenzoate), and only one thiol group per molecule of subunit is modified in the native enzyme. Antisera to each enzyme inhibit that enzyme, but do not cross-react. CPT activity in subcellular fractions can now be identified by titration with these antibodies. The soluble CPT from bovine liver is probably peroxisomal in origin, but, although antigenically similar, it differs from the peroxisomal carnitine octanoyltransferase found in rat and mouse liver in its specificity for the longer-chain acyl-CoA substrates.

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.

Hepatic carnitine palmitoyltransferase turnover and translation rates in fed, starved, streptozotocin-diabetic and diethylhexyl phthalate-treated rats.

Hepatic carnitine palmitoyltransferase (CPT) turnover was studied in control and in non-ketotic hyperglycaemic streptozotocin-diabetic rats. The degradation constant (kd) and half-life (t1/2) did not appear to be altered by mild diabetes. The hepatic CPT (micrograms/g of liver) was not increased by the mild, non-ketotic, diabetes. However, the total hepatic CPT (micrograms/liver) was 37% greater in the diabetic animals, owing to the increased liver weight. This resulted from a 40% increase in the synthesis constant (ks). Hepatic CPT activity (total detergent-solubilized) and translation rates were measured in fed, starved (48 h), non-ketotic diabetic, ketotic diabetic and diethylhexyl phthalate (DEHP)-treated rats. CPT activity (m units/mg of mitochondrial protein) was not significantly increased with non-ketotic diabetes (44% increase, but non-significant), but was increased approx. 2-fold with starvation and ketotic diabetes, and 3.5-fold with DEHP treatment. CPT expressed as units/liver was increased non-significantly (23%) in non-ketotic and starved rats, similar to the turnover study, but was significantly increased with ketotic diabetes and with DEHP treatment. mRNA-translation activity for CPT was elevated in all states to a somewhat greater extent than was activity. It was concluded that protein synthesis as a product of increased CPT-mRNA translation activity is a major means of long-term regulation.

Characterization of the mitochondrial carnitine palmitoyltransferase enzyme system. II. Use of detergents and antibodies.

Exposure of rat liver mitochondrial membranes to octyl glucoside, Triton X-100, or Tween 20 solubilized an active and tetradecylglycidyl-CoA (TG-CoA)-insensitive carnitine palmitoyltransferase (presumed to be carnitine palmitoyltransferase II). The residual membranes after octyl glucoside or Triton X-100 treatment were devoid of all transferase activity. By contrast, Tween 20-extracted membranes were still rich in transferase; this was completely blocked by TG-CoA and thus was presumed to be carnitine palmitoyltransferase I. The residual carnitine palmitoyltransferase activity disappeared from the membranes upon subsequent addition of octyl glucoside or Triton X-100 and could not be recovered in the supernatant fraction. Antibody raised against purified rat liver transferase II (Mr 80,000) recognized only this protein in immunoblots from untreated liver mitochondrial membranes containing both transferases I and II. Tween 20-extracted membranes, which contained only transferase I, did not react with the antibody. Purified transferase II from skeletal muscle (also of Mr 80,000) was readily recognized by the antiserum, suggesting antigenic similarity with the liver enzyme. These and other studies on the effects of detergents on the mitochondrial [3H]TG-CoA binding protein provide further support for the model of carnitine palmitoyltransferase proposed in the preceding paper. They suggest that: 1) carnitine palmitoyltransferases I and II in rat liver are immunologically distinct proteins; 2) transferase I is more firmly anchored into its membrane environment than transferase II; 3) association of carnitine palmitoyltransferase I with a membrane component(s) is necessary for catalytic activity. While carnitine palmitoyltransferase I is a different protein in liver and muscle, it seems likely that both tissues share the same transferase II.

Characterization of hepatic carnitine palmitoyltransferase. Use of bromoacyl derivatives and antibodies.

Carnitine palmitoyltransferase (CPT) is a mitochondrial-inner-membrane enzyme, with activities located on both the outer and inner sides of the membrane. The inhibition of CPT by bromopalmitate derivatives was studied in intact hepatic mitochondria (representing CPT-A activity, the outer enzyme), in inverted submitochondrial vesicles (representing CPT-B, the inner enzyme), and in purified hepatic CPT. Bromopalmitoyl-CoA had an I50 (concentration giving 50% inhibition of CPT activity) of 0.63 +/- 0.08 microM in intact mitochondria and 2.44 +/- 0.86 microM in inverted vesicles. Preincubation of mitochondria with bromopalmitoyl-CoA decreased V max. for both CPT-A and CPT-B. Sonication decreased sensitivity to bromopalmitoyl-CoA, and solubilization with Triton abolished sensitivity at the concentrations used (0-10 microM). Purified CPT had a bromopalmitoyl-CoA I50 of 353 microM in aqueous buffer, 67 microM in 20% dimethyl sulphoxide, 45 microM in phosphatidylcholine liposomes and 26 microM in cardiolipin liposomes. Increasing [carnitine] at constant bromopalmitoyl-CoA concentrations or increasing [bromopalmitoyl-CoA] in the preincubation resulted in increased inhibition of purified CPT. 2-Tetradecylglycidyl-CoA and malonyl-CoA did not offer measurable protection against bromopalmitoyl-CoA inhibition of the purified CPT, suggesting a different site of interaction of bromopalmitoyl-CoA with CPT. The data suggest that the sensitivity of CPT to bromopalmitoyl-CoA may be modulated by membrane environment and assay conditions.

Purification and properties of carnitine octanoyltransferase and carnitine palmitoyltransferase from rat liver.

The activities of carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) in rat liver were markedly increased by administration of di(2-ethyl-hexyl)phthalate. COT and CPT were purified from the enzyme-induced rat liver. COT was a 66,000-dalton polypeptide. The molecular weight of native CPT was 280,000--320,000 daltons, and the enzyme consisted of 69,200-dalton polypeptides. CAT, COT, and CPT were immunologically different. COT exhibited activity with all of the substrates tested (acyl-CoA's and acylcarnitines of saturated fatty acids having carbon chain lengths of C2--C20), though maximum activity was observed with hexanoyl derivatives. CPT exhibited catalytic activity with medium- and long-chain acyl derivatives. 2-Bromo-palmitoyl-CoA inactivated COT but not CPT. Malonyl-CoA inhibited CPT but not COT. CPT was confined to mitochondria, whereas COT was found in peroxisomes and the soluble compartment but not in mitochondria.