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Carnitine palmitoyltransferase activities (EC 2.3.1.-) of rat liver mitochondria.

ABSTRACT: 1. A continuously recording and sensitive fluorimetric assay is described for carnitine palmitoyltransferase. This assay has been applied to whole or disintegrated mitochondria and to soluble protein fractions. 2. When rat liver mitochondria had been disintegrated by ultrasound, the specific activity of carnitine palmitoyltransferase was 15-20m-units/mg of protein. Only one-fifth of this activity was assayable (with added substrates) before mitochondrial disintegration. 3. It is concluded that there are two carnitine palmitoyltransferase activities in rat liver mitochondria, of which one (type I) is relatively superficial in location and catalyses an acyl-group transfer between added CoA and carnitine, whereas the other (type II) is less superficial and catalyses an acyl-group transfer in unbroken mitochondria between added carnitine and intramitochondrial CoA. The existence of two distinct carnitine palmitoyltransferases was predicted by Fritz & Yue (1963). 4. In unbroken mitochondria, type I transferase is accessible to the inhibitor 2-bromostearoyl-CoA whereas the type II transferase is inaccessible. 5. A major part of the total carnitine palmitoyltransferase activity of rat liver mitochondria is membrane-bound and of type II. 6. These observations, when considered in conjunction with the penetration of mitochondria by CoASH or carnitine, indicate that the type II transferase is attached to the inner mitochondrial membrane.

SUBMITTER: Yates DW

PROVIDER: S-EPMC1179386 | biostudies-other | 1970 Sep

REPOSITORIES: biostudies-other

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

Project description: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.

| S-EPMC2937350 | biostudies-literature

Carnitine palmitoyltransferase 1C regulates cancer cell senescence through mitochondria-associated metabolic reprograming.

Project description:Cellular senescence is a fundamental biological process that has profound implications in cancer development and therapeutics, but the underlying mechanisms remain elusive. Here we show that carnitine palmitoyltransferase 1C (CPT1C), an enzyme that catalyzes carnitinylation of fatty acids for transport into mitochondria for ?-oxidation, plays a major role in the regulation of cancer cell senescence through mitochondria-associated metabolic reprograming. Metabolomics analysis suggested alterations in mitochondria activity, as revealed by the marked decrease in acylcarnitines in senescent human pancreatic carcinoma PANC-1 cells, indicating low CPT1C activity. Direct analyses of mRNA and protein show that CPT1C is significantly reduced in senescent cells. Furthermore, abnormal mitochondrial function was observed in senescent PANC-1 cells, leading to lower cell survival under metabolic stress and suppressed tumorigenesis in a mouse xenograft model. Knock-down of CPT1C in PANC-1 cells induced mitochondrial dysfunction, caused senescence-like growth suppression and cellular senescence, suppressed cell survival under metabolic stress, and inhibited tumorigenesis in vivo. Further, CPT1C knock-down suppressed xenograft tumor growth in situ. Silencing of CPT1C in five other tumor cell lines also caused cellular senescence. On the contrary, gain-of-function of CPT1C reversed PANC-1 cell senescence and enhanced mitochondrial function. This study identifies CPT1C as a novel biomarker and key regulator of cancer cell senescence through mitochondria-associated metabolic reprograming, and suggests that inhibition of CPT1C may represent a new therapeutic strategy for cancer treatment through induction of tumor senescence.

| S-EPMC5864250 | biostudies-literature

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

Project description: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.

| S-EPMC1147987 | biostudies-other

Interacting effects of L-carnitine and malonyl-CoA on rat liver carnitine palmitoyltransferase.

Project description:Malonyl-CoA significantly increased the Km for L-carnitine of overt carnitine palmitoyltransferase in liver mitochondria from fed rats. This effect was observed when the molar palmitoyl-CoA/albumin concentration ratio was low (0.125-1.0), but not when it was higher (2.0). In the absence of malonyl-CoA, the Km for L-carnitine increased with increasing palmitoyl-CoA/albumin ratios. Malonyl-CoA did not increase the Km for L-carnitine in liver mitochondria from 24h-starved rats or in heart mitochondria from fed animals. The Km for L-carnitine of the latent form of carnitine palmitoyltransferase was 3-4 times that for the overt form of the enzyme. At low ratios of palmitoyl-CoA/albumin (0.5), the concentration of malonyl-CoA causing a 50% inhibition of overt carnitine palmitoyltransferase activity was decreased by 30% when assays with liver mitochondria from fed rats were performed at 100 microM-instead of 400 microM-carnitine. Such a decrease was not observed with liver mitochondria from starved animals. L-Carnitine displaced [14C]malonyl-CoA from liver mitochondrial binding sites. D-Carnitine was without effect. L-Carnitine did not displace [14C]malonyl-CoA from heart mitochondria. It is concluded that, under appropriate conditions, malonyl-CoA may decrease the effectiveness of L-carnitine as a substrate for the enzyme and that L-carnitine may decrease the effectiveness of malonyl-CoA to regulate the enzyme.

| S-EPMC1152599 | biostudies-other

Acute liver carnitine palmitoyltransferase I overexpression recapitulates reduced palmitate oxidation of cardiac hypertrophy.

Project description:Muscle carnitine palmitoyltransferase I is predominant in the heart, but the liver isoform (liver carnitine palmitoyltransferase I [L-CPT1]) is elevated in hearts with low long chain fatty acid oxidation, such as fetal and hypertrophied hearts.This work examined the effect of acute L-CPT1 expression on the regulation of palmitate oxidation and energy metabolism in intact functioning rat hearts for comparison with findings in hypertrophied hearts.L-CPT1 was expressed in vivo in rat hearts by coronary perfusion of Adv.cmv.L-CPT1 (L-CPT1, n=15) vs. phosphate-buffered saline (PBS) infusion (PBS, n=7) or empty virus (empty, n=5). L-CPT1 was elevated 5-fold at 72 hours after Adv.cmv.L-CPT1 infusion (P<0.05), but muscle carnitine palmitoyltransferase I was unaffected. Despite similar tricarboxylic acid cycle rates, palmitate oxidation rates were reduced with L-CPT1 (1.12 ± 0.29 ?mol/min per gram of dry weight, mean±SE) vs. PBS (1.6 ± 0.34). Acetyl CoA production from palmitate was reduced with L-CPT1 (69 ± 0.02%; P<0.05; PBS=79 ± 0.01%; empty=81 ± 0.02%), similar to what occurs in hypertrophied hearts, and with no difference in malonyl CoA content. Glucose oxidation was elevated with L-CPT1 (by 60%). Surprisingly, L-CPT1 hearts contained elevated atrial natriuretic peptide, indicating induction of hypertrophic signaling.The results link L-CPT1 expression to reduced palmitate oxidation in a nondiseased adult heart, recapitulating the phenotype of reduced long chain fatty acid oxidation in cardiac hypertrophy. The implications are that L-CPT1 expression induces metabolic remodeling hypertrophic signaling and that regulatory factors beyond malonyl CoA in the heart regulate long chain fatty acid oxidation via L-CPT1.

| S-EPMC3537878 | biostudies-literature

Cycloheximide blocks changes in rat liver carnitine palmitoyltransferase 1 activity in starvation.

Project description:Starvation (24h) increased the maximum activity of carnitine palmitoyltransferase 1 in rat liver and increased the concentration of malonyl-CoA required to cause 50% inhibition of the enzyme (I50). Re-feeding (24h) with a standard cube diet or a high-carbohydrate diet reversed both of these changes, whereas re-feeding with a high-fat diet did not. Administration of cycloheximide (200 micrograms/100 g body wt.) blocked the increases in carnitine palmitoyltransferase 1 activity and I50 on starvation. It is suggested that increase in carnitine palmitoyltransferase 1 activity in starvation may involve synthesis of new enzyme.

| S-EPMC1144414 | biostudies-other

Regulation by oestrogen of carnitine palmitoyltransferase in hepatic mitochondria.

Project description:Treatment of female rats with ethynyloestradiol decreased both the activity of carnitine palmitoyltransferase and its Ki value for malonyl-CoA compared with pair-fed control rats. In starved rats, carnitine palmitoyltransferase activity and its Ki value for malonyl-CoA were both elevated as expected. Oestrogen treatment had no effect on carnitine palmitoyltransferase activity in the starved state, but the Ki value for malonyl-CoA was substantially decreased by oestrogen. Treatment with a more physiological concentration of ethynyloestradiol (15 micrograms/kg body wt. per day) also produced a decrease in the Ki value for malonyl-CoA. These data suggest that the oestrogen may produce its anti-ketogenic effect by increasing the affinity of carnitine palmitoyltransferase for its physiological inhibitor, malonyl-CoA.

| S-EPMC1147025 | biostudies-other

Target size analysis by radiation inactivation of carnitine palmitoyltransferase activity and malonyl-CoA binding in outer membranes from rat liver mitochondria.

Project description:The functional molecular sizes of the protein(s) mediating the carnitine palmitoyltransferase I (CPT I) activity and the [14C]malonyl-CoA binding in purified outer-membrane preparations from rat liver mitochondria were determined by radiation-inactivation analysis. In all preparations tested the dose-dependent decay in [14C]malonyl-CoA binding was less steep than that for CPT I activity, suggesting that the protein involved in malonyl-CoA binding may be smaller than that catalysing the CPT I activity. The respective sizes computed from simultaneous analysis for molecular-size standards exposed under identical conditions were 60,000 and 83,000 DA for malonyl-CoA binding and CPT I activity respectively. In irradiated membranes the sensitivity of CPT activity to malonyl-CoA inhibition was increased, as judged by malonyl-CoA inhibition curves for the activity in control and in irradiated membranes that had received 20 Mrad radiation and in which CPT activity had decayed by 60%. Possible correlations between these data and other recent observations on the CPT system are discussed.

| S-EPMC1133394 | biostudies-other

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

Project description: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.

| S-EPMC1148689 | biostudies-other

Cholestatic Jaundice Associated with Carnitine Palmitoyltransferase IA Deficiency.

Project description:Liver dysfunction usually accompanies metabolic decompensation in fatty acid oxidation disorders, including carnitine palmitoyltransferase (CPT) Ia deficiency. Typically, the liver is enlarged with raised plasma transaminase activities and steatosis on histological examination. In contrast, cholestatic jaundice is rare, having only been reported in long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency. We report a 3-year-old boy with CPT Ia deficiency who developed hepatomegaly and cholestatic jaundice following a viral illness. No cause for the jaundice could be found, apart from the fatty acid oxidation disorder. Liver histology showed diffuse, predominately macrovesicular steatosis, hepatocellular and canalicular cholestasis but no bile duct paucity or evidence of large duct obstruction. The liver dysfunction resolved in 4-7 weeks.

| S-EPMC3575043 | biostudies-literature

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