Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.1.21 (CPT)
4,580 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A procedure for the purification of the rat liver microsomal carnitine octanoyltransferase (COT) that catalyzes the reversible formation of medium-chain and long-chain acylcarnitines from acyl-coenzyme A is described. The K0.5 for L-carnitine is 0.6 mM and the K0.5 for both decanoyl-CoA and palmitoyl-CoA is 0.6 microM. The Vmax with decanoyl-CoA is approximately fourfold greater than the Vmax with palmitoyl-CoA. The enzyme is monomeric, sodium dodecyl sulfate-polyacrylamide gel electrophoresis gives a molecular weight of 50,100, and molecular sieving gives a molecular weight of 54,300. Purified COT does not cross-react with either antimitochondrial carnitine palmitoyltransferase or antiperoxisomal COT antibodies. It also does not form a covalent adduct when incubated with etomoxiryl-CoA. Microsomal COT is a different protein than either mitochondrial carnitine palmitoyltransferase or peroxisomal COT.
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PMID:Purification of the medium-chain/long-chain (COT/CPT) carnitine acyltransferase of rat liver microsomes. 142 10

Although the malonyl-CoA sensitivity of peroxisomal carnitine octanoyltransferase (COT) is reportedly lost on solubilization, we show that malonyl-CoA does inhibit the purified enzyme. Assay conditions such as buffer composition, pH, acyl-CoA substrate and the presence or absence of BSA can affect the observed inhibition. When assayed in the absence of BSA, COT shows simple competitive inhibition by malonyl-CoA. The Ki value for inhibition of purified COT is high (106 microM) compared with physiological concentrations (1-6 microM) and other short-chain acyl-CoA esters inhibit COT to the same degree. However, when COT is assayed in intact peroxisomes, the Ki for malonyl-CoA is almost 20-fold lower than found with the purified enzyme, whereas inhibition by other short-chain acyl-CoA esters does not change significantly. Several features of the inhibition of peroxisomal COT, including the specificity of malonyl-CoA over other short-chain acyl-CoA esters, resemble those of carnitine palmitoyltransferase (CPT)-I, suggesting that the regulation of COT and CPT-I in parallel may be necessary for the control of cellular fatty acid metabolism.
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PMID:Malonyl-CoA inhibition of peroxisomal carnitine octanoyltransferase. 153 May 96

We recently reported that purified carnitine acetyltransferase is competitively inhibited by bile acids (Sekas, G. and Paul, H.S. (1989) Anal. Biochem. 179, 262-267). In the present study, we initially investigated the effect of bile acids on carnitine acyltransferases in rat hepatic peroxisomes. Activities of carnitine acetyltransferase, carnitine octanoyltransferase, and carnitine palmitoyltransferase were progressively inhibited by increasing concentrations of chenodeoxycholic acid. Kinetic studies revealed that the inhibition by chenodeoxycholic acid was competitive with respect to carnitine with an apparent Ki of 890 microM for carnitine acetyltransferase, 650 microM for carnitine octanoyltransferase and 600 microM for carnitine palmitoyltransferase. We then investigated whether bile acids inhibit the activities of these enzymes ex vivo. The hepatic concentration of bile acids was increased by inducing cholestasis by bile duct ligation. Cholestasis reduced the activity of carnitine acetyltransferase, carnitine octanoyltransferase, and carnitine palmitoyltransferase to 66 +/- 2%, 64 +/- 3%, and 40 +/- 2%, of the control, respectively. The inhibition for each of these enzymes was proportional to the degree of cholestasis. The effect of cholestasis appeared specific for carnitine acyltransferases since the activity of catalase, another peroxisomal enzyme, was not affected by cholestasis. We conclude that bile acids inhibit the activities of carnitine acyltransferases in hepatic peroxisomes. This inhibition by bile acids may be of significance in cholestatic liver disease.
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PMID:Inhibition of carnitine acyltransferase activities by bile acids in rat liver peroxisomes. 157 63

The effects of etomoxiryl-CoA on purified carnitine acyltransferases and on carnitine acyl-transferases of rat heart mitochondria and rat liver microsomes were determined. At nanomolar concentrations, the data agreed with that of other investigators who have shown that etomoxiryl-CoA must be binding to a high affinity site with specific inhibition of mitochondrial carnitine palmitoyltransferase (CPTo). Micromolar amounts of etomoxiryl-CoA inhibited both short- and long-chain carnitine acyltransferases. The concentrations of etomoxiryl-CoA required for 50% inhibition of the different carnitine acetyltransferases and microsomal and peroxisomal carnitine octanoyltransferase were in the low micromolar range. Mixed-type and uncompetitive inhibition kinetics were obtained, depending on the source of purified enzyme. When purified rat heart CPT was incubated with etomoxiryl-CoA, it increased the K0.5 and decreased the Hill coefficient for acyl-CoA. Both proteins and phospholipids of mitochondria and microsomes formed covalent adducts of [3H]etomoxir, with the predominant labeling in phospholipids. None of the purified enzymes formed covalent adducts when incubated with [3H]etomoxiryl-CoA, in contrast to intact mitochondria or microsomes. The major 3H-labeled protein for rat heart mitochondria had a molecular weight of 81,000 +/- 4000, and the major proteins from microsomes had a molecular weight of 51,000-57,000. Malonyl-CoA prevented most of the tritum incorporation into the 81,000 Da protein of mitochondria, but it had little effect on incorporation of tritiated etomoxir into the 51,000-57,000 Da proteins of microsomes. When 50 microM etomoxiryl-CoA was added to microsomes and to mitochondria that had been incubated with radioactive etomoxiryl-CoA, much of the radioactive etomoxir disappeared from the major microsomal proteins, but virtually none was displaced from the mitochondrial protein. Thus, at least two different types of covalent etomoxir complexes were formed. This pulse-chase experiment showed that the mitochondrial protein-etomoxir complex was not turned over, consistent with other data showing that etomoxir inhibited carnitine palmitoyltransferase. In contrast, the major protein-etomoxir complex in microsomes was turned over during the pulse-chase experiment.
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PMID:Effect of etomoxiryl-CoA on different carnitine acyltransferases. 173 21

Carnitine acyltransferase activities in the hearts of normal and dystrophic, sedentary and swim exercised hamsters were studied, in order to analyze the relationship between carnitine metabolism and exercise in cardiomyopathy. After 12 weeks, the mean specific activities of cardiac carnitine acetyltransferase (CAT), carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) were significantly higher in the dystrophic sedentary group, relative to the normal sedentary group (p less than 0.05). There was no significant effect of exercise on the mean specific activity of the carnitine acyltransferases, compared to the dystrophic or normal sedentary controls. Thus, the improvements in cardiac histopathology due to exercise noted previously are not associated with altered carnitine acyltransferase activity.
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PMID:Cardiac carnitine acyltransferase activities in exercised normal and dystrophic hamsters. 178 40

Carnitine acyltransferase activities were studied in normal human skeletal muscle and in muscle of three patients with carnitine palmitoyltransferase deficiency. Carnitine acetyltransferase (CAT), carnitine octanoyltransferase (COT), and carnitine palmitoyltransferase (CPT) were differentiated (i) by the use of the substrates acetyl-CoA, octanoyl-CoA, lauroyl-CoA, and palmitoyl-CoA, (ii) by the inhibitors malonyl-CoA, chlorpromazine, and dithio-bis-nitrobenzoic acid (DTNB), and (iii) by the solubilities of the carnitine acyltransferase activities after centrifugation at 48,000 g for 30 min. The results are consistent with the notion of three different carnitine acyltransferases in human skeletal muscle: a membrane-bound malonyl-CoA-sensitive CPT, a soluble malonyl-CoA-insensitive CAT, and a malonyl-CoA-sensitive COT that is not attached to the mitochondrial membrane. The different solubilities of the carnitine acyltransferases allow a clear differentiation of CPT from CAT and COT in homogenates of previously frozen muscle biopsies whereas a separate determination of CAT and COT is only partially possible. In patients with CPT deficiency total CPT activity was within the normal range but was abnormally inhibited by malonyl-CoA and chlorpromazine. Activities of carnitine acyltransferases with the substrates acetyl-CoA and octanoyl-CoA were normal indicating that the biochemical defect in CPT deficiency is confined to CPT without compensatory changes of CAT and COT.
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PMID:Carnitine acyltransferases in normal human skeletal muscle and in muscle of patients with carnitine palmitoyltransferase deficiency. 182 3

The effects of dehydroepiandrosterone (DHEA) and clofibrate on mitochondrial and peroxisomal proliferation and carnitine acyltransferases [mitochondrial carnitine palmitoyltransferase (CPT) and peroxisomal carnitine octanoyltransferase (COT)] were measured in lean and obese female Zucker rats. DHEA increased total hepatic mitochondrial protein twofold; clofibrate increased total hepatic peroxisomal protein more than fivefold. Both DHEA and clofibrate administration increased enzyme activities, immunoreactive protein, messenger RNA levels and transcription rates for the carnitine acyltransferases. Transcription rates and messenger RNA concentration for both carnitine acyltransferases correlated with the increases in activity. These data suggest that the hepatic CPT and COT in female Zucker rats are regulated primarily at the transcriptional level by DHEA and clofibrate.
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PMID:Regulation of carnitine acyltransferase synthesis in lean and obese Zucker rats by dehydroepiandrosterone and clofibrate. 182 32

Salicylyl-CoA and benzoyl-CoA were good inhibitors of carnitine acetyltransferase (CAT), competing with acetyl-CoA with Ki values of 7.5 and 22 microM respectively in the forward direction and with CoA in the reverse reaction with similar Ki values. They were also competitive inhibitors of carnitine octanoyltransferase (Ki = 261 and 295 microM respectively), but were only weakly inhibitory to carnitine palmitoyltransferase. Inhibition of energy production by salicylate may result from the inhibition of CAT by salicylyl-CoA.
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PMID:Effect of carboxylic acid xenobiotics and their metabolites on the activity of carnitine acyltransferases. 195 85

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.
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PMID:The medium-chain carnitine acyltransferase activity associated with rat liver microsomes is malonyl-CoA sensitive. 235 18

We report the isolation and characterization of a full-length cDNA encoding rat liver carnitine palmitoyltransferase II (CPT II). Beginning with the purified protein CNBr fragments were generated and sequenced. Corresponding oligonucleotides were used to screen a rat liver cDNA library constructed in the plasmid cloning vector, pcDV. The clone ultimately obtained consisted of a 62 nucleotide 5'-untranslated region, a single open reading frame of 1,974 bases predicting a protein of 658 amino acids (Mr = 74,119), and a 3'-untranslated segment of 260 nucleotides followed by the poly (A) tail. The identity of the cDNA was confirmed by the findings that (a) the open reading frame encoded all three peptides found in the original protein; (b) a fourth peptide synthesized from a portion of the deduced amino acid sequence and used to immunize a rabbit resulted in the generation of an antibody that recognized pure CPT II on a Western blot; (c) in vitro transcription and translation of the cDNA (ligated into pBlue-script KS (+] generated a protein that was specifically immunoprecipitated by anti-CPT II antibody and having a Mr slightly greater than that of mature CPT II; (d) transfection of COS cells with the cDNA subcloned into the expression vector, pCMV4, resulted in a 6-fold induction of mitochondrial CPT II catalytic activity. It seems likely that the de novo synthesized enzyme gains entry into the mitochondrion via a targeting peptide that is subsequently cleaved. The mature protein probably associates (relatively loosely) with the inner membrane through a limited number of membrane spanning domains. The predicted amino acid sequence of CPT II shows strong identity with those of two other acyltransferases, namely, rat liver peroxisomal carnitine octanoyltransferase and porcine choline acetyltransferase.
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PMID:Cloning, sequencing, and expression of a cDNA encoding rat liver mitochondrial carnitine palmitoyltransferase II. 235 18


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