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Ciarán N. Cronin - One of the best experts on this subject based on the ideXlab platform.
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The conserved serine-threonine-serine motif of the Carnitine Acyltransferases is involved in Carnitine binding and transition-state stabilization: a site-directed mutagenesis study.
Biochemical and biophysical research communications, 1997Co-Authors: Ciarán N. CroninAbstract:Abstract There has been speculation that the Carnitine Acyltransferase reaction mechanism may involve the formation of an acyl-serine intermediate. A serine–threonine–serine (STS) motif that is conserved throughout the Carnitine Acyltransferase family, and is present also in the choline acetyltransferases, contains the only two conserved serines. The functional role of this motif in Carnitine octanoyltransferase was probed by using a site-directed mutagenesis strategy to generate all seven possible alanine substitutions: single, double and triple mutants. Kinetic analyses of these mutant enzymes demonstrated that the STS motif is not essential for catalysis, thereby excluding an acyl-serine intermediate from the reaction mechanism. The kinetic analyses support, however, substantial roles for the STS motif in Carnitine binding and transition-state stabilization.
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cDNA cloning, recombinant expression, and site-directed mutagenesis of bovine liver Carnitine octanoyltransferase--Arg505 binds the carboxylate group of Carnitine.
European journal of biochemistry, 1997Co-Authors: Ciarán N. CroninAbstract:The cDNA for bovine liver Carnitine octanoyltransferase (COT) has been cloned by a combination of lambda gt11 library screening and 3' rapid amplification of cDNA ends (3'-RACE). The cDNA comprises 338 bases of 5' non-coding sequence, a reading frame of 1839 bases including the stop codon, and 820 bases of 3' non-coding DNA. The deduced amino acid sequence of 612 residues predicts a protein with a calculated mass of 70263 Da and pI 6.28. The enzyme was expressed in recombinant soluble form in Escherichia coli and was purified by a two-step procedure to near-homogeneity with a yield of purified protein of 2-3 mg/l culture. Recombinant COT had similar kinetic properties to those of the enzyme isolated directly from beef liver. Arg505 in COT, conserved in all reported Carnitine Acyltransferase sequences but replaced by asparagine or isoleucine in the choline acetyltransferases, was converted to asparagine by site-directed mutagenesis. This single mutation resulted in a greater than 1650-fold increase in the Km value for COT towards Carnitine, but had little effect on the value of k(cat) or the Km value for the acyl-CoA substrate. In addition, although choline was an extremely poor substrate for COT, the k(cat)/Km ratio towards this substrate was increased fourfold as a result of the mutation. These data support the notion that Arg505 in COT, and other Carnitine Acyltransferases, contributes to substrate binding by forming a salt bridge with the carboxylate moiety of Carnitine.
Pascal Degrace - One of the best experts on this subject based on the ideXlab platform.
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Mitochondrial respiration on rumenic and linoleic acids.
Biochemical Society Transactions, 2001Co-Authors: Pierre Clouet, Laurent Demizieux, Joseph Gresti, Pascal DegraceAbstract:Rumenic acid ( cis -9, trans -11-C 18:2 ) represents approx. 80% of conjugated linoleic acid (CLA) in dairy products. CLA has been shown to exert beneficial effects on health, but little work has been devoted to the ability to oxidize CLA isomers and the role of these isomers in the modulation of β-oxidation flux. In the present study, respiration on rumenic acid was compared with that on linoleic acid ( cis -9, cis -12-C 18:2 ) with the use of rat liver mitochondria. In state-3, respiration was decreased by half with rumenic acid in comparison with linoleic acid. In the uncoupled state, respiration on CLA remained 30% lower. The lower ability to oxidize CLA was investigated through characterization of the enzymic steps. Rumenic acid was 33% less activated by acyl-CoA synthase than was linoleic acid. However, after such activation, the transfer of both acyl moieties to Carnitine by Carnitine Acyltransferase I (CAT I) was of the same order. Moreover, CAT II activity was comparable with either isomer. After prior incubation with rumenic acid, oxidation of octanoic acid by re-isolated mitochondria was unimpaired, but that of palmitoleic acid was impaired unless linoleic acid was used in the prior incubation. The slower respiration on cis -9, trans -11-C 18:2 is suggested to arise from lower Carnitine-acylCarnitine translocase activity towards the acylCarnitine form, causing an upstream increase in the corresponding acyl-CoA.
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Mitochondrial respiration on rumenic and linoleic acids.
Biochemical Society transactions, 2001Co-Authors: Pierre Clouet, Laurent Demizieux, Joseph Gresti, Pascal DegraceAbstract:Rumenic acid (cis-9, trans-11-C(18:2)) represents approx. 80% of conjugated linoleic acid (CLA) in dairy products. CLA has been shown to exert beneficial effects on health, but little work has been devoted to the ability to oxidize CLA isomers and the role of these isomers in the modulation of beta-oxidation flux. In the present study, respiration on rumenic acid was compared with that on linoleic acid (cis-9, cis-12-C(18:2)) with the use of rat liver mitochondria. In state-3, respiration was decreased by half with rumenic acid in comparison with linoleic acid. In the uncoupled state, respiration on CLA remained 30% lower. The lower ability to oxidize CLA was investigated through characterization of the enzymic steps. Rumenic acid was 33% less activated by acyl-CoA synthase than was linoleic acid. However, after such activation, the transfer of both acyl moieties to Carnitine by Carnitine Acyltransferase I (CAT I) was of the same order. Moreover, CAT II activity was comparable with either isomer. After prior incubation with rumenic acid, oxidation of octanoic acid by re-isolated mitochondria was unimpaired, but that of palmitoleic acid was impaired unless linoleic acid was used in the prior incubation. The slower respiration on cis-9, trans-11-C(18:2) is suggested to arise from lower Carnitine-acylCarnitine translocase activity towards the acylCarnitine form, causing an upstream increase in the corresponding acyl-CoA.
E D Saggerson - One of the best experts on this subject based on the ideXlab platform.
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Microsomal malonyl-CoA-sensitive Carnitine Acyltransferase.
Biochemical Society Transactions, 2001Co-Authors: Neil M. Broadway, Richard J. Pease, Graeme M. Birdsey, Nigel Turner, Majid Shayeghi, E D SaggersonAbstract:Liver microsomes contain two Carnitine Acyltransferase activities. One of these has properties closely corresponding to those of 88 kDa mitochondrial Carnitine palmitoyltransferase-1 (CPT-1). Antisera against CPT-1 cross-react with an 88 kDa microsomal protein, suggesting that CPT-1 may be targeted to both microsomal and mitochondrial membranes. However, no experiments using cDNAs corresponding to CPT-1 involving in vitro translation with microsomes or involving in vivo COS-1 cell transfection provided any evidence to support this hypothesis.
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Effect of membrane environment on the activity and inhibitability by malonyl-CoA of the Carnitine Acyltransferase of hepatic microsomal membranes
Biochemical Journal, 1997Co-Authors: Neil M. Broadway, E D SaggersonAbstract:We have investigated the extent to which membrane environment affects the catalytic properties of the malonyl-CoA-sensitive Carnitine Acyltransferase of liver microsomal membranes. Arrhenius-type plots of activity were linear in the absence and presence of malonyl-CoA (2.5 μ M). Sensitivity to malonyl-CoA increased with decreasing assay temperature. Partly purified enzyme displayed an increased K 0.5 (substrate concentration supporting half the maximal reaction rate) for myristoyl-CoA and a reduced sensitivity to malonyl-CoA compared with the enzyme in situ in membranes. Reconstitution with liposomes of a range of compositions restored the K 0.5 for myristoyl-CoA to values similar to that seen in native membranes. The lipid requirements for restoration of sensitivity to malonyl-CoA were more stringent. When animals were starved for 24 h the specific activity of Carnitine Acyltransferase in microsomal membrane residues was increased 3.3-fold, whereas sensitivity to malonyl-CoA was decreased to 1/2.8. When enzymes partly purified from fed and starved animals were reconstituted into crude soybean phosphatidylcholine liposomes there was no difference in sensitivity to malonyl-CoA. When partly purified enzyme from fed rats was reconstituted into liposomes prepared from microsomal membrane lipids from fed animals it was 2.2-fold more sensitive to malonyl-CoA than when reconstituted with liposomes prepared from microsomal membrane lipids from starved animals. This suggests that the physiological changes in sensitivity to malonyl-CoA are mediated via changes in membrane lipid composition rather than via modification of the enzyme protein itself. The increased specific actvity of Acyltransferase observed on starvation could not be attributed to changes in membrane lipid composition.
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Solubilization and separation of two distinct Carnitine Acyltransferases from hepatic microsomes: characterization of the malonyl-CoA-sensitive enzyme.
Biochemical Journal, 1995Co-Authors: Neil M. Broadway, E D SaggersonAbstract:Conditions have been developed for the solubilization of hepatic microsomal Carnitine Acyltransferase activity in good yield, with excellent long-term stability and with retention of malonyl-CoA sensitivity. Solubilized microsomal Carnitine Acyltransferase activity can be separated into malonyl-CoA-sensitive and -insensitive activities either by gel filtration on Superdex 200 or by anion-exchange chromatography on Resource Q. On gel filtration the apparent molecular masses of the malonyl-CoA-sensitive and -insensitive activities are approx. 300 kDa and 60 kDa respectively. The malonyl-CoA-sensitive and -insensitive activities have different fatty-acyl-chain-length specificities and different stabilities in the detergent octyl glucoside. Together these findings indicate that the malonyl-CoA-sensitive and -insensitive activities are due to different enzymes. The malonyl-CoA sensitivity of the inhibitable enzyme is markedly increased on reconstitution into soybean L-alpha-lecithin liposomes, demonstrating that phospholipids play a crucial role in the inhibition by this metabolite. Evidence is also provided that the malonyl-CoA-sensitive microsomal Carnitine Acyltransferase is a different enzyme from the malonyl-CoA-sensitive Carnitine palmitoyltransferase found in the mitochondrial outer membrane. The possible physiological role of the two microsomal Acyltransferases is discussed.
Ousmane Diallo - One of the best experts on this subject based on the ideXlab platform.
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increased missense mutation burden of fatty acid metabolism related genes in nunavik inuit population
PLOS ONE, 2015Co-Authors: Sirui Zhou, Lan Xiong, Pingxing Xie, Amirthagowri Ambalavanan, Cynthia V. Bourassa, Dan Spiegelman, Maude Turcotte Gauthier, Edouard Henrion, Alexandre Dionnelaporte, Ousmane DialloAbstract:Background Nunavik Inuit (northern Quebec, Canada) reside along the arctic coastline where for generations their daily energy intake has mainly been derived from animal fat. Given this particular diet it has been hypothesized that natural selection would lead to population specific allele frequency differences and unique variants in genes related to fatty acid metabolism. A group of genes, namely CPT1A, CPT1B, CPT1C, CPT2, CRAT and CROT, encode for three Carnitine Acyltransferases that are important for the oxidation of fatty acids, a critical step in their metabolism. Methods Exome sequencing and SNP array genotyping were used to examine the genetic variations in the six genes encoding for the Carnitine Acyltransferases in 113 Nunavik Inuit individuals. Results Altogether ten missense variants were found in genes CPT1A, CPT1B, CPT1C, CPT2 and CRAT, including three novel variants and one Inuit specific variant CPT1A p.P479L (rs80356779). The latter has the highest frequency (0.955) compared to other Inuit populations. We found that by comparison to Asians or Europeans, the Nunavik Inuit have an increased mutation burden in CPT1A, CPT2 and CRAT; there is also a high level of population differentiation based on Carnitine Acyltransferase gene variations between Nunavik Inuit and Asians. Conclusion The increased number and frequency of deleterious variants in these fatty acid metabolism genes in Nunavik Inuit may be the result of genetic adaptation to their diet and/or the extremely cold climate. In addition, the identification of these variants may help to understand some of the specific health risks of Nunavik Inuit.
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Genomic features of Carnitine Acyltransferase genes.
2015Co-Authors: Sirui Zhou, Lan Xiong, Pingxing Xie, Amirthagowri Ambalavanan, Cynthia V. Bourassa, Alexandre Dionne-laporte, Dan Spiegelman, Maude Turcotte Gauthier, Edouard Henrion, Ousmane DialloAbstract:Genomic features of Carnitine Acyltransferase genes.
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Coding variants of Carnitine Acyltransferase genes discovered in Nunavik Inuit.
2015Co-Authors: Sirui Zhou, Lan Xiong, Pingxing Xie, Amirthagowri Ambalavanan, Cynthia V. Bourassa, Alexandre Dionne-laporte, Dan Spiegelman, Maude Turcotte Gauthier, Edouard Henrion, Ousmane DialloAbstract:Coding variants of Carnitine Acyltransferase genes discovered in Nunavik Inuit.
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Variant frequencies and deleterious score predictions of Carnitine Acyltransferase genes.
2015Co-Authors: Sirui Zhou, Lan Xiong, Pingxing Xie, Amirthagowri Ambalavanan, Cynthia V. Bourassa, Alexandre Dionne-laporte, Dan Spiegelman, Maude Turcotte Gauthier, Edouard Henrion, Ousmane DialloAbstract:Variant frequencies and deleterious score predictions of Carnitine Acyltransferase genes.
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Deleterious scores of all rare missense variants in Carnitine Acyltransferase genes found in Nunavik Inuit and 1KGP Asians.
2015Co-Authors: Sirui Zhou, Lan Xiong, Pingxing Xie, Amirthagowri Ambalavanan, Cynthia V. Bourassa, Alexandre Dionne-laporte, Dan Spiegelman, Maude Turcotte Gauthier, Edouard Henrion, Ousmane DialloAbstract:Deleterious scores of all rare missense variants in Carnitine Acyltransferase genes found in Nunavik Inuit and 1KGP Asians.
Neil M. Broadway - One of the best experts on this subject based on the ideXlab platform.
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Microsomal malonyl-CoA-sensitive Carnitine Acyltransferase.
Biochemical Society Transactions, 2001Co-Authors: Neil M. Broadway, Richard J. Pease, Graeme M. Birdsey, Nigel Turner, Majid Shayeghi, E D SaggersonAbstract:Liver microsomes contain two Carnitine Acyltransferase activities. One of these has properties closely corresponding to those of 88 kDa mitochondrial Carnitine palmitoyltransferase-1 (CPT-1). Antisera against CPT-1 cross-react with an 88 kDa microsomal protein, suggesting that CPT-1 may be targeted to both microsomal and mitochondrial membranes. However, no experiments using cDNAs corresponding to CPT-1 involving in vitro translation with microsomes or involving in vivo COS-1 cell transfection provided any evidence to support this hypothesis.
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Effect of membrane environment on the activity and inhibitability by malonyl-CoA of the Carnitine Acyltransferase of hepatic microsomal membranes
Biochemical Journal, 1997Co-Authors: Neil M. Broadway, E D SaggersonAbstract:We have investigated the extent to which membrane environment affects the catalytic properties of the malonyl-CoA-sensitive Carnitine Acyltransferase of liver microsomal membranes. Arrhenius-type plots of activity were linear in the absence and presence of malonyl-CoA (2.5 μ M). Sensitivity to malonyl-CoA increased with decreasing assay temperature. Partly purified enzyme displayed an increased K 0.5 (substrate concentration supporting half the maximal reaction rate) for myristoyl-CoA and a reduced sensitivity to malonyl-CoA compared with the enzyme in situ in membranes. Reconstitution with liposomes of a range of compositions restored the K 0.5 for myristoyl-CoA to values similar to that seen in native membranes. The lipid requirements for restoration of sensitivity to malonyl-CoA were more stringent. When animals were starved for 24 h the specific activity of Carnitine Acyltransferase in microsomal membrane residues was increased 3.3-fold, whereas sensitivity to malonyl-CoA was decreased to 1/2.8. When enzymes partly purified from fed and starved animals were reconstituted into crude soybean phosphatidylcholine liposomes there was no difference in sensitivity to malonyl-CoA. When partly purified enzyme from fed rats was reconstituted into liposomes prepared from microsomal membrane lipids from fed animals it was 2.2-fold more sensitive to malonyl-CoA than when reconstituted with liposomes prepared from microsomal membrane lipids from starved animals. This suggests that the physiological changes in sensitivity to malonyl-CoA are mediated via changes in membrane lipid composition rather than via modification of the enzyme protein itself. The increased specific actvity of Acyltransferase observed on starvation could not be attributed to changes in membrane lipid composition.
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Solubilization and separation of two distinct Carnitine Acyltransferases from hepatic microsomes: characterization of the malonyl-CoA-sensitive enzyme.
Biochemical Journal, 1995Co-Authors: Neil M. Broadway, E D SaggersonAbstract:Conditions have been developed for the solubilization of hepatic microsomal Carnitine Acyltransferase activity in good yield, with excellent long-term stability and with retention of malonyl-CoA sensitivity. Solubilized microsomal Carnitine Acyltransferase activity can be separated into malonyl-CoA-sensitive and -insensitive activities either by gel filtration on Superdex 200 or by anion-exchange chromatography on Resource Q. On gel filtration the apparent molecular masses of the malonyl-CoA-sensitive and -insensitive activities are approx. 300 kDa and 60 kDa respectively. The malonyl-CoA-sensitive and -insensitive activities have different fatty-acyl-chain-length specificities and different stabilities in the detergent octyl glucoside. Together these findings indicate that the malonyl-CoA-sensitive and -insensitive activities are due to different enzymes. The malonyl-CoA sensitivity of the inhibitable enzyme is markedly increased on reconstitution into soybean L-alpha-lecithin liposomes, demonstrating that phospholipids play a crucial role in the inhibition by this metabolite. Evidence is also provided that the malonyl-CoA-sensitive microsomal Carnitine Acyltransferase is a different enzyme from the malonyl-CoA-sensitive Carnitine palmitoyltransferase found in the mitochondrial outer membrane. The possible physiological role of the two microsomal Acyltransferases is discussed.
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Inhibition of liver microsomal Carnitine Acyltransferases by sulphonylurea drugs.
FEBS letters, 1995Co-Authors: Neil M. Broadway, E.david SaggersonAbstract:The sulphonylureas glibenclamide and tolbutamide inhibited Carnitine Acyltransferase activities in rat liver microsomes. Glibenclamide was a more potent inhibitor than tolbutamide. The effect of tolbutamide on the malonyl-CoA-inhibitable transferase was influenced by the phospholipid/detergent environment whereas the effect of glibenclamide was not. Glibenclamide was a more potent inhibitor of the malonyl-CoA-inhibitable transferase than of the malonyl-CoA-insensitive enzyme. The extent of inhibition of the malonyl-CoA-inhibitable transferase by tolbutamide was similar to its effect on VLDL triacylglycerol secretion as reported by Wiggins and Gibbons [Biochem. J. 284 (1992) 457–462] possibly supporting the suggestion that microsomal Carnitine Acyltransferases are involved in VLDL triacylglycerol assembly/secretion.