Cloning and expression of the liver and muscle isoforms of ovine carnitine palmitoyltransferase 1: residues within the n-terminus of the muscle isoform influence the kinetic properties of the enzyme

N.T. Price, V.N. Jackson, F.R. Van der Leij, J.M. Cameron, M. Travers, B. Bartelds, N.C. Huijkman, V.A. Zammit

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Fatty acid and ketone body metabolism differ considerably between monogastric and ruminant species. The regulation of the key enzymes involved may differ accordingly. Carnitine palmitoyltransferase 1 (CPT 1) is the key locus for the control of long-chain fatty acid b-oxidation and liver ketogenesis. Previously we showed that CPT 1 kinetics in sheep and rat liver mitochondria differ. We cloned cDNAs for both isoforms [liver- (L-) and muscle- (M-)] of ovine CPT 1 in order to elucidate the structural features of these proteins and their genes (CPT1A and CPT1B). Their deduced amino acid sequences show a high degree of conservation compared with orthologues from other mammalian species, with the notable exception of the N-terminus of ovine M-CPT 1. These differences were also present in bovine M-CPT 1, whose N-terminal sequence we determined. In addition, the 5´-end of the sheep CPT1B cDNA suggested a different promoter architecture when compared with previously characterized CPT1B genes. Northern blotting revealed differences in tissue distribution for both CPT1A and CPT1B transcripts compared with other species. In particular, ovine CPT1B mRNA was less tissue restricted, and the predominant transcript in the pancreas was CPT1B. Expression in yeast allowed kinetic characterization of the two native enzymes, and of a chimaera in which the distinctive N-terminal segment of ovine M-CPT 1 was replaced with that from rat M-CPT 1. The ovine N-terminal segment influences the kinetics of the enzyme for both its substrates, such that the Km for palmitoyl-CoA is decreased and that for carnitine is increased for the chimaera, relative to the parental ovine M-CPT 1.
Original languageEnglish
Pages (from-to)871-879
Number of pages9
JournalBiochemical Journal
Volume372
DOIs
Publication statusPublished - 2003

Fingerprint

Carnitine O-Palmitoyltransferase
Cloning
Liver
Muscle
Organism Cloning
Sheep
Protein Isoforms
Muscles
Kinetics
Enzymes
Rats
Fatty Acids
Complementary DNA
Tissue
Palmitoyl Coenzyme A
Ketone Bodies
Mitochondria
Internal-External Control
Carnitine
Liver Mitochondrion

Keywords

  • acyltransferase
  • malonyl-CoA
  • fatty acid metabolism

Cite this

Price, N.T. ; Jackson, V.N. ; Van der Leij, F.R. ; Cameron, J.M. ; Travers, M. ; Bartelds, B. ; Huijkman, N.C. ; Zammit, V.A. / Cloning and expression of the liver and muscle isoforms of ovine carnitine palmitoyltransferase 1: residues within the n-terminus of the muscle isoform influence the kinetic properties of the enzyme. In: Biochemical Journal. 2003 ; Vol. 372. pp. 871-879.
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abstract = "Fatty acid and ketone body metabolism differ considerably between monogastric and ruminant species. The regulation of the key enzymes involved may differ accordingly. Carnitine palmitoyltransferase 1 (CPT 1) is the key locus for the control of long-chain fatty acid b-oxidation and liver ketogenesis. Previously we showed that CPT 1 kinetics in sheep and rat liver mitochondria differ. We cloned cDNAs for both isoforms [liver- (L-) and muscle- (M-)] of ovine CPT 1 in order to elucidate the structural features of these proteins and their genes (CPT1A and CPT1B). Their deduced amino acid sequences show a high degree of conservation compared with orthologues from other mammalian species, with the notable exception of the N-terminus of ovine M-CPT 1. These differences were also present in bovine M-CPT 1, whose N-terminal sequence we determined. In addition, the 5´-end of the sheep CPT1B cDNA suggested a different promoter architecture when compared with previously characterized CPT1B genes. Northern blotting revealed differences in tissue distribution for both CPT1A and CPT1B transcripts compared with other species. In particular, ovine CPT1B mRNA was less tissue restricted, and the predominant transcript in the pancreas was CPT1B. Expression in yeast allowed kinetic characterization of the two native enzymes, and of a chimaera in which the distinctive N-terminal segment of ovine M-CPT 1 was replaced with that from rat M-CPT 1. The ovine N-terminal segment influences the kinetics of the enzyme for both its substrates, such that the Km for palmitoyl-CoA is decreased and that for carnitine is increased for the chimaera, relative to the parental ovine M-CPT 1.",
keywords = "acyltransferase, malonyl-CoA, fatty acid metabolism",
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Cloning and expression of the liver and muscle isoforms of ovine carnitine palmitoyltransferase 1: residues within the n-terminus of the muscle isoform influence the kinetic properties of the enzyme. / Price, N.T.; Jackson, V.N.; Van der Leij, F.R.; Cameron, J.M.; Travers, M.; Bartelds, B.; Huijkman, N.C.; Zammit, V.A.

In: Biochemical Journal, Vol. 372, 2003, p. 871-879.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Cloning and expression of the liver and muscle isoforms of ovine carnitine palmitoyltransferase 1: residues within the n-terminus of the muscle isoform influence the kinetic properties of the enzyme

AU - Price, N.T.

AU - Jackson, V.N.

AU - Van der Leij, F.R.

AU - Cameron, J.M.

AU - Travers, M.

AU - Bartelds, B.

AU - Huijkman, N.C.

AU - Zammit, V.A.

PY - 2003

Y1 - 2003

N2 - Fatty acid and ketone body metabolism differ considerably between monogastric and ruminant species. The regulation of the key enzymes involved may differ accordingly. Carnitine palmitoyltransferase 1 (CPT 1) is the key locus for the control of long-chain fatty acid b-oxidation and liver ketogenesis. Previously we showed that CPT 1 kinetics in sheep and rat liver mitochondria differ. We cloned cDNAs for both isoforms [liver- (L-) and muscle- (M-)] of ovine CPT 1 in order to elucidate the structural features of these proteins and their genes (CPT1A and CPT1B). Their deduced amino acid sequences show a high degree of conservation compared with orthologues from other mammalian species, with the notable exception of the N-terminus of ovine M-CPT 1. These differences were also present in bovine M-CPT 1, whose N-terminal sequence we determined. In addition, the 5´-end of the sheep CPT1B cDNA suggested a different promoter architecture when compared with previously characterized CPT1B genes. Northern blotting revealed differences in tissue distribution for both CPT1A and CPT1B transcripts compared with other species. In particular, ovine CPT1B mRNA was less tissue restricted, and the predominant transcript in the pancreas was CPT1B. Expression in yeast allowed kinetic characterization of the two native enzymes, and of a chimaera in which the distinctive N-terminal segment of ovine M-CPT 1 was replaced with that from rat M-CPT 1. The ovine N-terminal segment influences the kinetics of the enzyme for both its substrates, such that the Km for palmitoyl-CoA is decreased and that for carnitine is increased for the chimaera, relative to the parental ovine M-CPT 1.

AB - Fatty acid and ketone body metabolism differ considerably between monogastric and ruminant species. The regulation of the key enzymes involved may differ accordingly. Carnitine palmitoyltransferase 1 (CPT 1) is the key locus for the control of long-chain fatty acid b-oxidation and liver ketogenesis. Previously we showed that CPT 1 kinetics in sheep and rat liver mitochondria differ. We cloned cDNAs for both isoforms [liver- (L-) and muscle- (M-)] of ovine CPT 1 in order to elucidate the structural features of these proteins and their genes (CPT1A and CPT1B). Their deduced amino acid sequences show a high degree of conservation compared with orthologues from other mammalian species, with the notable exception of the N-terminus of ovine M-CPT 1. These differences were also present in bovine M-CPT 1, whose N-terminal sequence we determined. In addition, the 5´-end of the sheep CPT1B cDNA suggested a different promoter architecture when compared with previously characterized CPT1B genes. Northern blotting revealed differences in tissue distribution for both CPT1A and CPT1B transcripts compared with other species. In particular, ovine CPT1B mRNA was less tissue restricted, and the predominant transcript in the pancreas was CPT1B. Expression in yeast allowed kinetic characterization of the two native enzymes, and of a chimaera in which the distinctive N-terminal segment of ovine M-CPT 1 was replaced with that from rat M-CPT 1. The ovine N-terminal segment influences the kinetics of the enzyme for both its substrates, such that the Km for palmitoyl-CoA is decreased and that for carnitine is increased for the chimaera, relative to the parental ovine M-CPT 1.

KW - acyltransferase

KW - malonyl-CoA

KW - fatty acid metabolism

U2 - 10.1042/BJ20030086

DO - 10.1042/BJ20030086

M3 - Article

VL - 372

SP - 871

EP - 879

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

ER -