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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. and Jackson, V.N. and Van der Leij, F.R. and Cameron, J.M. and Travers, M. and Bartelds, B. and Huijkman, N.C. and Zammit, V.A. (2003) 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. Biochemical Journal, 372. pp. 871-879. ISSN 0264-6021

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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.