A functional gene encoding carnitine palmitoyltransferase 1 and its transcriptional and kinetic regulation during...

Carnitine palmitoyltransferase 1 (CPT1) plays an essential role in maintaining energy supply via fatty acid oxidation, especially under fasting. In this study, the complete cDNA sequence of cpt1a was cloned from liver of large yellow croaker (Larimichthys crocea), with an open reading frame of 2319 bp encoding a protein of 772 amino acids. Bioinformatics analysis predicted the presence of conserved functional motifs and amino acid residues. The highest mRNA expression of cpt1a was observed in the liver. Phylogenetic tree clearly shows that CPT1A protein is a homologue of mammalian CPT1A. Recombinant protein rCPT1A showed catalytic activity, with Michaelis constant (km) (≈1.38 mM) and maximal reaction rates (Vmax) for carnitine (≈12.66 nmols/min/mg protein). The cpt1a mRNA expression dramatically increased and CPT1 activity remained unchanged after fasting. Fasting did not significantly change Vmax and free carnitine (FC) content in liver. Interestingly, catalytic efficiency (Vmax/Km) and FC/Km increased in fish fasted for 4 days, implying FC contents might be enough to ensure the optimal fatty acid oxidation. Contrarily, both indicators declined when fish fasted for 12 days. The present results demonstrated cpt1a has a biological function and showed that the transcriptional and kinetic regulation of CPT1 during fasting, emphasizing that fasting-induced fatty acid oxidation depends on changes in kinetic properties instead of CPT1 activity and transcription.