A Functional Gene Encoding Carnitine Palmitoyltransferase 1 And Its Kinetic Regulation During Fasting In Large Yellow Croaker

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 cpt1 a 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 cpt1 a was expressed in liver,kidney,gill,muscle,intestine,heart,brain and spleen.The highest mRNA expression of cpt1 a was observed in the liver.Phylogenetic tree clearly shows that CPT1 A protein is a homologue of mammalian CPT1 A.Recombinant protein rCPT1 A showed catalytic activity,with Michaelis constant(Km)(?1.38 mM)and maximal reaction rates(Vmax)for carnitine(? 12.66 nmols/min/mg protein).The cpt1 a 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 that cpt1 a 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.During short-term fasting,the fish liver may have a strong capacity for ensuring the optimal catalytic efficiency through regulating carnitine-dependent Km rather than gene transcription and enzyme avtivity.However,long-term starvation may have a negative effect on CPT1 catalytic efficiency.