Molecular cloning and sequence analysis of the rat liver carnitine octanoyltransferase cDNA, its natural gene and the gene promoter

Long-chain acyl-CoA esters have potent specific actions (e.g. on gene transcription, membrane trafficking) as well as non-specific ones (e.g. on phospholipid bilayers). They are synthesized on the cytosolic aspects of several intracellular membranes, to give rise to (a) cytosolic pool(s) to which a variety of enzymes and processes have access, including some localized in the nucleus. Their concentration in cells is highly regulated, interconversion with corresponding acylcarnitines being the most important mechanism involved. This reaction is catalysed by cytosol-accessible carnitine long-chain acyl (palmitoyl) transferase activities that are themselves located on multiple membrane systems. Regulation of these activities is through the inhibitory action of malonyl-CoA. Hence the existence of a potent malonyl-CoA-acyl-CoA axis through which many processes involved in the maintenance of mammalian cell function are regulated. The molecular, topographical and physiological interactions that make this possible are described and discussed.

Section: Movement Of Acyl Moieties Across Membranes For β-Oxidationmentioning

confidence: 99%

The malonyl-CoA–long-chain acyl-CoA axis in the maintenance of mammalian cell function

Zammit

1999

“…There is considerable evidence for the presence of additional CPT activities in peroxisomes and microsomes based upon cDNA cloning, protein purification and antibody studies [15][16][17]. L-CPT I gene expression and enzyme activity increase dramatically in the liver at birth [18].…”

Section: Introductionmentioning

confidence: 99%

Cloning and characterization of the promoter for the liver isoform of the rat carnitine palmitoyltransferase I (L-CPT I) gene

Park

Steffen

Song

et al. 1998

Carnitine palmitoyltransferase I (CPT I) catalyses the transfer of long chain fatty acids to carnitine for translocation across the mitochondrial inner membrane. The cDNAs of two isoforms of CPT I, termed the hepatic and muscle isoforms, have been cloned. Expression of the hepatic CPT I gene (L-CPT I) is subject to developmental, hormonal and tissue specific regulation. We have cloned the promoter of the L-CPT I gene from a rat genomic library. In the L-CPT I gene, there are two exons 5ʹ to the exon containing the ATG that initiates translation. Exon 1 and the 5ʹ end of exon 2 contain sequences that were not previously described in the rat L-CPT I cDNA. There is an alternatively spliced form of the L-CPT I mRNA in which exon 2 is skipped. The proximal promoter of the L-CPT I gene is extremely GC rich and does not contain a TATA box. There are several putative Sp1 binding sites near the transcriptional start site. A 190 base pair fragment of the promoter can efficiently drive transcription of luciferase and CAT (chloramphenicol acetyltransferase) reporter genes transiently transfected into HepG2 cells. Sequences in both the first intron and the promoter contribute to basal expression. Our results provide the foundation for further studies into the regulation of L-CPTI gene expression.

“…Comparison of this region of beef liver COT with the sequence for rat liver COT shows a high degree of homology but little similarly to the other carnitine acyltransferases [6,14,15]. Indeed, the 14 residues beginning at residue 350 are completely identical…”

Section: The Site Of Modification Of Cot By Fdnbmentioning

confidence: 96%

Active sites residues of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT-II)

a'BHÁIRD

Yankovskaya

Ramsay

1998

The carnitine acyltransferases which catalyse the reversible transfer of fatty acyl groups between carnitine and coenzyme A have been proposed to contain a catalytic histidine. Here, the chemical reactivity of active site groups has been used to demonstrate differences between the active sites of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase-II (CPT-II). Treatment of CPT-II with the histidineselective reagent, diethyl pyrocarbonate (DEPC), resulted in simple linear pseudo-first-order kinetics. The reversal of the inhibition by hydroxylamine and the pK a (7n1) of the modified residue indicated that the residue was a histidine. The order of the inactivation kinetics showed that 1 mol of histidine was modified per mol of CPT-II.