Molecular Cloning of cDNA Encoding Rat Very Long-chain Acyl-CoA Synthetase

Abstract

The cDNA encoding rat very long-chain acyl-CoA synthetase (VLACS) was cloned, using degenerative primers synthesized according to the partial amino acid sequences of the peptide fragments of the purified rat liver enzyme. The longest cDNA insert was 2972 base pairs with a 1860-base pair open reading frame encoding 620 amino acids. The calculated molecular mass of 70,692 daltons was consistent with size of the purified enzyme. In Northern blot analysis, a single band was detected at the position of about 3 kilobases, corresponding to the size of the cloned cDNA. cDNA-directed expression in Escherichia coli resulted in accumulation of expressed protein, as an inclusion body. An antibody was raised using this expressed protein to characterize the cDNA and the enzyme. The subcellular localization of VLACS in peroxisomes and microsomes was demonstrated in Western blot analysis. The specific activity and the substrate specificity of the cDNA expressed enzyme in COS-1 cells were consistent with those of the purified rat enzyme. The predicted amino acid sequence of VLACS had a high sequence similarity to fatty acid transport protein (Schaffer, J. E., and Lodish, H. F. (1994) Cell 79, 427-436), and was considered to have domains for adenylation and thioester formation. The entire structure of VLACS was dissimilar to that of longchain acyl-CoA synthetase

Molecular cloning and expression of rat liver bile acid CoA ligase

“…Figure 5 shows the phylogenetic tree of the comparison of the translation of rBAL with the sequences of six very long-chain acyl-CoA synthetases (VLACS) and two very long-chain acyl-CoA synthetase related proteins. rBAL is 65.2-65.5% similar and 42.3-43.9% identical to VLACS proteins cloned from rat (14), mouse (15), and human (16,17). rBAL is most homologous to a mouse VLACSrelated protein (87.2% identity, 93.3% similarity) (18).…”

Characterization of the Acyl-CoA Synthetase Activity of Purified Murine Fatty Acid Transport Protein 1

“…Sequence analysis of cloned FATP family members revealed two sequence motifs indicative of acyl-CoA synthetases. Subsequent studies have demonstrated that the overexpression of FATP1, FATP2, or FATP4 in COS1 cells increases cell-associated acyl-CoA synthetase activities (11,26,27). Moreover, mutation of serine 250 to alanine significantly reduced fatty acid uptake, suggesting that esterification of fatty acids is coupled to influx (28).…”

Evidence for the physical association of mitochondrial fatty acid oxidation and oxidative phosphorylation complexes

“…Rotenone is a specific inhibitor of complex I. 3-Mercaptopropionic acid (MPA) inhibits long chain ACADs (30). Kinetic parameters for cytochrome c reduction were calculated using a nonlinear regression algorithm as previously described (29,31).…”