title: Characterization of recombinant long-chain rat acyl-CoA synthetase isoforms 3 and 6: identification of a novel variant of isoform 6.
abstract: The metabolism of long-chain fatty acids in brain and their incorporation into signaling molecules such as diacylglycerol and LPA and into structural components of membranes, including myelin, requires activation by long-chain acyl-CoA synthetase (ACSL). Because ACSL3 and ACSL6 are the predominant ACSL isoforms in brain, we cloned and characterized these isoforms from rat brain and identified a novel ACSL6 clone (ACSL6_v2). ACSL6_v2 and the previously reported ACSL6_v1 represent splice variants that include exon 13 or 14, respectively. Homologue sequences of both of these variants are present in the human and mouse databases. ACSL3, ACSL6_v1, and ACSL6_v2 with Flag-epitopes at the C-termini were expressed in Escherichia coli and purified on Flag-affinity columns. The three recombinant proteins were characterized. Compared to ACSL4, another brain isoform, ACSL3, ACSL6_v1, and ACSL6_v2 showed similarities in kinetic values for CoA, palmitate, and arachidonate, but their apparent Km values for oleate were 4- to 6-fold lower than for ACSL4. In a direct competition assay with palmitate, all the polyunsaturated fatty acids tested were strong competitors only for ACSL4 with IC50 values of 0.5 to 5 microM. DHA was also strongly preferred by ACSL6_v2. The apparent Km value for ATP of ACSL6_v1 was 8-fold higher than that of ACSL6_v2. ACSL3 and the two variants of ACSL6 were more resistant than ACSL4 to heat inactivation. Despite the high amino acid identity between ACSL3 and ACSL4, rosiglitazone inhibited only ACSL4. Triacsin C, an inhibitor of ACSL1 and ACSL4, also inhibited ACSL3, but did not inhibit the ACSL6 variants. These data further document important differences in the closely related ACSL isoforms and show that amino acid changes near the consensus nucleotide binding site alter function in the two splice variants of ACSL6.
authors.affiliation: Department of Nutrition, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: Cynthia G
authors.initials: CG
authors.lastname: Van Horn
authors.forename: Jorge M
authors.initials: JM
authors.lastname: Caviglia
authors.forename: Lei O
authors.initials: LO
authors.lastname: Li
authors.forename: Shuli
authors.initials: S
authors.lastname: Wang
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Granger
authors.forename: Rosalind A
authors.initials: RA
authors.lastname: Coleman
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000378:metabolism; D006358:Hot Temperature; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D017830:Octoxynol / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D000077154:Rosiglitazone; D045162:Thiazolidinediones / Q000737:chemistry; D014226:Triazenes / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D005231:Fatty Acids, Unsaturated; D007527:Isoenzymes; D011994:Recombinant Proteins; D045162:Thiazolidinediones; D014226:Triazenes; D000077154:Rosiglitazone; C034613:triacsin C; D017830:Octoxynol; C500899:Acsl6 protein, rat; D003066:Coenzyme A Ligases