We established a convenient
assay method for measuring elongation of very
long chain fatty acids (ELOVLs) using a Unifilter-96 GF/C plate. The Unifilter GF/C plate preferentially interacts with hydrophobic end products of ELOVLs (i.e.,
long chain fatty acid), with minimal malonyl-CoA (C2 unit donor for
fatty acid elongation) interaction. This new method results in the quick separation and detection of [(14)C] incorporated end products (e.g., [(14)C] palmitoyl-CoA) from reaction mixtures containing excessive amounts of [(14)C] malonyl-CoA. In the Unifilter-96 GF/C plate
assay, recombinantly expressed human ELOVLs (i.e., ELOVL1,-2,-3,-5 and -6) displayed appreciable
assay windows (>2-fold vs. mock-transfected control), enabling us to conduct comprehensive
substrate profiling of ELOVLs. The
substrate concentration profile of ELOVL6 in the Unifilter-96 GF/C plate
assay is consistent with that obtained from the conventional
liquid extraction method, thus, supporting the reliability of the Unifilter-96 GF/C plate
assay. We then examined the
substrate specificities of ELOVLs in a comprehensive fashion. As previously reported, ELOVL1, -3 and -6 preferably elongated the saturated fatty acyl-CoAs while ELOVL2 and
ELOVL5 preferentially elongated the
polyunsaturated fatty acyl-CoAs. This further confirms the Unifilter-96 GF/C plate
assay reliability. Taken together, our newly developed
assay provides a convenient and comprehensive
assay platform for ELOVLs, allowing investigators to conduct high
density screening and characterization of ELOVLs chemical tools.
