Premium
Development of a High‐Density Assay for Long‐Chain Fatty Acyl‐CoA Elongases
Author(s) -
Kitazawa Hidefumi,
Miyamoto Yasuhisa,
Shimamura Ken,
Nagumo Akira,
Tokita Shigeru
Publication year - 2009
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/s11745-009-3320-8
Subject(s) - malonyl coa , fatty acid , elongation , substrate (aquarium) , chemistry , clinical chemistry , polyunsaturated fatty acid , chromatography , long chain , biochemistry , beta oxidation , biology , materials science , ecology , polymer science , metallurgy , ultimate tensile strength
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.