Development of a high-density assay for long-chain fatty acyl-CoA elongases.
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.
Kitazawa
Hidefumi
H
Department of Metabolic Disorder Research, Tsukuba Research Institute, BANYU Pharmaceutical Co., Ltd., Okubo 3, Tsukuba, Ibaraki, Japan.
Miyamoto
Yasuhisa
Y
Shimamura
Ken
K
Nagumo
Akira
A
Tokita
Shigeru
S
D000123:Acetyltransferases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D000081031:Fatty Acid Elongases; D006801:Humans; D015592:Immunoradiometric Assay / Q000379:methods; D008316:Malonyl Coenzyme A / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D014162:Transfection
D000214:Acyl Coenzyme A; C000630949:ELOVL1 protein, human; C000630967:ELOVL2 protein, human; C000630954:ELOVL5 protein, human; C000630955:ELOVL6 protein, human; D010171:Palmitoyl Coenzyme A; D008316:Malonyl Coenzyme A; D000123:Acetyltransferases; D000081031:Fatty Acid Elongases