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Development of a High-Density Assay for Long-Chain Fatty Acyl-CoA Elongases

  • Methods
  • Published:
Lipids

Abstract

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 [14C] incorporated end products (e.g., [14C] palmitoyl-CoA) from reaction mixtures containing excessive amounts of [14C] 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.

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Abbreviations

BSA:

Bovine serum albumin

CoA:

Coenzyme A

EDTA:

Ethylenediaminetetraacetic acid

ELOVL:

Elongase of long chain fatty acyl-CoA

LCFA:

Long chain fatty acid

NADPH:

Reduced nicotinamide adenine dinucleotide phosphate

SDS:

Sodium lauryl sulfate

TBS:

Tris-buffered saline

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Authors and Affiliations

  1. Department of Metabolic Disorder Research, Tsukuba Research Institute, BANYU Pharmaceutical Co., Ltd., Okubo 3, Tsukuba, Ibaraki, 300-2611, Japan

    Hidefumi Kitazawa, Yasuhisa Miyamoto, Ken Shimamura, Akira Nagumo & Shigeru Tokita

Authors
  1. Hidefumi Kitazawa
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  2. Yasuhisa Miyamoto
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  3. Ken Shimamura
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  4. Akira Nagumo
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  5. Shigeru Tokita
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Corresponding author

Correspondence to Shigeru Tokita.

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H. Kitazawa and Y. Miyamoto were contributed equally to this work.

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Kitazawa, H., Miyamoto, Y., Shimamura, K. et al. Development of a High-Density Assay for Long-Chain Fatty Acyl-CoA Elongases. Lipids 44, 765–773 (2009). https://doi.org/10.1007/s11745-009-3320-8

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  • DOI: https://doi.org/10.1007/s11745-009-3320-8

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