An acyl-CoA dehydrogenase microplate activity assay using recombinant porcine electron transfer flavoprotein.
Acyl-CoA dehydrogenase
Electron transfer flavoprotein
Enzyme activity assay
Fatty acid oxidation
Mitochondria
Journal
Analytical biochemistry
ISSN: 1096-0309
Titre abrégé: Anal Biochem
Pays: United States
ID NLM: 0370535
Informations de publication
Date de publication:
15 09 2019
15 09 2019
Historique:
received:
26
04
2019
revised:
03
06
2019
accepted:
03
06
2019
pubmed:
14
6
2019
medline:
20
3
2020
entrez:
14
6
2019
Statut:
ppublish
Résumé
Acyl-CoA dehydrogenases (ACADs) play key roles in the mitochondrial catabolism of fatty acids and branched-chain amino acids. All nine characterized ACAD enzymes use electron transfer flavoprotein (ETF) as their redox partner. The gold standard for measuring ACAD activity is the anaerobic ETF fluorescence reduction assay, which follows the decrease of pig ETF fluorescence as it accepts electrons from an ACAD in vitro. Although first described 35 years ago, the assay has not been widely used due to the need to maintain an anaerobic assay environment and to purify ETF from pig liver mitochondria. Here, we present a method for expressing recombinant pig ETF in E coli and purifying it to homogeneity. The recombinant protein is virtually pure after one chromatography step, bears higher intrinsic fluorescence than the native enzyme, and provides enhanced activity in the ETF fluorescence reduction assay. Finally, we present a simplified protocol for removing molecular oxygen that allows adaption of the assay to a 96-well plate format. The availability of recombinant pig ETF and the microplate version of the ACAD activity assay will allow wide application of the assay for both basic research and clinical diagnostics.
Identifiants
pubmed: 31194945
pii: S0003-2697(19)30408-7
doi: 10.1016/j.ab.2019.06.003
pmc: PMC6661201
mid: NIHMS1532453
pii:
doi:
Substances chimiques
Electron-Transferring Flavoproteins
0
Fatty Acids
0
Recombinant Proteins
0
Acyl-CoA Dehydrogenases
EC 1.3.-
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
113332Subventions
Organisme : NIDDK NIH HHS
ID : R01 DK045482
Pays : United States
Organisme : NIDDK NIH HHS
ID : R01 DK078775
Pays : United States
Organisme : NIDDK NIH HHS
ID : R01 DK090242
Pays : United States
Organisme : NIDDK NIH HHS
ID : R01 DK109907
Pays : United States
Informations de copyright
Copyright © 2019 Elsevier Inc. All rights reserved.
Références
Biochim Biophys Acta. 2000 May 31;1485(2-3):121-8
pubmed: 10832093
Methods Enzymol. 2000;324:241-58
pubmed: 10989435
Biochemistry. 2002 Sep 17;41(37):11126-33
pubmed: 12220177
Mol Genet Metab. 2002 Sep-Oct;77(1-2):68-79
pubmed: 12359132
Eur J Biochem. 2004 Feb;271(3):494-508
pubmed: 14728676
J Biol Chem. 2005 Sep 16;280(37):32309-16
pubmed: 16020546
Am J Hum Genet. 2007 Jul;81(1):87-103
pubmed: 17564966
Cardiovasc Res. 2008 Jul 15;79(2):331-40
pubmed: 18339649
Obesity (Silver Spring). 2009 Jan;17(1):196-8
pubmed: 18948967
Biochim Biophys Acta. 2009 Aug;1791(8):806-15
pubmed: 19465148
PLoS One. 2009 Jul 30;4(7):e6449
pubmed: 19649258
Nature. 2010 Mar 4;464(7285):121-5
pubmed: 20203611
J Inherit Metab Dis. 2010 Oct;33(5):479-94
pubmed: 20490924
Hum Mutat. 2013 Mar;34(3):515-24
pubmed: 23281178
Anal Biochem. 1990 May 1;186(2):280-4
pubmed: 2363500
Cell Rep. 2013 Aug 29;4(4):842-51
pubmed: 23954790
J Biol Chem. 2013 Nov 22;288(47):33837-47
pubmed: 24121500
Cell Metab. 2013 Dec 3;18(6):920-33
pubmed: 24315375
Mol Cell Proteomics. 2014 Mar;13(3):811-22
pubmed: 24403596
J Biol Chem. 2014 Apr 11;289(15):10668-79
pubmed: 24591516
Cell Rep. 2014 Sep 25;8(6):1930-1942
pubmed: 25242319
PLoS One. 2015 Mar 26;10(3):e0122297
pubmed: 25811481
Sci Rep. 2015 Mar 31;5:9520
pubmed: 25825284
Annu Rev Physiol. 2016;78:23-44
pubmed: 26474213
FEBS J. 2016 Oct;283(19):3587-3603
pubmed: 27486859
Breast Cancer Res Treat. 2016 Nov;160(2):371-383
pubmed: 27722841
J Biol Chem. 2017 Jun 16;292(24):10239-10249
pubmed: 28458255
Orphanet J Rare Dis. 2018 Jul 19;13(1):120
pubmed: 30025539
J Inherit Metab Dis. 1987;10 Suppl 1:159-200
pubmed: 3119938
JIMD Rep. 2019 Mar 26;47(1):30-34
pubmed: 31240164
Biochem J. 1988 Nov 1;255(3):869-76
pubmed: 3145738
Biochem Med. 1985 Feb;33(1):38-44
pubmed: 3994700
J Biol Chem. 1985 Oct 25;260(24):12998-3002
pubmed: 4055729
Biochemistry. 1985 Nov 19;24(24):6830-9
pubmed: 4074729
Biochem J. 1984 May 1;219(3):1043-7
pubmed: 6743239
Biochem J. 1983 Feb 1;209(2):541-5
pubmed: 6847633
J Biol Chem. 1983 Jun 25;258(12):7563-9
pubmed: 6863254
Anal Biochem. 1993 Nov 1;214(2):528-34
pubmed: 8109745
Biochem Med Metab Biol. 1993 Jun;49(3):351-62
pubmed: 8347379
Eur J Biochem. 1993 May 1;213(3):1003-8
pubmed: 8504797
Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14355-60
pubmed: 8962055
Biochim Biophys Acta. 1998 Jul 31;1393(1):35-40
pubmed: 9714723
Neurology. 1999 Jan 15;52(2):366-72
pubmed: 9932958