Binding of fluorescent fatty acids to bovine liver non-specific lipid-transfer protein (nsL-TP) was assessed by measuring fluorescence resonance energy transfer (FRET) between the single tryptophan residue of nsL-TP and the fluorophore. Upon addition of pyrene dodecanoic acid (Pyr-C12) and cis-parinaric acid to nsL-TP, FRET was observed indicating that these fatty acids were accommodated in the lipid binding site closely positioned to the tryptophan residue. Substantial binding was observed only when these fatty acids were presented in the monomeric form complexed to β-cyclodextrin. As shown by time-resolved fluorescence measurements, translocation of Pyr-C12 from the Pyr-C12–β-cyclodextrin complex to nsL-TP changed dramatically the direct molecular environment of the pyrene moiety: i.e. the fluorescence lifetime of the directly excited pyrene increased at least by 25% and a distinct rotational correlation time of 7 ns was observed. In order to evaluate the affinity of nsL-TP for intermediates of the β-oxidation pathway, a binding assay was developed based on the ability of fatty acyl derivatives to displace Pyr-C12 from the lipid binding site as reflected by the reduction of FRET. Hexadecanoyl-CoA and 2-hexadecenoyl-CoA were found to bind readily to nsL-TP, whereas 3-hydroxyhexadecanoyl-CoA and 3-ketohexadecanoyl-CoA bound poorly. The highest affinities were observed for the very-long-chain fatty acyl-CoA esters (24:0-CoA, 26:0-CoA) and their enoyl derivatives (24:1-CoA, 26:1-CoA). Binding of non-esterified hexadecanoic acid and tetracosanoic acid (24:0) was negligible.
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Research Article|
March 25 1999
High-affinity binding of very-long-chain fatty acyl-CoA esters to the peroxisomal non-specific lipid-transfer protein (sterol carrier protein-2)
Tobias B. DANSEN;
Tobias B. DANSEN
*Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
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Jan WESTERMAN;
Jan WESTERMAN
*Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
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Fred S. WOUTERS;
Fred S. WOUTERS
*Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
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Ronald J. A. WANDERS;
Ronald J. A. WANDERS
†Departments of Clinical Chemistry and Pediatrics, Academical Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
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Arie van HOEK;
Arie van HOEK
‡MicroSpectroscopy Center, Department of Biomolecular Sciences, Wageningen Agricultural University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands
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Theodorus W. J. GADELLA, Jr.;
Theodorus W. J. GADELLA, Jr.
‡MicroSpectroscopy Center, Department of Biomolecular Sciences, Wageningen Agricultural University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands
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Karel W. A. WIRTZ
Karel W. A. WIRTZ
1
*Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
1To whom correspondence should be addressed (e-mail k.w.a.wirtz@chem.uu.nl).
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Publisher: Portland Press Ltd
Received:
September 18 1998
Revision Received:
December 07 1998
Accepted:
January 20 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 339 (1): 193–199.
Article history
Received:
September 18 1998
Revision Received:
December 07 1998
Accepted:
January 20 1999
Citation
Tobias B. DANSEN, Jan WESTERMAN, Fred S. WOUTERS, Ronald J. A. WANDERS, Arie van HOEK, Theodorus W. J. GADELLA, Karel W. A. WIRTZ; High-affinity binding of very-long-chain fatty acyl-CoA esters to the peroxisomal non-specific lipid-transfer protein (sterol carrier protein-2). Biochem J 1 April 1999; 339 (1): 193–199. doi: https://doi.org/10.1042/bj3390193
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