Binding of fatty acids to the uncoupling protein from brown adipose tissue mitochondria.
The uncoupling protein 1 (UCP1) is a H(+) carrier which plays a key role in heat generation in brown adipose tissue. The H(+) transport activity of UCP1 is activated by long-chain fatty acids and inhibited by purine nucleotides. While nucleotide binding has been well characterized, the interaction of fatty acid with UCP1 remains unknown. Here I demonstrate the binding of fatty acids by competition with a fluorescent nucleotide probe 2(')-O-dansyl guanosine 5(')-triphosphate (GTP), which has been shown previously to bind at the nucleotide binding site in UCP1. Fatty acids but not their esters competitively inhibit the binding of 2(')-O-dansyl GTP to UCP1. The fatty acid effect was enhanced at higher pH, suggesting the binding of fatty acid anion to UCP1. The inhibition constants K(i) were determined by fluorescence titrations for various fatty acids. Short-chain (C<8) fatty acids display no affinity, whereas medium-chain (C10-14) and unsaturated C18 fatty acids exhibit stronger affinity (K(i)=65 microM, for elaidic acid). This specificity profile agrees with previous functional data obtained in both proteoliposomes and mitochondria, suggesting a possible physiological role of this fatty acid binding site.
Huang
Shu-Gui
SG
Institute of Physical Biochemistry, University of Munich, Schillerstrasse 44, Munich D-80336, Germany. fhuang@agyinc.com
D000273:Adipose Tissue / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005227:Fatty Acids / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D010100:Oxygen / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D005227:Fatty Acids; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D000071256:Uncoupling Protein 1; D006859:Hydrogen; D006160:Guanosine Triphosphate; D010100:Oxygen