A fluorescently labeled intestinal fatty acid binding protein. Interactions with fatty acids and its use in monitoring free fatty acids.
The fatty acid-binding protein from rat intestine (I-FABP) has been covalently modified with the fluorescent compound Acrylodan. Acrylodan was found to label Lys27, one of the few amino acid residues found by x-ray diffraction studies to change orientation upon fatty acid (FA) binding to I-FABP. Binding of FA to this Acrylodan-modified I-FABP (ADIFAB) induces a large shift in fluorescence emission wavelength from 432 to 505 nm. As a consequence, the ratio of emission intensities provides a direct measure of the concentration of FA bound to the protein. Binding of FA is well described by single site equilibrium for FA concentrations below the critical micelle concentration. ADIFAB dissociation constants (Kd) determined at 37 degrees C and at concentrations below the critical micelle concentration for oleate, palmitate, linoleate, arachidonate, and linolenate were, respectively, 0.28, 0.33, 0.97, 1.6, and 2.5 microM. The variation of these Kd values with FA molecular species is highly correlated with the solubility of the FA in water, suggesting that all these FA bind with a similar conformation in the I-FABP binding site. The ADIFAB response together with the measured equilibrium constants allows a direct determination of the concentration of long chain free fatty acid (FFA) in the concentration range, depending upon the FA molecular species, between 1 nM and > 20 microM. As an example of its use as a probe to measure FFA levels, ADIFAB is used here to monitor the time course for FFA release from IgE receptor- and ionomycin-activated rat basophilic leukemia (RBL) cells.
Richieri
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GV
Division of Membrane Biology, Medical Biology Inst., La Jolla, CA 92037.
Ogata
R T
RT
Kleinfeld
A M
AM
D015081:2-Naphthylamine / Q000031:analogs & derivatives*; D000818:Animals; D000831:Animals, Newborn; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D005230:Fatty Acids, Nonesterified / Q000032:analysis* / Q000378:metabolism*; D005456:Fluorescent Dyes; D015723:Gene Library; D007413:Intestinal Mucosa / Q000378:metabolism; D008823:Micelles; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D002352:Carrier Proteins; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D005230:Fatty Acids, Nonesterified; D005456:Fluorescent Dyes; D008823:Micelles; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C038518:acrylodan; D015081:2-Naphthylamine